CN104138603A - Application of microRNA-7 for inhibiting prostate tumor growth and prostate tumor progress - Google Patents

Abstract

The invention relates to the application of microRNA-7 to inhibit the growth of prostate tumor and its tumor progression. In the present invention, by constructing a prostate cancer cell subclone cell line stably overexpressing miR-7, and establishing a corresponding nude mouse subcutaneous tumor-bearing animal model, it is verified in vitro and in vivo that miR-7 inhibits the growth of prostate tumor cells and prostate cancer stem cells. The role of "stem maintenance" capacity. The present invention found that after stably overexpressing miR-7, it can target and inhibit the expression of PI3K/Akt signaling pathway, reduce the activity of Akt, and significantly down-regulate the phosphorylation level of its downstream cycle regulatory protein p21, and promote the non-phosphorylated p21 protein Into the nucleus, thereby realizing the arrest of cell cycle progression in tumor cells, and inhibiting the proliferation ability of tumor cells. The present invention verifies that miR-7 has the function of inhibiting prostate tumor growth and tumor progression both in vivo and in vitro. It is suggested that natural nucleic acid molecules such as miR-7, as a new type of small molecule drug, have broad application prospects in the clinical treatment of prostate cancer.

Description

Application microRNA-7 suppresses tumor of prostate growth and tumor progression thereof

Technical field

The invention belongs to biomedicine field, be specifically related to a kind of Microrna, microRNA-7 (miR-7).In prostate gland cancer cell, stablized and expressed the miR-7 growth of inhibition tumor cell effectively.Meanwhile, the ability maintaining by suppressing prostate cancer stem cells dryness, stablized and expressed the tumor progression that miR-7 can suppress carcinoma of prostate effectively.Pointed out miR-7 can be applicable in the target gene therapy for carcinoma of prostate as natural acid micromolecular medicine.

Background technology

Carcinoma of prostate is the modal malignant tumor of male, and wherein 95% falls ill in more than 60 years old old group.Along with the intensification of China's population aging degree, approximately there are every year ten thousand carcinoma of prostate of 7-8 to increase case newly, sickness rate, with the speed increment of annual 25%-30%, becomes the fastest malignant tumor of sickness rate increase.In Beijing, Shanghai, the coastal flourishing city such as Guangzhou, carcinoma of prostate has become the major disease of harm elderly men health.Therefore, understand its tumorigenic mechanism in depth comprehensively and maybe can provide for relevant clinical treatment brand-new thinking.Existing research shows, the clinical manifestation of carcinoma of prostate and current tumor stem cell (cancer stem cells, CSCs) prevailing theoretical consistent [1].Traditional view thinks, tumor is to be formed by somatic mutation, and each tumor cell can unrestrictedly be grown.But this cannot explain that tumor cell seems to have unlimited vitality and be not the phenomenon that all tumor cells can unrestrictedly be grown.Routine treatment can not be killed (comprising chemotherapy, radiotherapy, hormonotherapy etc.) all tumor cells of tumor tissues conventionally clinically.Can not obviously there is different biological characteristicses with the killed tumor cell of energy by killed tumor cell.That is to say, tumor tissues contains dissimilar tumor cell, and different tumor cells has different molecular biosciences characteristics.Tumor tissues is inhomogenous on cell and molecular level.Wherein most cells is responsive to routine treatment, and those peanuts to the insensitive cell of routine treatment, there is high oncogenicity, high animal migration, its growth, transfer and the feature of recurrence and the fundamental characteristics of stem cell are quite similar, be called as " tumor stem cell ", Here it is " tumor stem cell " theoretical [2].Tumor stem cell theory has obtained support in acute myeloblastic leukemia, breast carcinoma, the cerebral tumor, pulmonary carcinoma, renal carcinoma, colon cancer, cancer of pancreas and melanomatous research work [3-10]: the existence of tumor stem cell, the power of its dryness, and closely related with the tumor progressions such as power, the conversion of epithelium mesenchyme and tumor prognosis and recurrence of tumor opposing chemicotherapy ability.This is theoretical for the present invention re-recognizes origin and the essence of tumor of prostate, and clinical treatment provides new direction and visual angle.

For prostate cancer stem cells, study its Basic Biological Character and relevant regulatory mechanism will contribute to the present invention to be further familiar with the generation evolution of carcinoma of prostate.Wherein, " dryness maintains " (maintenance of pluripotency) ability is that prostate cancer stem cells maintains self quantity, keeps the essential condition [11] of self renewal and differentiation capability.Existing research shows the regulation and control relevant [12-16] of the multiple factor and signal path and prostate cancer stem cells " dryness maintains " ability.Nearest research further points out that again microRNA (miRNA) has brought into play important effect [17-24] in the regulation process of prostate cancer stem cells " dryness maintains ".

MicroRNA (miRNA) is the little RNA of a kind of endogenous non-coding, and existing research shows, miRNA has the effect [24-26] that development occurs modulate tumor in comprising the kinds of tumors of carcinoma of prostate.MicroRNA-7 (miR-7) is newfound a kind of miRNA in recent years.In the kinds of tumors such as hepatocarcinoma, colorectal cancer, head and neck cancer, breast carcinoma, gastric cancer, neuroma, confirmed that miR-7 can bring into play significant cancer suppressing action [25,27-34].But whether miR-7 brings into play the effect of antioncogene in carcinoma of prostate, whether by regulating and controlling " dryness maintains " of prostate cancer stem cells, participate in the regulation and control of carcinoma of prostate tumor progression, and relevant mechanism of action does not all still understand.

Based on this, the present invention stablized the prostate gland cancer cell subclonal cell line of expressing miR-7 by structure, and set up corresponding nude mouse tumor-bearing model, in vitro, in body assessment miR-7 for the growth inhibited effect of prostate gland cancer cell, and for the regulating and controlling effect of prostate cancer stem cells " dryness maintains " ability and illustrate its possible mechanism of action.The present invention finds to stablize in prostate gland cancer cell and expresses miR-7 and effectively suppressed the growth of tumor cell in vitro, in body.MiR-7 can suppress " dryness maintains " ability of prostate cancer stem cells effectively simultaneously, points out it to have the ability that suppresses tumor progression by regulation and control prostate cancer stem cells dryness.This invention has positive reference for improving existing carcinoma of prostate clinical diagnosis and therapeutic scheme,, miR-7 be can be applicable in the target gene therapy for carcinoma of prostate as natural acid micromolecular medicine meanwhile.

Summary of the invention

The object of the invention is to apply the growth that miR-7 suppresses prostate tumor cells, suppress tumor progression (maintaining ability by weakening the dryness of prostate cancer stem cells).

In order to achieve the above object, the invention provides the effect of microRNA-7 aspect the expression inhibitor as PI3K/Akt signal path.

The present invention also provides the effect of microRNA-7 in the targeting inhibitor as KLF4.

The present invention also provides the application of microRNA-7 in the medicine of preparation inhibition tumor of prostate growth.

The present invention also provides the application of microRNA-7 in the medicine of preparation retardance prostate tumor cells cycle progression.

The present invention stablized the prostate gland cancer cell subclonal cell line of expressing miR-7 by structure, and set up the subcutaneous tumor-bearing model of corresponding nude mouse, in vitro and in body, verified that miR-7 suppresses the effect of prostate tumor cells growth and prostate cancer stem cells " dryness maintains " ability.The present invention finds to stablize after expression miR-7, can targeting suppress the expression of PI3K/Akt signal path, reduce the activity of Akt, and significantly lower the phosphorylation level of its downstream cycle regulatory protein p21, promoted the core that enters of unphosphorylated p21 albumen, thereby in tumor cell, realized the retardance of cell cycle progression, suppressed the multiplication capacity of tumor cell.

The present invention finds first and has identified the target gene KLF4 (Kr ü ppel-like factor4) of a brand-new miR-7 in prostate cancer cell line.KLF4 belongs to KLF transcription factor family, and the member of this family participates in the expression of several genes, and these genes relate to cell differentiation, growth and the regulate several biological processes such as apoptosis, and with the generation evolution closely related [35,36] of tumor.In addition, KLF4 has been proved with " dryness maintains " of embryonic stem cell closely related.Suppress KLF4 expression and can weaken embryonic stem cell " dryness maintains " ability [37].In carcinoma of prostate, the present invention finds that expression that miR-7 suppresses KLF4 by targeting has weakened " dryness maintains " ability of prostate cancer stem cells first.In stablizing the prostate gland cancer cell subclonal cell line of expressing miR-7, by cell surface marker thing CD44, CD133 sorting prostate cancer stem cells (CD44+/CD133+), balling-up ability in vitro of the prostate cancer stem cells that the present invention finds gained and the tumor of the one-tenth again ability in body significantly decline, and the known dryness factor (KLF4, Oct4, Sox2, Nanog) expression significantly suppressed.

