CN107432876A - The 5p of Diagnosis of Non-Small Cell Lung label microRNA 769 and the application in medicine and diagnostic kit - Google Patents
The 5p of Diagnosis of Non-Small Cell Lung label microRNA 769 and the application in medicine and diagnostic kit Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, discloses one group of 5p of molecular marked compound microRNA 769 for being used for the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy.The present invention further discloses application of the molecular marked compound in the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy diagnostic reagent or kit is prepared.The invention also discloses the 5p of microRNA 769 to prepare with the application in suppression tumour medicine and the 5p of microRNA 769 expression conditions in preparation with the application in suppressing tumor prognosis or target therapeutic agent.The present invention utilizes Protocols in Molecular Biology, can effectively carry out molecular level detection to judge that the mark of transfer, prognosis and targeted therapy occurs for Patients with Non-small-cell Lung, and then these diseases provide facility for individualized treatment.The development to follow-up clinical research has important directive significance simultaneously.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically, is related to a group mark gene microRNA-769-5p
Application as Diagnosis of Non-Small Cell Lung label and in medicine and diagnostic kit.
Background technology
Lung cancer is always one of most dangerous malignant tumour, its high incidence and high case fatality rate be cause Now Domestic or even
In world wide the main reason for Cancer death.Lung cancer is broadly divided into two major classes according to histological type：One kind is ED-SCLC,
Account for the 15% of lung cancer ratio；Another kind of is that non-small cell lung cancer accounts for 85%.Although have necessarily for the research aspect of lung cancer
Progress, include early diagnosis, Neoadjuvant chemotherapy, even the radiotherapy application of targeted therapy of patients with lung cancer, but lung cancer morbidity is hidden
Hide, be already belonging to middle and advanced stage most of when patients with lung cancer is medical, miss best occasion for the treatment, it is sensitive, special, effectively plus lacking
Diagnostic and therapeutic method, the total 5 years survival rates still as little as 15% of patients with lung cancer, even if being early stage when medical, but because of recurrence rate
Very high, therapeutic effect is also bad.According to statistics, it is annual global because the lethal patient of lung cancer is more than 1,000,000, and with annual 1200000
New hair cases of lung cancer be incremented by, serious threat is caused to human health and life.Most area in China, the morbidity of lung cancer
Rate and the death rate are in continue ascendant trend, and men and women's lung cancer morbidity rate and the death rate occupy all kinds of cancer umber ones, if not taking corresponding
Measure is controlled by, and will turn into global first lung cancer big country to China in 2025 is estimated.Therefore explore effectively diagnose, treat with
And the method for prevention lung cancer is particularly important.
Using Protocols in Molecular Biology, molecular level detection can be effectively carried out to judge that malignant tumor patient occurs to turn
The mark of shifting, prognosis and targeted therapy.And then these diseases provide facility for individualized treatment.
MicroRNA is the single-stranded non-coding RNA of small fragment for being about 19-22 nucleotides.It is matched somebody with somebody by defective base
The complementary series of 3 ' non-translational regions to binding its target gene controls gene expression, so that target gene is being transcribed or translated
Horizontal expression is lowered.Recent microRNA such as turns into from growing cancer at the important and revolutionary of many pathophysiological processes
Conservative regulation factor.In different malignant tumours, same microRNA effect may be different.One microRNA can be with
There is the target gene for much belonging to different paths, and a gene can be regulated and controled by several microRNA.Scientist's Development Technology is come
The identification special gene expression mark related to neoplasm staging and patient's prognosis, to improve prognosis and treatment.People use complete
Face transcription is studied to identify the prognosis mark based on gene expression, and still, none can apply to clinic so far.With warp
The mRNA express spectras of allusion quotation are compared, and microRNA tumour express spectras seem more accurately determine the classification of tumors subtypes.It is more next
More evidence support entity tumours has special microRNA marks.At the same time, it has been found that increasing
MicroRNA target gene plays an important role in lung cancer generation, such as let-7, miR-34 family and miR-17-92 clusters.Mesh
Before, identification of the scientist to microRNA target gene is still limited.In molecular framework, turn into after ripe microRNA is powered
The silencing complex (miRISC) of microRNA inductions.The complex contains (a kind of huge protein man of Argonaute families
Race), hold the complementary site of non-translational region to react with being usually located at target gene 3 '.Current model thinks microRNA and its target base
The reaction of cause originates from one section of 6 to 8 nucleotides short-movie section for being referred to as " Seed Sequences " positioned at microRNA 5 ' ends.
Target gene can be reconfigured to especially be spaced by the silencing complex of MicroRNA inductions, the retardance of processing translation wherein and mRNA
Decay.Many researchs confirm the mRNA unstabilitys of microRNA inductions.Combined calculation predicts that the measurement of mRNA express spectras represents
The effective ways of identification function microRNA- target relations.In non-small cell lung cancer, many different microRNA regulations are different
Often, there may be carcinogenic or cancer suppressing action, and predict prognosis.Identification can predict that the microRNA of patients with lung cancer prognosis is a weight
It was found that this shows that microRNA may play an important role in tumour progression.Existing clinicopathologic stage is in prediction patient
There is its limitation on brain metastes.Transfer generation and prognosis with similar clinical pathological characteristic or identical patients with lung cancer by stages
May be significantly different.Molecular marked compound can help doctor to differentiate high-risk patient, to give individual's treatment, so as to change
Kind existence.
Tumor cells mark (Tumor Marker) is chemicals existing for reflection tumour.They are not present in
Normal adult tissue and be detected in embryonic tissue, or the content in tumor tissues substantially exceeds the content in normal structure,
The presence of which or quantitative change can prompt the property of tumour, so as to understanding the tissue generation of tumour, cell differentiation, cell function,
To help the diagnosis of tumour, classification, Index for diagnosis and treatment to instruct.
The content of the invention
In order to solve the early detection of current Patients with Non-small-cell Lung hardly possible, the difficult technical barrier of early intervention, the present invention
A group mark gene microRNA-769-5p is provided as Diagnosis of Non-Small Cell Lung label and in medicine and diagnostic reagent
Application in box.
In order to solve the above-mentioned technical problem, the present invention is addressed by following technical proposals：
Prepared containing material as described below with the application in suppressing tumour medicine：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
The microRNA-769-5p sequences are：UGAGACCUCUGGGUUCUGAGCU.
Preferably, the suppression tumour shifts to suppress non-small cell lung carcinogenesis, diffusion.
Present invention also offers the expression conditions of the material to prepare with suppression tumor prognosis or targeted therapy
Application in medicine：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
Preferably, the tumour is non-small cell lung cancer.
Further object of the present invention is used for the molecular marked compound of the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy,
Described molecular marked compound is microRNA-769-5p.
Further, the molecular marked compound is preparing the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy diagnosis
Application in reagent or kit.
Further, the prognosis monitors for detection, curative effect evaluation or transfer and relapse.
Molecular marked compound is microRNA-769-5p, abbreviation miR-769-5p.
Compared with prior art, advantageous effects of the invention are as follows：
1) Protocols in Molecular Biology is utilized, can effectively carry out molecular level detection to judge Patients with Non-small-cell Lung
The mark of transfer, prognosis and targeted therapy occurs, and then these diseases provide facility for individualized treatment.
2) the invention discloses a kind of gene microRNA-769-5p related to Patients with Non-small-cell Lung, one is gone forward side by side
Step confirms that the molecular marked compound of transfer, prognosis and targeted therapy can occur as Patients with Non-small-cell Lung for said gene, not only
Accuracy greatly improves, while has important directive significance to the development of follow-up clinical research.
Brief description of the drawings
Fig. 1 is that expressions of the microRNA-769-5p of the present invention in non-small cell tissue is normal significantly lower than by cancer
Lung tissue figure.
Fig. 2 is that miR-769-5p expressions of the present invention are schemed with clinical stages in obvious negative correlation, and miR-769-5p is in III
Expression in+IV phase Non-Small Cell Lung Carcinomas is less than the expression in I+II phase Non-Small Cell Lung Carcinomas.
Fig. 3 is that miR-769-5p expressions of the present invention are schemed with lymphatic metastasis in obvious negative correlation, and miR-769-5p exists
Expression in lymphatic metastasis Non-Small Cell Lung Carcinoma is less than in without lymphatic metastasis Non-Small Cell Lung Carcinoma
Expression.
Fig. 4 is that survival analysis of the present invention shows that miR-769-5p low expression group patient's (n=70) life cycles are substantially less than height
Expression group figure.
Fig. 5 is that survival analysis of the present invention shows that miR-769-5p low expression group patient's (n=93) life cycles are substantially less than height
Expression group figure.
Fig. 6 is that the high expression of miR-769-5p of the present invention can suppress non-small cell lung cancer cell strain cell vegetative map.
Fig. 7 is that the high expression of miR-769-5p of the present invention can suppress non-small cell lung cancer cell strain cell plates Clone formation energy
Try hard to.
Fig. 8 is that the high expression of miR-769-5p of the present invention can suppress non-small cell lung cancer cell strain cell invasion transfer figure.