The present invention has built the luciferase assay of identifying miRNA target molecules: by conventional molecular biology method, it is skeleton [40] that the phEW-luc plasmid of expressing Firefly luciferase gene is take in the present invention, by restricted enzyme BglII single endonuclease digestion, (this sentences miR-7 is example to the sequence that the miRNA complete complementary with needs research of insertion synthetic matches, but be not limited to this), build positive control report carrier.This sequence, except two ends comprise BglII enzyme action recognition site, holds at 5 ' end and 3 ' the enzyme action recognition site (sequence four) of also introducing respectively restricted enzyme NheI and EcoRV.By NheI/EcoRV double digestion, can remove positive control sequence and by the linearisation of plasmid skeleton.By conventional PCR method, clone or the 3'UTR sequence of synthetic target gene mRNA, at its 5 ' end and 3 ', hold the enzyme action recognition site of introducing respectively restricted enzyme NheI (or its isocaudarner XbaI) and EcoRV (or other produce the restricted enzyme of flat end) simultaneously.By this fragment of double digestion, then be connected with linearizing plasmid skeleton, build the report carrier that contains target gene mRNA3'UTR sequence.By report carrier or positive control carrier,, need the secondary precursor molecule pre-miRNA of miRNA of research, (this sentences pre-miR-7 is example with expressing the internal reference plasmid pRL-cmv (being purchased from U.S. promega company) of Renilla luciferase gene, but be not limited to this) or the analogies mimic of its ripe molecule, three uses the cell line (this prostate cancer cell line PC3 that sentences people is example, but is not limited to this) in the method cotransfection people source of liposome transfection.After transfection 18～24 hours, use Dual-Luciferase detection kit (being purchased from U.S. promega company) to detect and calculate the activity of relative fluorescence element enzyme, whether the target gene of investigating expection is subject to studied miRNA regulation and control really, and (this sentences miR-7 and target gene KLF4 is example, but be not limited to this, refer to embodiment 2).In the present invention, use this system, the present invention has verified the target gene KLF4 of a miR-7 first in carcinoma of prostate.

The present invention has assessed miR-7 and as natural nucleic acid small-molecule drug, has been applied to the feasibility of the target gene therapy of carcinoma of prostate, for further clinical practice lays the foundation.Specifically, by building an expression vector at eukaryotic expression miR-7, the method for using puromycin resistance and the EGFP fluorescence positive to combine screening builds stablized the carcinoma of prostate subclonal cell line of expressing miR-7.Take PC3 as example, and in its subclonal cell line PC3-miR-7, the stably express amount of miR-7 has increased nearly 26 times with respect to contrast PC3-null.After setting up that corresponding mice is subcutaneous and planting tumor model, find, the tumor tissues in PC3-miR-7 source, to inoculate latter 30 days its gross tumor volumes be matched group 0.5 times, tumor is heavily 0.44 times of contrast.Wherein, for expression vector, puromycin resistance and the EGFP fluorescence positive of eukaryotic cell expression miRNA combine screening build stablized express the method for miRNA subclonal cell line, for the identification of luciferase assay and the subcutaneous foundation of planting tumor animal model of mice of miRNA target molecules, except being applicable to assess the effect that miR-7 suppresses tumor of prostate growth, also can be applicable to assess the effect of other miRNA modulate tumor processes, be with a wide range of applications.

The present invention obtains prostate cancer stem cells by the cell surface molecule of the specific prostate cancer stem cells of labelling by fluidic cell sorting: for the subclonal cell line of In vitro culture, the present invention is by 0.05% conventional pancreatin (containing 0.02%EDTA) digestion, the method for centrifugal collecting cell in complete medium and after pancreatin; For subcutaneous, plant the tumor tissues that tumor obtains, the present invention is by mechanical shearing, IV Collagenase Type (1 mg/ml)+trypsinization (0.01%), the method for centrifugal collecting cell in complete medium and after pancreatin, the most at last～10 ⁸individual cell with minimal medium, be resuspended in EP pipe and standardize solution to 100 microlitres.Add each 5 micrograms of people's the CD44 antibody [38] of APC labelling and the people's of PE labelling CD133 antibody [39] (two kinds of antibody be all purchased from German beautiful Tian Ni MACS company) simultaneously, 4 ℃ of lucifuges are hatched 40～50 minutes, centrifugal removal supernatant, with after the resuspended rinsing of PBS 1～2 time, with the PBS containing 1%FBS, be resuspended to suitable concentration and carry out fluidic cell sorting.Obtain CD44 ⁺cD133 ⁺two positive cells and CD44 ^-cD133 ^-jack to jack adapter sexual cell.By detecting external balling-up capability analysis (referring to embodiment 8) after the expression (referring to embodiment 7), gradient dilution of two groups of cell dryness correlation factors, subcutaneous one-tenth tumor capability analysis (referring to embodiment 9), in conjunction with existing relevant report, checking CD44 ⁺cD133 ⁺two positive cells are prostate cancer stem cells (S cell), CD44 ^-cD133 ^-jack to jack adapter sexual cell is the non-cancer stem cell that differentiation degree is higher (NS cell).Result shows that stablized the ratio of prostate cancer stem cells (PC3-miR-7-S) after expression miR-7 drops to 0.75 times of matched group (PC3-null-S).The cell of sorting gained is carried out to gradient dilution and again become tumor, find that the tumor in PC3-miR-7-S source is in latter 40 days (inoculum concentrations 10 of inoculation ²individual cell) just have visible strumae, than the tumor of PC3-null-S source (contrast), occur strumae time retardation 10 days.To latter 70 days of inoculation, its volume is matched group 0.29 times, tumor is heavily 0.31 times of contrast.For the cell mass of jack to jack adapter (PC3-null-NS, PC3-miR-7-NS), carry out also finding the tumor that PC3-miR-7-NS originates after this experiment equally, inoculate latter 50 days (inoculum concentrations 10 ⁵individual cell), volume is matched group 0.36 times, tumor is heavily 0.45 times of contrast.Wherein, be used for evaluating the subcutaneous tumor model of planting of gradient dilution mice of tumor stem cell " dryness is strong and weak ", except being applicable to assess the effect that miR-7 suppresses prostate cancer stem cells " dryness maintains " ability, also can be applicable to assess other miRNA " dryness regulation and control " effect to different tumor stem cells, (referring to embodiment 9) is equally with a wide range of applications.

The present invention has set up subclonal cell line and the subcutaneous one-tenth tumor of prostate cancer stem cells mouse model: will contrast and subclonal cell line (respectively dilution be 10 ⁶or the CD44 obtaining after sorting individual cell/50 microlitre), ^-cD133 ^-(dilution is 10 to jack to jack adapter sexual cell ⁵individual cell/50 microlitre, 10 ⁴two gradients of individual cell/50 microlitre) and CD44 ⁺cD133 ⁺(dilution is 10 to two positive cells ³individual cell/50 microlitre, 10 ²two gradients of individual cell/50 microlitre) be resuspended in minimal medium, after mixing with isopyknic metrogel (being purchased from U.S. BD Bioscience company) on ice, use insulin syringe to be injected in the large leg outer side of the male nude mouse of athymism in 5 week age (being purchased from Si Laike laboratory animal company) subcutaneous, set up corresponding subcutaneous one-tenth tumor mouse model.Under SPF grade standard animal feeding room and independent purification feeding system condition, raise.At reasonable time point, observe, measure the volume of mice subcutaneous tumors piece.Treat that volume approaches 1cm ³time put to death mice, extract tumor tissues, for follow-up correlation analysis.For having inoculated CD44 ^-cD133 ^-jack to jack adapter sexual cell or CD44 ⁺cD133 ⁺the tumor tissues obtaining after two positive cells, be digested to unicellular rear available CD44, CD133 serve as a mark thing sorting prostate cancer stem cells (two sun) and the higher non-cancer stem cell (jack to jack adapter) of differentiation degree again, carry out the second gradient dilution of taking turns and become tumor analysis, by that analogy.The dryness that this model can be used for long-range assessment tumor stem cell maintains ability (referring to embodiment 9).

Compared with prior art, the invention has the beneficial effects as follows:

The present invention has all verified that miR-7 has the effect that suppresses tumor of prostate growth and tumor progression (suppressing prostate cancer stem cells " dryness maintains " ability) in vivo and in vitro.Pointed out this class natural acid molecule of miR-7 to have broad application prospects in the clinical treatment of carcinoma of prostate as novel small-molecule drug.

Accompanying drawing explanation

Fig. 1: use Dual-Luciferase reporting system to identify the mutual work between miRNA and target gene mRNA3'UTR.Figure top is the sequence sketch plan of ripe KLF4mRNA, points out the identification binding site (shown in black triangle) that partly has two miR-7 at its 3'UTR by sequence alignment.Figure below is the result that Dual-Luciferase reporting system detects, under the condition that shows to exist at miR-7, by the mutual work of miR-7 and KLF43'UTR, can effectively reduce the expression of Firefly luciferase, contrast phase specific energy with scramble RNA and significantly reduce luciferase activity (p < 0.01).This results suggest miR-7 can do mutually with KLF43'UTR specifically, and on post-transcriptional level, regulate and control its expression.

Fig. 2: identify and stablized the carcinoma of prostate subclonal cell line of expressing miR-7.(a) stablized after expression miR-7, and used the method for TaqMan MicroRNA Assay to identify that the expression of miR-7 in PC3-miR-7 rises to 26 times of contrast.(b) by qRT-PCR method, find, stablized after expression miR-7, its target gene KLF4, and downstream p21 and cyclinD1; A known target gene p100 δ, and the expression of downstream Akt and mTOR significantly reduces compared with the control.(c) by the method for protein immunoblot, find, on protein level, the expression of related gene and the expression of mRNA are consistent, stablize to express expression and the activated state of related gene after miR-7 and all significantly decline.Simultaneously, because p-Akt (activated state of reflection Akt) significantly declines, cause the amount of the p-p21 (being positioned in Cytoplasm) in downstream to decline thereupon, thereby the amount that enters nuclear unphosphorylated p21 is increased relatively, pointed out and stablely may cause cell cycle arrest after crossing expression miR-7.GAPDH is cytoplasm protein internal reference, and TBP is Nuclear extract internal reference.＊＊：p＜0.01。

Fig. 3: stablized expression miR-7 and can realize cell cycle arrest in prostate cancer cell line.(a) by Hoechst method and fluidic cell sorting, find after cell cycle synchronization, PC3-miR-7 cell needs 12 hours from the recovering state (G0/G1 phase cell proportion < 70%) of serum starvation, and contrast PC3-null cell only needs 8 hours.S phase (b) and G2/M phase (c) cell proportion change and to show, PC3-miR-7 cell completes a cell cycle need to be approximately 14 hours, and PC3-null cell takes 12 hours.Results suggest in prostate gland cancer cell, stablized after expression miR-7, can cause cell cycle arrest, and then the multiplication capacity of inhibition tumor cell.

Fig. 4: stablized expression miR-7 and can significantly suppress the propagation of prostate gland cancer cell in vitro.(a) by CCK8 method, detect, after expression miR-7 is stablized in discovery, the light absorption value of cell at 450nm place significantly reduced than contrast.Pointed out stable cross expression miR-7 can be in vitro, the propagation of inhibition tumor cell under the state of cell attachment growth.(b) after cell synchronization, recovering serum supplies with, after 16 hours, by immunofluorescence method, detect the cell proliferation related expression because of ki-67, discovery was stablized and is expressed the expression of ki-67 after miR-7 and significantly reduce, and had pointed out stable mistake to express miR-7 and can significantly suppress in vitro the propagation of the prostate gland cancer cell of adherent growth.Micro-imaging magnification ratio is 200 times, and scale length is 20 microns.＊＊：p＜0.01。

Fig. 5: stablized express miR-7 can significantly suppress in vitro prostate gland cancer cell from wall energy for growth.By three-dimensional, from wall culture experiment, find to stablize after expression miR-7, the ability that prostate gland cancer cell forms maxicell ball (80 microns of diameter >) significantly reduces.Figure has shown in left side two kinds of representational cell ball micro-imagings that surely turn cell line source, and amplification is respectively 100 times and 400 times, and scale length is 50 microns.Figure right side is the statistical result of cell ball quantity.＊＊：p＜0.01。

Fig. 6: stablized and express the growth that miR-7 suppresses tumor of prostate in vivo.(a) by setting up the subcutaneous tumor model of planting of nude mouse, at lateral leg subcutaneous vaccination PC3-null, 30 days gross tumor volumes of PC3-miR-7 Continuous Observation, change respectively, after expression miR-7 is stablized in discovery, tumor was significantly suppressed in subcutaneous growth.Figure left side is the statistical result of growth curve, and right side is to put to death mice after 30 days, from the tumor tissues of subcutaneous taking-up.(b) tumor tissues of taking-up is taken to weight, find that the weight of the tumor tissues that PC3-miR-7 originates is significantly lower than contrast.(c) from the tumor tissues in PC3-null, PC3-miR-7 source, extract total RNA respectively, method by TaqMan MicroRNA Assay identifies, finds that the expression of miR-7 in the tumor tissues in PC3-miR-7 source still maintains the more than 5 times of contrast.The expression of the relevant target gene of finding other by qRT-PCR method in the tumor tissues in PC3-miR-7 source be remarkable expression in contrast lower than it all.From the tumor tissues in PC3-null, PC3-miR-7 source, extract total protein respectively and carry out protein immunoblot detection, obtain equally the result consistent with mRNA level.(d) respectively the tumor tissues in PC3-null, PC3-miR-7 source is carried out to specimens paraffin embedding slices and does H & E dyeing, discovery has had obvious tumor-blood-vessel growth (black arrow indicating area) in the tumor tissues (T indicating area) in PC3-null source, and infiltrate (* indicating area) with subcutaneous fat to a certain degree, and in the tumor tissues in PC3-miR-7 source, only have a small amount of erythrocyte to infiltrate (black arrow indicating area), and there is homogeneous, well-defined connective tissue between skin (S indicating area) and tumor tissues.Figure left side is for amplifying the micro-imaging of 100 times, and right side is 400 times of micro-imagings in region in the square frame of left side, and scale length is 20 microns.＊：p＜0.05，＊＊：p＜0.01。

Fig. 7: carry out respectively airflow classification prostate cancer stem cells (CD44 from Subcutaneous tumor tissue, subclonal cell line ⁺cD133 ⁺).(a) by PC3-null, PC3-miR-7 sub-clone cell (three groups of figure tops), and unicellular (three groups of the figure belows) of the tumor tissues digestion gained in both subcutaneous vaccinations source carry out fluidic cell sorting after dying altogether by the people CD44 antibody of APC labelling and the people CD133 antibody of PE labelling, find the CD44 directly obtaining from the cell of external adherent growth ⁺cD133 ⁺cell proportion there is no significant difference in both, but enter the subcutaneous tumor of planting, from tissue, digests CD44 in gained cell ⁺cD133 ⁺cell proportion significantly drops to 0.75 times of contrast after expressing miR-7 stablizing, and has pointed out stable mistake to express to have weakened in vivo after miR-7 the dryness of prostate cancer stem cells to maintain ability.(b) cell for sorting gained carries out after rejection tablet, by immunofluorescence dyeing, detect the coherency factor (with KLF4, Nanog is example, but be not limited to this) expression, find it is no matter to digest again sorting (two row below figure) from directly sorting the cell of external adherent growth (figure top two row) or through subcutaneous after planting tumor, KLF4 and Nanog location and the expression in nucleus all significantly reduced after stablizing expression mIR-7, pointed out stable mistake to express the expression that has suppressed dryness related gene after miR-7, thereby the dryness that has reduced prostate cancer stem cells maintains ability.Micro-imaging amplification is 200 times, and scale length is 10 microns.

Fig. 8: vitro detection was stablized the impact of expression miR-7 on prostate cancer stem cells " dryness maintains " ability.To under PC3-null, PC3-miR-7 sub-clone cell skin, plant after tumor sorting CD44 from tumor tissues ⁺cD133 ⁺prostate cancer stem cells carries out three-dimensional from wall growth, and the impact of expression miR-7 on prostate cancer stem cells dryness stablized in assessment in vitro.Within 15 days, add up afterwards the quantity of the cell ball of different-diameter, discovery was stablized after expression miR-7, major diameter (80 microns of >) the cell ball number forming only accounts for total approximately 10%, significantly lower than contrast (35%), having pointed out stable mistake to express miR-7 can be by suppressing the dryness of prostate cancer stem cells, and performance suppresses the effect of tumor growth.Figure left side is representational cell ball micro-imaging, and amplification is 400 times, and scale length is 50 microns; Figure right side is the statistical result of cell ball quantity, * *: P < 0.01.