Fig. 9 is the multiplication capacity figure that miR-769-5p low expressions of the present invention can promote non-small cell lung cancer cell strain cell.
Figure 10 is the plate clone shape that miR-769-5p low expressions of the present invention can promote non-small cell lung cancer cell strain cell
Into can try hard to.
Figure 11 is that miR-769-5p low expressions of the present invention can promote the invasion and attack of non-small cell lung cancer cell strain cell to shift energy
Try hard to.
Figure 12 is after miR-769-5p of the present invention lowers expression, hence it is evident that promotes the growth figure of transplanted tumor in nude mice.
Figure 13 is after miR-769-5p of the present invention lowers expression, hence it is evident that promotes the generation of non-small cell lung cancer metastatic tumor
Figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further elaborated, but embodiment does not form any limit to the present invention
System.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The experimental method of unreceipted specific condition in embodiment, usually this area conventional method.
The method that the mark of the transfer of non-small cell lung carcinogenesis, prognosis and targeted therapy is found in this implementation is as follows：
First, patient and sample：
Looked back 2004.7-2009.6, after complete excision 93 paraffin specimens of proved by pathology non-small cell lung cancer and
It is preoperative do not receive chemicotherapy or other for tumour treatment patient, last follow up time is in August, 2014.
Collect the tissue specimen and distance of the Patients with Non-small-cell Lung of operation on December 30, -2015 years on the 1st January in 2013
Lung tissue paraffin specimen by tumor tissues ＞ 5cm cancer, totally 70, last follow up time is in July, 2017.
Clinical stages, all patient's arts are carried out according to the TNM stage standard of International Union Against Cancer (UICC) formulation in 2010
Preceding not receive any treatment, postoperative all patients receive adjuvant chemotherapy of patients.
2nd, in situ hybridization：
It is conventional to dewax to water, phosphate buffer solution (PBS, main component K2HPO4、KH2PO 3min × 2) are washed；0.1M
Glycine is incubated 5min × 2；0.3%Triton X-100 (PBS) handle 15min, and PBS washes 3min × 3；(concentration is Proteinase K
20ug/ml) 37 DEG C (first preheating) 20min, PBS wash 3min × 3；4% paraformaldehyde fixes 5min, and PBS washes 3min × 3；
0.25% acetic anhydride (0.1M triethanolamines, pH8.0) handles 10min, and PBS washes 3min × 3；At -20 DEG C, respectively with 70%,
85% and 100% ethanol postincubation 5min, is dehydrated, is finally air-dried；Probe is added dropwise, and (4 probes press 1 respectively：250 dilutions,
Each section is added on request, and 50ul/ opens), slide is capped, warm box is placed in, 48 DEG C of hybridized overnights, is 16-18h；Post-hybridization washing,
SSC cushioning liquid (main component is sodium chloride, sodium citrate, hydrochloric acid) capping piece, 37 DEG C of SSC cushioning liquid 5min × 3, SSC
15min × 2 time；Section, which is placed in buffer buffer solutions (main component is sodium chloride, potassium chloride, magnesium chloride, bicarbonate radical), soaks
5min；Each tissue 0 DEG C of incubation 15min in buffer buffer solutions；1 is pressed with buffer buffer solutions：1000 dilution digoxin
DNA marker antibody, 50ul antibody diluents, 37 DEG C of incubation 1h are added dropwise；10minX2 is washed with buffer buffer solutions；Delayed with buffer
Fliud flushing weighing apparatus section 5min；Nitrite ion is prepared, 1 is pressed with buffer buffer solutions：50 dilution tetrazolyl nitro blue stostes, prepared before use,
Lucifuge.After nitrite ion is added dropwise in section, colour developing situation is observed after lucifuge colour developing 2-3h, such as develops the color shallower, can develop the color overnight.With
Buffer buffer solutions wash color development stopping, and washing is several times；Core fast red is added dropwise to redye, 37 DEG C, dyes 5-10min, washing is several times；It hurry up
Speed is dehydrated transparent, neutral gum mounting, micro- sem observation.
3rd, RNA extractions, quality testing, chip hybridization and data processing
(1), limonene dewaxing treatment paraffin-embedded tissue
The pretreatment of paraffin-embedded tissues：It is optional to need thickness to be not more than 50 μ if paraffin-embedded tissue is sliced
M section ten or so, removes surrounding paraffin, is placed in 1.5mL centrifuge tubes as far as possible.
Tissue can gently be scraped off if a monoblock and be placed in 1.5mL centrifugations by paraffin-embedded tissue with sharp cutter
Pipe, avoids scraping paraffin as far as possible.The tissue mass scraped is controlled within 100mg.