Fig. 9: the gradient dilution again subcutaneous tumor mouse model of planting is evaluated in vivo and stablized the inhibitory action of expression miR-7 to prostate cancer stem cells " dryness maintains " ability.(a) gradient dilution subcutaneous tactful sketch plan of planting tumor mouse model again.(b) by statistics Subcutaneous tumor growth curve, find for CD44 ^-cD133 ^-cell (PC3-null-NS, PC3-miR-7-NS), 10 ⁵during the inoculum concentration of individual cell, stablized and express miR-7 and suppressed significantly non-tumor stem cell regrowing in vivo; For CD44 ⁺cD133 ⁺cell (PC3-null-S, PC3-miR-7-S), 10 ²during the inoculum concentration of individual cell, stablized express miR-7 equally can be by suppressing the dryness of prostate cancer stem cells, effectively suppressed the growth in vivo of tumor tissues that tumor stem cell originates.(c) inoculating respectively latter 50 days (PC3-null-NS, PC3-miR-7-NS), and 70 days (PC3-null-S, PC3-miR-7-S), getting tumor tissues weighs, expression miR-7 was stablized in discovery, tumor tissues for non-tumor stem cell and tumor stem cell source, its tumor weight, all significantly lower than contrast, has pointed out miR-7 can suppress by suppressing the dryness performance of tumor stem cell the effect of tumor growth and tumor progression.＊＊：p＜0.01。

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.

Embodiment 1

Use Dual-Luciferase reporting system to identify the mutual work between miRNA and target gene mRNA3'UTR: PC3 cell (is bought to the cell bank in the Chinese Academy of Sciences, as example, but be not limited to this) in 24 orifice plates, use DMEM complete medium (containing 10% hyclone, be purchased from U.S. Gibco company) adhere-wall culture, when cell density reaches 70%～80%, use lipofectamine2000 transfection reagent (being purchased from U.S. Life Technology company) to proceed as follows: 1) to get 3 porocytes as Duplicate Samples, (positive control is herein the sequence of matching with miR-7 complete complementary to the positive control of transfection simultaneously report carrier, be sequence four), the internal reference carrier pRL-cmv (being purchased from U.S. promega company) and the contrast scramble RNA that express Renilla luciferase gene (are purchased from U.S. Life Technology company, data are shown in white caption in the miR-7target row of Fig. 1 left side), 2) get 3 porocytes as Duplicate Samples, (this sentences pre-miR-7 is example to the secondary precursor molecule pre-miRNA of the miRNA of the above-mentioned positive control report carrier of transfection simultaneously, above-mentioned internal reference carrier pRL-cmv and need research, but be not limited to this, be purchased from U.S. Life Technology company, data are shown in black caption in the miR-7target row of Fig. 1 left side), 3) get 3 porocytes as Duplicate Samples, (this sentences KLF43'UTR is example to the report carrier that transfection simultaneously comprises target gene mRNA3'UTR sequence, see sequence five, but be not limited to this), above-mentioned internal reference carrier pRL-cmv and above-mentioned contrast scramble RNA (data are shown in white caption in Fig. 1 right side full-length KLF43'UTR row), 4) get 3 porocytes as Duplicate Samples, report carrier, above-mentioned internal reference carrier pRL-cmv and the pre-miR-7 molecule of the above-mentioned KLF43'UTR of the comprising sequence of transfection simultaneously (data are shown in black caption in Fig. 1 right side full-length KLF43'UTR row), the description that concrete operations flow process provides according to transfection reagent manufacturer is carried out.After transfection 18～24 hours, use Dual-Luciferase detection kit (being purchased from U.S. promega company) on luciferase cold light instrument (BERTHOLD Technology), to detect and calculate the active R1/R2 of relative fluorescence element enzyme.Wherein R1 represents the activity of the Firefly luciferase that report carrier is expressed, and R2 represents the activity (Fig. 1) of the Renilla luciferase of internal reference vector expression.Result shows, when having pre-miR-7 to exist, can on post-transcriptional level, lower the expression of Firefly luciferase by doing mutually with KLF43'UTR, while causing the activity of its relative fluorescence element enzyme to compare with scramble RNA, significantly reduces.This results suggest KLF4 is the possible new target drone gene of of miR-7 in carcinoma of prostate.

Embodiment 2:

(1) build the expression vector for stable eukaryotic expression miRNA: take pEGP-miR plasmid as skeleton (being purchased from U.S. Cell BioLab company), by conventional restricted enzyme BamHI/NheI double digestion, by plasmid linearization.(these three kinds of different precursor molecule pri-miR-7-1, pri-miR-7-2, pri-miR-7-3 that sentence miR-7 are example to precursor molecule sequence by the required miRNA of synthetic, but be not limited to this, see sequence one, two, three), the recognition site of simultaneously introducing respectively restricted enzyme BamHI and NheI (can be also XbaI, be the isocaudarner of NheI) at 5 ' end and the 3 ' end of sequence.The fragment obtaining is carried out to conventional restricted enzyme BamHI/NheI (can be also BamHI/XbaI) double digestion, and conventional DNA attended operation, (this sentences pEGP-miR-7-1 to build expression vector for stable eukaryotic expression miRNA, pEGP-miR-7-2, pEGP-miR-7-3 is example, but be not limited to this, three all can realize the expression of ripe miR-7 molecule effectively, below to take the result that pEGP-miR-7-1 obtains be example explanation).

(2) method of combining screening by puromycin resistance and the EGFP fluorescence positive builds stablized the carcinoma of prostate subclonal cell line of expressing miRNA: by conventional liposome transfection method, (in the present invention, adopt lipofectamine2000 transfection reagent, be purchased from U.S. Life Technology company, but be not limited to this) expression vector (this sentences pEGP-miR-7-1 is example, but is not limited to this) or the control plasmid pEGP-null (being purchased from U.S. Cell BioLab company) that stablized expression miRNA are proceeded in prostate cancer cell line respectively.The prostate cancer cell line that relates to is including but not limited to PC3, DU145, LNCaP (all can successfully realize and stablize the subclonal cell line structure of expressing miR-7 in three, the subclonal cell line building in PC3 of take is below example explanation, and PC3 prostate cancer cell line Ke Cong Chinese Academy of Sciences cell obtains.After transfection 72 hours, by the conventional attached cell cultural method that goes down to posterity, cell is proceeded to amplification culture to cell density in the cultivation dish of diameter 10cm and reach 70%～80%.Add puromycin (being purchased from U.S. sigma company) to final concentration 0.5 mcg/ml, within 2～3 days, change liquid once, continue to give puromycin 10 days, observation of cell no longer continues to stop puromycin screening after death.Collect the cell of survival, by conventional fluidic cell method for separating, filter out EGFP positive cell (fusion rotein that contains puromycin resistance gene and EGFP in pEGP-miR plasmid skeleton, this fusion rotein and miRNA precursor molecule share same promoter).This method of combining screening is compared to traditional single tolerant gene expression screening technique, can effectively avoid the false positive results causing by force because of the spontaneous drug resistance of prostate cancer cell line, the cell with puromycin resistance and the EGFP fluorescence positive is only stablized the carcinoma of prostate sub-clone cell of expressing miRNA.By the sub-clone cell obtaining at 75cm ²in Tissue Culture Flask, amplification culture continuous passage are 3 times, maintain during this time the content of final concentration 0.5 mcg/ml puromycin, again by fluidic cell method for separating, check, its EGFP fluorescence positive rate, higher than 95%, can be confirmed this sub-clone cell construction success.If EGFP fluorescence positive rate is too low, can repeat above-mentioned screening process, by the cell for the enrichment EGFP fluorescence positive, until set up required subclonal cell line PC3-miR-7.

(3) identify and stablized the carcinoma of prostate subclonal cell line of expressing miR-7: this sentences PC3-miR-7 cell is example, but is not limited to this.Use DMEM complete medium (containing 10% hyclone in cell, be purchased from U.S. Gibco company) in adhere-wall culture and 6 orifice plates, when cell density reaches 70%～80%, remove culture medium, use Trizol (being purchased from U.S. Life Technology company) to carry out conventional total RNA extracting.Use the specific reverse transcription primer of miR-7 (being purchased from U.S. Life Technology company), miRNA reverse transcription test kit (being purchased from U.S. Life Technology company), the standard step providing according to manufacturer is carried out the reverse transcription of miR-7.Use the same method U44 (little nRNA, snRNA, internal reference) is carried out to specific reverse transcription.Using the reverse transcription product obtaining as template, use respectively the special probe of miR-7, and the special probe (two kinds of probes are all purchased from U.S. Life Technology company) of U44, adopt TaqMan MicroRNA Assay method (to have dedicated kit, be purchased from U.S. Life Technology company), the standardization program providing according to test kit on real-time PCR is carried out the expression of miR-7 of quantitative analysis maturation, and (Fig. 2 is a).Result shows, with respect to control cells, is PC3-null, and in PC3-miR-7, the expression of ripe miR-7 has improved 26 times.Simultaneously, the present invention uses conventional mRNA reverse transcription box (be purchased from Japanese TAKARA company) to carry out reverse transcription to the total RNA extracting, use SYBR-Green reagent (be purchased from Japanese TOYOBO company) to KLF4, p21, cyclin D1, p110delta (catalytic subunit of PI3K), Akt, the mRNA level of mTOR gene is carried out qRT-PCR analysis (Fig. 2 b).Result shows the expression of the target gene KLF4 of miR-7 and the p21 in downstream, cyclin D1; The target gene p110delta[Hepatology of known miR-7, Fang YX] and the Akt in downstream, the expression of mTOR all significantly declines in PC3-miR-7.Meanwhile, from PC3-null and PC3-miR-7 cell, by conventional method, extract respectively total protein, Nuclear extract and cytoplasm protein respectively, by protein immunoblot method, detected the expressing quantity (Fig. 2 c) of said gene.Result is consistent with the quantitative analysis of mRNA level, stablized after expression miR-7, KLF4 in total protein, p110delta, Akt, p-Akt, the expression of mTOR significantly reduces, wherein the content of the activated form p-Akt of Akt is significantly suppressed, and causes the content of p-p21 in Cytoplasm to decline, and has promoted location and the enrichment (antibody be purchased from U.S. SantaCruz company HuoCST company) of p21 in nucleus.This enrichment meeting causes cell cycle arrest in the G1-S phase, has pointed out stablely may extend cell cycle after crossing expression miR-7, and has therefore affected the propagation of cell.

Embodiment 3:

Stablize expression miR-7 and can at prostate cancer cell line, (take PC3-miR-7 cell as example, but be not limited to this) in realize cell cycle arrest: by PC3-miR-7 and contrast PC3-null, by conventional cell culture processes adhere-wall culture in 12 orifice plates, every kind of cell need repeat 3 at the time point of each detection and answer holes.Within in the present embodiment every 2 hours, detect the once cell quantity ratio of each cell period (G0/G1 phase, S phase, G2/M phase), continuous detecting 48 hours, totally 24 time points.When cell density reaches 60%～70%, complete medium is replaced to the minimal medium of serum-free, by serum starvation 30～36 hours, make cell cycle be synchronized to the G0 phase, and in quiescent condition.Recover to start to detect after serum supply, the starting point of recovering serum supply of take is 0 hour, timing successively.In detection time, put and digest collection by conventional cell dissociation collection method, use 0.05% pancreatin (containing 0.02%EDTA) to carry out after cell dissociation, in and pancreatin centrifugal collecting cell, by the resuspended rear centrifugal remaining culture medium of once removing of 1 * PBS, collected cell precipitation is resuspended and be transferred in 5 milliliters of fluidic cell sorting loading pipes with 500 microlitre 1 * PBS.The Hoechst (being purchased from U.S. Molecular Probe company) that adds 1 microlitre 10 mg/ml concentration, 37 ℃ of lucifuges are hatched 10 minutes, by flow cell sorter, carry out cell cycle analysis, use ModfitLT software to carry out data analysis.The ability of recovering (G0/G1 phase ratio is lower than 70%) to finding to stablize cell after expression miR-7 after data statistics from serum starvation state weakens, and recover to need 8 hours, and PC3-miR-7 need within 12 hours, (Fig. 3 a) in contrast PC3-null.Secondly, from the ratio in each period, change, contrast PC3-null completes a cell cycle and needs 12～13 hours after serum starvation recovers, PC3-miR-7 needs 14 hours (Fig. 3 a-3c) under the same conditions, pointed out stable mistake to express the cell cycle that miR-7 can block prostate gland cancer cell effectively, and propagation that can this inhibition tumor cell.

Embodiment 4:

By CCK8 proliferation experiment and the detection of Ki-67 staining, stablized expression miR-7 prostate cancer cell line (be take to PC3-miR-7 cell as example, but be not limited to this) impact of in-vitro multiplication ability: by the conventional cell dissociation collection method in embodiment 3, the contrast PC3-null collecting, PC3-miR-7 cell are resuspended in EP pipe with complete medium and are settled to 1 milliliter.Use blood counting chamber to carry out cell counting, get in the 24 every holes of orifice plate and inoculate 5000 cells, continuous detecting one week, detects 5 multiple holes every day.During detection, in 500 microlitre complete mediums, add 50 microlitre CCK8 reagent (be purchased from green the skies company) to mix, 37 ℃ of lucifuges are hatched 2 hours.Get 20 microlitre supernatant in 96 orifice plates, use microplate reader, at 450nm place, detect absorption value.The testing result of one week is carried out to statistical analysis, and (Fig. 4 a), find that PC3-miR-7 cell is significantly less than PC3-null cell through reacted absorbance, pointed out stable cross expression miR-7 can be in vitro, the propagation of inhibition tumor cell under the state of cell attachment growth.Simultaneously, the present invention gets 12 orifice plates and puts into cell climbing sheet, on slide, inoculate 5000 cells (200 microlitre volume), after cell is completely adherent, remove supernatant, in hole, add complete medium, by the method for serum starvation, carry out synchronization (seeing embodiment 3), after cell synchronization, recovering serum supplies with, in the supply of recovery serum, after 16 hours, take out slide, by conventional cell immunofluorescent staining method, detect the expression (Fig. 4 b) of this propagation related gene of Ki-67 (antibody is purchased from U.S. CST company) in cell.The Ki-67 expression of finding PC3-miR-7 cell by fluorescence microscopy Microscopic observation, significantly lower than contrast PC3-null cell, has again been pointed out stable mistake to express miR-7 and can significantly have been suppressed prostate tumor cells propagation in vitro.

Embodiment 5:

By dimensional culture, test in vitro to detect and stablized expression miR-7 prostate cancer cell line (be take to PC3-miR-7 cell as example, but be not limited to this) from the impact of wall energy for growth: by the conventional cell dissociation collection method in embodiment 3, the contrast PC3-null collecting, PC3-miR-7 cell are resuspended in EP pipe with minimal medium and are settled to 1 milliliter, and it is standby that use blood counting chamber carries out cell counting.By the matrigel that contains somatomedin and Geltrex (both are all purchased from U.S. BD Bioscience company) in mixing in 1: 1 ratio on ice.In 96 orifice plates, be added dropwise to mixed liquor 20 microlitres/hole, spread rapidly after even and it was fully solidified in standing 30 minutes in 37 ℃ of cell culture incubators.Cell concentration is diluted to 200 cell/40 microlitres, drips in the substrate of solidifying, in 37 ℃ of cell culture incubators, hatch 1 hour.Prepare during this time upper strata substrate: the matrigel that contains somatomedin is diluted to 4% (volume ratio) with minimal medium.100 microlitre upper strata substrate are dripped in existing cell suspension, in 37 ℃ of cell culture incubators, continue to cultivate.After 3 days, change for the first time liquid, within every 2 days afterwards, change liquid once.While changing liquid, from liquid level, draw after 80 microlitres (considering moisture evaporation in incubator), be added dropwise to freshly prepd upper strata substrate.3 multiple holes of every group of inoculation, cell is inoculated latter 15 days, the quantity (Fig. 5) of using phase contrast microscope to take pictures and add up the cell ball of different-diameter size.Result shows, the plastidogenetic diameter of PC3-miR-7 is in the quantity of the cell ball of 20～80 microns, and the quantity of 80 microns of above super large cell balls is compared the cell ball quantity that PC3-null cell forms under the same conditions and significantly reduced.Prompting, under the state of dimensional culture, was stablized the ability that mIR-7 has inhibition prostate tumor cells propagation equally of expressing.

Embodiment 6:

By setting up the subcutaneous tumor model of planting of nude mouse, evaluate in vivo to stablize and express the inhibitory action of miR-7 to tumor of prostate growth: by the conventional cell dissociation collection method in embodiment 3, the contrast PC3-null collecting, PC3-miR-7 cell are resuspended in EP pipe with minimal medium and are settled to l milliliter, and it is standby that use blood counting chamber carries out cell counting.Cell is settled to 10 ⁶individual cell/50 microlitre, mixes with isopyknic matrigel that contains somatomedin (being purchased from U.S. BD Bioscience company) on ice.Be selected to the male nude mouse of athymism in 5 week age (being purchased from Si Laike laboratory animal company) and set up animal model.Use insulin syringe the cell suspension after mixing to be injected at respectively to subcutaneous (the left side injection contrast PC3-null cell in this example of the large leg outer side of mice, right side injection PC3-miR-7 cell), repeat 5 mices as Duplicate Samples, whole process operates in the Biohazard Safety Equipment of SPF level Animal House.Experiment mice after injection is raised by normal experiment veterinary method in SPF level Animal House, every volume (volume=length * wide * height * 6/ π) of observing and use vernier caliper measurement Subcutaneous tumor for 5 days.Continuous record 30 days after injection, (Fig. 6 a) to add up and draw the growth curve of gross tumor volume.Simultaneously, after experiment mice is put to death, obtain Subcutaneous tumor tissue and weigh (Fig. 6 b), after portion of tissue is rapidly cooling in liquid nitrogen, at-80 ℃ of deep coolings, preserve, be respectively used to extract total RNA, total protein, in tissue, detect the expression (Fig. 6 c) of related gene; Portion of tissue is fixing in 4% paraformaldehyde, for specimens paraffin embedding slices and HE pathological staining (Fig. 6 d); Portion of tissue is shredded rear with IV Collagenase Type digestion tissue, for the fluidic cell sorting (seeing embodiment 7) of prostate cancer stem cells.Result shows, the tumor tissues in PC3-miR-7 source, to inoculate latter 30 days its gross tumor volumes be matched group 0.5 times, tumor is heavily 0.44 times of contrast.By preceding method, extract total RNA, related gene is carried out to reverse transcription, adopt respectively TaqMan MicroRNA Assay method and conventional qRT-PCR method (seeing embodiment 2) to detect miR-7, KLF4, p110delta, Akt, mTOR, and the expression of cyclin D1 (take these genes as example, but be not limited to this).Find 5 times in the tumor tissues that is expressed as contrast PC3-null source of miR-7 in the tumor tissues in PC3-miR-7 source, it is 0.33 times that the expression of KLF4 and cyclin D1 is all less than 0.5 times, p110delta.Akt, the expression of mTOR also all reduces (Fig. 6 c).While detecting on protein level by protein immunoblot, also obtain consistent result (Fig. 6 c).Tissue is carried out to HE dyeing rear (Fig. 6 d), in the tumor tissues in discovery contrast PC3-null source, have obvious tumor-blood-vessel growth, and infiltrate with subcutaneus adipose tissue to a certain degree.And in the tumor tissues in PC3-miR-7 source, between tumor cell and mouse skin, by subcutaneous connective tissue, isolated, both boundaries are clearly demarcated, have no obvious tumor-blood-vessel growth in tumor tissues.The above results has all pointed out stable mistake to express miR-7 can suppress the cell proliferation of carcinoma of prostate in vivo effectively.

Embodiment 7:

From Subcutaneous tumor tissue, subclonal cell line, carry out respectively airflow classification prostate cancer stem cells (CD44 ⁺cD133 ⁺): after respectively the tumor tissues in the tumor tissues in contrast PC3-null source and PC3-miR-7 source being shredded, add 4 milliliters of IV Collagenase Types, final concentration is 1 mg/ml (being purchased from U.S. Life Technology company).In 37 ℃ of water-bath vibration casees, hatch after 30 minutes, add 1 milliliter of 0.05% pancreatin (containing 0.02%EDTA), final concentration is 0.01%, continue to hatch 5 minutes, and by cell dissociation, be further unicellular.Add isopyknic complete medium cessation reaction centrifugal collecting cell.Use the resuspended rear centrifugal rinsing of carrying out of 1 * PBS, repeat twice.With in the resuspended EP of the transferring to pipe of 1 * PBS and be settled to 1 milliliter, use blood counting chamber to carry out cell counting in cell.Cell is pressed to 10 ⁸individual cell/100 microlitre divides and is filled in EP pipe, add each 5 micrograms of people's the CD44 antibody [38] of APC labelling and the people's of PE labelling CD133 antibody [39] (two kinds of antibody be all purchased from German beautiful Tian Ni MACS company) simultaneously, 4 ℃ of lucifuges are hatched 40～50 minutes, centrifugal removal supernatant, with after the resuspended rinsing of PBS 1～2 time, with the PBS containing 1%FBS, be resuspended to suitable concentration and carry out fluidic cell sorting.Adopt the sorting of the APC-PE dual pathways to obtain CD44 ⁺cD133 ⁺two positive cells and CD44 ^-cD133 ^-(Fig. 7 a) for jack to jack adapter sexual cell.For adhere-wall culture PC3-null and PC3-miR-7 cell, by conventional digestion collection method, count and be settled to after identical cell concentration, according to identical method, carry out antibody incubation and subsequent operation, and the result CD44 obtaining with identical separation condition ⁺cD133 ⁺two positive cells and CD44 ^-cD133 ^-(Fig. 7 a) for jack to jack adapter sexual cell.By the cell obtaining carry out successively centrifugal rejection tablet, 4% paraformaldehyde is fixed and the differential expression of two groups of cell dryness correlation factors of routine immunization fluoroscopic examination (Fig. 7 b).Result shows, no matter is from the direct sorting of subclonal cell line, or plants sorting tumor from the subcutaneous different of inoculation separately, stablizes CD44 after expression miR-7 ⁺cD133 ⁺the ratio that two positive cells (being prostate cancer stem cells) account for general cell all declines to some extent.The cell of acquisition is carried out after rejection tablet, by conventional immunofluorescence dyeing, detect the expression of dryness correlation factor (with KLF4, Nanog is example, but is not limited to this), find to stablize CD44 after expression miR-7 ⁺cD133 ⁺in two positive cells, the expression of these factors is all lower than contrast, can weaken by suppressing the expression performance of these important dryness factors the effect of prostate cancer stem cells " dryness maintains " ability after having pointed out stable mistake to express miR-7.

Embodiment 8:

By dimensional culture, test in vitro and detect and stablized the impact of expression miR-7 on prostate cancer stem cells " dryness maintains " ability: according to the method for embodiment 5, by the CD44 obtaining in the tumor tissues in contrast PC3-null or PC3-miR-7 source ⁺cD133 ⁺two positive cells (being prostate cancer stem cells) carry out external three-dimensional to be cultivated from wall, all inoculates 3 multiple holes for every group.Inoculate latter 15 days, the quantity (Fig. 8) of using phase contrast microscope to take pictures and add up the cell ball of different-diameter size.Result shows, stablized after expression PC3-miR-7, the cell ball overwhelming majority diameters that form are between 20～80 microns, and the quantity of 80 microns of super large cell balls only accounts for 10%, significantly lower than the quantity of super large cell ball in contrast (PC3-null source be 35%).Pointed out in vitro, stablized and express the dryness that has weakened prostate cancer stem cells after PC3-miR-7.

Embodiment 9:

By set up gradient dilution again the subcutaneous tumor mouse model of planting evaluate in vivo and stablized the inhibitory action of expression miR-7 to prostate cancer stem cells " dryness maintains " ability: will contrast PC3-null or the tumor tissues in PC3-miR-7 source in the CD44 that obtains ⁺cD133 ⁺two positive cells (being prostate cancer stem cells, difference called after PC3-null-S, PC3-miR7-S) and CD44 ^-cD133 ^-jack to jack adapter sexual cell (non-cancer stem cell, respectively called after PC3-null-NS, PC3-miR7-NS) is used blood counting chamber to carry out with the minimal medium dilution standardize solution of serum-free, arriving different concentration after cell counting: for CD44 ^-cD133 ^-the dilution of jack to jack adapter sexual cell is 10 ⁵individual cell/50 microlitre, 10 ⁴two gradients of individual cell/50 microlitre; For CD44 ⁺cD133 ⁺two positive cell dilutions are 10 ³individual cell/50 microlitre, 10 ²two gradients of individual cell/50 microlitre.On ice, cell suspension and isopyknic matrigel that contains somatomedin (being purchased from U.S. BD Bioscience company) after dilution are mixed.Be selected to the male nude mouse of athymism in 5 week age (being purchased from Si Laike laboratory animal company) and set up animal model.Use insulin injection syringe, the large leg outer side that PC3-null-NS, PC3-miR7-NS " cell-matrigel " mixed liquor is injected at respectively to mice is subcutaneous, and 10 ⁵dilution factor repeats 5 mices as Duplicate Samples; 10 ⁴dilution factor repeats 5 mices as Duplicate Samples.The large leg outer side that PC3-null-S, PC3-miR7-S " cell-matrigel " mixed liquor is injected at equally respectively to mice is subcutaneous, and 10 ³dilution factor repeats 5 mices as Duplicate Samples; 10 ²dilution factor repeats 5 mices, and as Duplicate Samples, (Fig. 9 a).Within every 5 days, observe and measure subcutaneous tumor volumes (method is with embodiment 6), PC3-null-NS, PC3-miR7-NS group Continuous Observation is put to death experiment mice in latter 50 days to inoculation, obtains Subcutaneous tumor tissue and weighs; PC3-null-S, PC3-miR7-S group Continuous Observation is put to death experiment mice in latter 70 days to inoculation, obtains Subcutaneous tumor tissue and weighs (Fig. 9 b, 9c).Result shows, the tumor tissues in PC3-miR7-NS source, 10 ⁵under individual cell inoculum concentration, inoculate latter 50 days its gross tumor volumes and be matched group (PC3-null-NS) 0.36 times, tumor is heavily 0.45 times of contrast (PC3-null-NS).The tumor tissues in PC3-miR7-S source, 10 ²under individual cell inoculum concentration, to inoculate latter 70 days its gross tumor volumes be matched group 0.29, and tumor is heavily 0.31 times of contrast.Pointed out miR-7 in the tumor cell of non-cancer stem cell, to there is the ability that suppresses its cell proliferation, for cancer stem cell, can also effectively suppress the ability of its " dryness maintains " simultaneously.

The result verification of above-described embodiment this natural acid quasi-molecule of miR-7 as novel small-molecule drug, in the clinical practice of carcinoma of prostate, have broad prospects, be a kind of desirable target gene drug candidate.

The related gene sequence exemplifying in the present invention:

Sequence one:

MiR-7 precursor molecule 1 (pri-miR-7-1): chromosome mapping 9q21.32

NR_029605.1

1ttggatgttg?gcctagttct?gtgtggaaga?ctagtgattt?tgttgttttt?agataactaa

61atcgacaaca?aatcacagtc?tgccatatgg?cacaggccat?gcctctacag

Sequence two:

MiR-7 precursor molecule 2 (pri-miR-7-2): chromosome mapping 15q26.1

NR_029606.1

1ctggatacag?agtggaccgg?ctggccccat?ctggaagact?agtgattttg?ttgttgtctt

61actgcgctca?acaacaaatc?ccagtctacc?taatggtgcc?agccatcgca

Sequence three:

MiR-7 precursor molecule 3 (pri-miR-7-3): chromosome mapping 19p13.3

NR_029607.1

1agattagagt?ggctgtggtc?tagtgctgtg?tggaagacta?gtgattttgt?tgttctgatg

61tactacgaca?acaagtcaca?gccggcctca?tagcgcagac?tcccttcgac

Sequence four:

The sequence of synthetic and ripe miR-7 complete complementary:

5’-AGA?TCT?GCT?AGC?CAA?CAA?AAT?CAC?TAG? TCT?TCC?AGA?TAT?CAG?ATC?T-3’

Wherein AGATCT is the recognition reaction site of restricted enzyme BglII; GCTAGC is the recognition reaction site of restricted enzyme NheI; GATATC is the recognition reaction site of restricted enzyme EcoRV.Underscore sequence tCT TCCthe complementary region of core identification sequence 5 '-GGUUGU-3 ' of expression and miR-7.

Sequence five:

That in the present invention, verifies has the 3'UTR sequence in the KLF4mRNA of mutual work with miR-7:

1atcccagaca?gtg gatatga?cccacactgc?cagaagagaa?ttcagtattt?t ttacttttc

61 acactgtctt?cccgatgagg?gaaggagccc?agccagaaag?cactacaatc?atggtcaagt

121tcccaactga?gtcatcttgt?gagtggataa?tcaggaaaaa?tgaggaatcc?aaaagacaaa

181aatcaaagaa?cagatggggt?ctgtgactgg?atcttctatc?attccaattc?taaatccgac

241ttgaatattc?ctggacttac?aaaatgccaa?gggggtgact?ggaagttgtg?gatatcaggg

301tataaattat?atccgtgagt?tgggggaggg?aagaccagaa?ttcccttgaa?ttgtgtattg

361atgcaatata?agcataaaag?atcaccttgt?attctcttta?ccttctaaaa?gccattatta

421tgatgttaga?agaagaggaa?gaaattcagg?tacagaaaac?atgtttaaat?agcctaaatg

481atggtgcttg?gtgagtcttg?gttctaaagg?taccaaacaa?ggaagccaaa?gttttcaaac

541tgctgcatac?tttgacaag g?aaaatctata?tttgtcttcc?gatcaacatt?tatgacctaa

601gtcaggtaat?atacctggtt?tacttcttta?gcatttttat?gcagacagtc?tgttatgcac

661tgtggtttca?gatgtgcaat?aatttgtaca?atggtttatt?cccaagtatg?ccttaagcag

721aacaaatgtg?tttttctata?tagttccttg?ccttaataaa?tatgtaatat?aaatttaagc

781aaacgtctat?tttgtatatt?tgtaaactac?aaagtaaaat?gaacattttg?tggagtttgt

841attttgcata?ctcaaggtga?gaattaagtt?ttaaataaac?ctataatatt?ttatctgaaa

Wherein underscore is partly can be by miR-7 identification calmodulin binding domain CaM.

The relevant references of quoting in the present invention:

[1]Trott?KR.Tumour?stem?cells:the?biological?concept?and?its?application?in?cancer?treatment.Radiother?Oncol.1994；30:1-5.

[2]Reya?T，Morrison?SJ，Clarke?MF，Weissman?IL.Stem?cells，cancer，and?cancer?stem?cells.Nature2001；414:105-111.

[3]Singh?SK，Hawkins?C，Clarke?ID，Squire?JA，Bayani?J，Hide?T，et?al.Identification?of?human?brain?tumour?initiating?cells.Nature2004；432:396-401.

[4]Lapidot?T，Sirard?C，Vormoor?J，Murdoch?B，Hoang?T，Caceres-Cortes?J，et?al.A?cell?initiating?human?acute?myeloid?leukaemia?after?transplantation?into?SCID?mice.Nature1994；367:645-648.

[5]Al-Hajj?M，Wicha?MS，Benito-Hernandez?A，Morrison?SJ，Clarke?MF.Prospective?identification?of?tumorigenic?breast?cancer?cells.Proc?Natl?Acad?Sci?U?S?A.2003；100:3983-3988.

[6]Kim?CF，Jackson?EL，Woolfenden?AE，Lawrence?S，Babar?I，Vogel?S，et?al.Identification?of?bronchioalveolar?stem?cells?in?normal?lung?and?lung?cancer.Cell2005；121:823-835.

[7]Florek?M，Haase?M，Marzesco?AM，Freund?D，Ehninger?G，Huttner?WB，et?al.Prominin-1/CD133，a?neural?and?hematopoietic?stem?cell?marker，is?expressed?in?adult?human?differentiated?cells?and?certain?types?of?kidney?cancer.Cell?Tissue?Res.2005；319:15-26.

[8]O′Brien?CA，Pollett?A，Gallinger?S，Dick?JE.A?human?colon?cancer?cell?capable?of?initiating?tumour?growth?in?immunodeficient?mice.Nature2007；445:106-110.

[9]Li?C，Heidt?DG，Dalerba?P，Burant?CF，Zhang?L，Adsay?V，et?al.Identification?of?pancreatic?cancer?stem?cells.Cancer?Res.2007；67:1030-1037.

[10]Schatton?T，Murphy?GF，Frank?NY，Yamaura?K，Waaga-Gasser?AM，Gasser?M，et?al.Identification?of?cells?initiating?human?melanomas.Nature2008；451:345-349.

[11]Lobo?NA，Shimono?Y，Qian?D，Clarke?MF.The?biology?of?cancer?stem?cells.Annu?Rev?Cell?Dev?Biol.2007；23:675-699.

[12]Wang?XD，Leow?CC，Zha?J，Tang?Z，Modrusan?Z，Radtke?F，et?al.Notch?signaling?is?required?for?normal?prostatic?epithelial?cell?proliferation?and?differentiation.Dev?Biol.2006；290:68-80.

[13]Shou?J，Ross?S，Koeppen?H，de?Sauvage?FJ，Gao?WQ.Dynamics?of?Notch?expression?during?murine?prostate?development?and?tumorigenesis.Cancer?Res.2001；61:7291-7297.

[14]Leong?KG，Gao?WQ.The?Notch?pathway?in?prostate?development?and?cancer.Differentiation2008，76(6):699-716.

[15]Reya?T，Clevers?H.Wnt?signalling?in?stem?cells?and?cancer.Nature2005；434:843-850.

[16]Wang?BE，Wang?XD，Ernst?JA，Polakis?P，Gao?WQ.Regulation?of?epithelial?branching?morphogenesis?and?cancer?cell?growth?of?the?prostate?by?Wnt?signaling.PLoS?One2008；3:e2186.

[17]Liu?C，Tang?DG.MicroRNA?regulation?of?cancer?stem?cells.Cancer?Res.2011；71:5950-5954.

[18]Liu?C，Kelnar?K，Liu?B，Chen?X，Calhoun-Davis?T，Li?H?et?al.The?microRNA?miR-34a?inhibits?prostate?cancer?stem?cells?and?metastasis?by?directly?repressing?CD44.Nat?Med.2011；17:211-215.

[19]Kong?D，Heath?E，Chen?W，Cher?ML，Powell?I，Heilbrun?L?et?al.Loss?of?let-7up-regulates?EZH2in?prostate?cancer?consistent?with?the?acquisition?of?cancer?stem?cell?signatures?that?are?attenuated?by?BR-DIM.PLoS?One2012；7:e33729.

[20]Liu?C，Kelnar?K，Vlassov?AV，Brown?D，Wang?J，Tang?DG.Distinct?microRNA?Expression?Profiles?in?Prostate?Cancer?Stem/Progenitor?Cells?and?Tumor-Suppressive?Functions?of?let-7.Cancer?Res.2012；72:3393-3404.

[21]Huang?S，Guo?W，Tang?Y，Ren?D，Zou?X，Peng?X.miR-143and?miR-145inhibit?stem?cell?characteristics?of?PC-3prostate?cancer?cells.Oncol?Rep.2012；28:1831-1837.

[22]Hsieh?IS，Chang?KC，Tsai?YT，Ke?JY，Lu?P?J，Lee?KH?et?al.MicroRNA-320suppresses?the?stem?cell-like?characteristics?of?prostate?cancer?cells?by?down-regulating?the?Wnt/beta-catenin?signaling?pathway.Carcinogenesis2012；e-pub?ahead?of?print19Dec2012.

[23]Saini?S，Majid?S，Shahryari?V，Arora?S，Yamamura?S，Chang?I?et?al.miRNA-708control?of?CD44(+)prostate?cancer-initiating?cells.Cancer?Res.2012；72:3618-3630.

[24]Fang?YX，Gao?WQ.Roles?of?microRNAs?during?prostatic?tumorigenesis?and?tumor?progression.Oncogene2014；33:135-147.

[25]Fang?YX，Xue?JL，Shen?Q，Chen?J，Tian?L.MicroRNA-7inhibits?tumor?growth?and?metastasis?by?targeting?the?phosphoinositide3-kinase/Akt?pathway?in?hepatocellular?carcinoma.Hepatology2012；55:1852-1862.

[26]Bonci?D，Coppola?V，Musumeci?M，Addario?A，Giuffrida?R，Memeo?L?et?al.The?miR-15a-miR-16-1cluster?controls?prostate?cancer?by?targeting?multiple?oncogenic?activities.Nat?Med.2008；14:1271-1277.

[27]Chakrabarti?M，Ai?W，Banik?NL，Ray?SK.Overexpression?of?miR-7-1Increases?Efficacy?of?Green?Tea?Polyphenols?for?Induction?of?Apoptosis?in?Human?Malignant?Neuroblastoma?SH-SY5Y?and?SK-N-DZ?Cells.Neurochem?Res.2013；38:420-432.

[28]Zhang?N，Li?X，Wu?CW，Dong?Y，Cai?M，Mok?MT，et?al.microRNA-7is?a?novel?inhibitor?of?YY1contributing?to?colorectal?tumorigenesis.Oncogene2012；e-pub?ahead?of?print3Dec2012；doi:10.1038/onc.2012.526.

[29]Kalinowski?FC，Giles?KM，Candy?PA，Ali?A，Ganda?C，Epis?MR，et?al.Regulation?of?epidermal?growth?factor?receptor?signaling?and?edotinib?sensitivity?in?head?and?neck?cancer?cells?by?miR-7.PLoS?One2012；7:e47067.

[30]Kong?X，Li?G，Yuan?Y，He?Y，Wu?X，Zhang?W，et?al.MicroRNA-7inhibits?epithelial-to-mesenchymal?transition?and?metastasis?of?breast?cancer?cells?via?targeting?FAK?expression.PLoS?One2012；7:e41523.

[31]Kong?D，Piao?YS，Yamashita?S，Oshima?H，Oguma?K，Fushida?S，et?al.Inflammation-induced?repression?of?tumor?suppressor?miR-7in?gastric?tumor?cells.Oncogene2012；31:3949-3960.

[32]Saydam?O，Senol?O，Würdinger?T，Mizrak?A，Ozdener?GB，Stemmer-Rachamimov?AO，et?al.miRNA-7attenuation?in?Schwannoma?tumors?stimulates?growth?by?upregulating?three?oncogenic?signaling?pathways.Cancer?Res.2011；71:852-861.

[33]Kefas?B，Godlewski?J，Comeau?L，Li?Y，Abounader?R，Hawkinson?M，et?al.microRNA-7inhibits?the?epidermal?growth?factor?receptor?and?the?Akt?pathway?and?is?down-regulated?in?glioblastoma.Cancer?Res.2008；68:3566-3572.

[34]Reddy?SD，Ohshiro?K，Rayala?SK，Kumar?R.MicroRNA-7，a?homeoboxD10target，inhibits?p21-activated?kinase1and?regulates?its?functions.Cancer?Res.2008；68:8195-8200.

[35]Dang?DT，Pevsner?J，Yang?VW.The?biology?of?the?mammalian?Kruppel-like?family?of?transcription?factors.Int.J.Biochem.Cell?Biol.2000；32:1103-1121.

[36]Black?AR，Black?JD，Azizkhan-Clifford?J.Sp1and?kruppel-like?factor?family?of?transcription?factors?in?cell?growth?regulation?and?cancer.J.Cell.Physiol.2001；188:143-160.

[37]Xu?N，Papagiannakopoulos?T，Pan?G，Thomson?JA，Kosik?KS.MicroRNA-145regulates?OCT4，SOX2，and?KLF4and?represses?pluripotency?in?human?embryonic?stem?cells.Cell2009；137:647-658.

[38]Liu?C，Kelnar?K，Liu?B，Chen?X，Calhoun-Davis?T，Li?H，et?al.The?microRNA?miR-34a?inhibits?prostate?cancer?stem?cells?and?metastasis?by?directly?repressing?CD44.Nat?Med.2011；17:211-215.

[39]Leong?KG，Wang?BE，Johnson?L，Gao?WQ.Generation?of?a?prostate?from?a?single?adult?stem?cell.Nature2008；456:804-808.

[40]Fang?YX，Zhang?XB，Wei?W，Liu?YW，Chen?JZ，Xue?JL，et?al.Development?of?chimeric?gene?regulators?for?cancer-specific?gene?therapy?with?both?transcriptional?and?translational?targeting.Mol?Biotechnol2010；45:71-81.

Claims (4)

1. The role of microRNA-7 as an expression inhibitor of PI3K/Akt signaling pathway. 2. The role of microRNA-7 as a targeted inhibitor of KLF4. 3. The application of microRNA-7 in the preparation of drugs for inhibiting the growth of prostate tumors. 4. The application of microRNA-7 in the preparation of drugs for blocking the cell cycle progression of prostate tumors.