Limonene dewaxing treatment：
1ml limonenes (Limonen) are added in centrifuge tube, 55 DEG C of concussions mix 5min.13,200rpm room temperatures centrifuge
2min, abandon supernatant.This step is carried out 3 times altogether.
1ml absolute ethyl alcohols are added into centrifuge tube, 55 DEG C of concussions mix 2min, 13,200rpm room temperatures centrifugation 2min, abandon
Clearly.Repeat this step once.
Supernatant is abandoned, the of short duration centrifugation of room temperature, is suctioned out remaining absolute ethyl alcohol with pipettor.
Centrifuge tube is placed in vacuum drying instrument, drained to tissue in the powdered of drying.
(2), RNA, microRNA of lung tissue sample extraction
1. organize RNA extraction
All processes are carried out in strict accordance with Trizol kit specifications, are operated under without RNase environment, and test tube used is inhaled
Head and solution are routinely with 1% pyrocarbonic acid diethyl ester (DEPC) water process (except Tris), ambient temperature overnight.
(1) each 50~l00mg of the cancerous lung tissue frozen, Carcinoma side normal tissue is taken respectively, is inserted and is previously added 0.5ml
In the 1.5mlEppendorf tubules of Trizol lysates, slurry is constantly ground into homogenizer, and adds Trizol cracking
Liquid is to 1ml.
Being incubated 5 minutes at (2) 15~30 DEG C is completely dissolved nucleoprotein complex, is imitated per 1ml Trizol reaction solution chlorinations
0.2ml, strong concussion 15 seconds, 15~30 DEG C are incubated 2~3 minutes.
(3) 4 DEG C of 12,000g are centrifuged 15 minutes, move supernatant into another Eppendorf tubules, volume about adds
The 60% of Trizol reaction solution volumes.
(4) RNA is precipitated：Add 0.5ml isopropanols per 1mlTrizol, lO minutes are incubated at 15~30 DEG C.
(5) 2~8 DEG C≤12,000g is centrifuged 10 minutes.
(6) supernatant is abandoned, 70% ethanol (tri-distilled water handled with DEPC is prepared) >=lml is rinsed 2 times.
(7) 2~8 DEG C≤7,500g is centrifuged 5 minutes.
(8) vacuum drains RNA, is dissolved in 50 μ lDEPC water.- 70 DEG C save backup.
(9) RNA is quantitative and electroresis appraisal
A. take l μ l RNA solutions to add 100 μ l water, after mixing with ultraviolet-uisible spectrophotometer determine A260, A280 and
A260/A280 ratios, and read RNA concentration.It should be 1.8~2.0.
B.1.2% denaturing formaldehyde agarose gel electrophoresis identification RNA mass：
1. 0.3% H of electrophoresis tank202Immersion 30 minutes, rinsed and dried with DEPC water.
2. glue (20ml), 0.24g containing agarose, without RNAase water 17.4ml, 10 × MOPS 2ml, 37% formaldehyde
0.6ml, EB lml, after Agarose water micro-wave oven heating and melting, add 10 × MOPS, treat that gel is cooled to 60 DEG C and adds formaldehyde again
And EB.Then the Casting of gels in glue groove, comb, horizontal positioned rear use to be solidified are plugged.
3. by gel prerunning 5min, voltage is reduced to 5V/cm.
4. taking appropriate RNA, add electrophoretic buffer (10 ×) 2 μ l, μ l of formaldehyde 3.5, formamide l0 μ l and mix, put 60 DEG C
10min is incubated, on ice quickly cooling.The sample-loading buffer for adding 3 μ l mixes, and takes and is loaded onto in right amount in gel loading wells.With time point RNA
Standard items.
5. electrophoresis is taken pictures after terminating under uviol lamp.
28S and the band of 18S two, and 28S should be seen after electrophoresis：18S ratios are more than 2, illustrate that RNA is undegraded.
2. the extraction of cell tiny RNA
The extraction of cell line microRNA (≤200nt) is carried to specifications with mirVanaTMmiRNA extracts kits
Take.Method is summarized as follows：
Cell (<Cracked after 107) collecting with 600 μ l cracking/combination buffer (take and organize in right amount (<250mg), in liquid nitrogen
Middle grind into powder, it is transferred in 600 μ l cracking/combination buffer and cracks), 60 μ lmiRNA homogenates are added, are put on ice
Put lOmin.600 μ l acid phenol/chloroform is added, 30-60s is acutely mixed and (is divided completely with 10000rpm centrifugations 5min after room temperature
Layer).Careful supernatant of drawing is transferred in new no RNase1.5ml centrifuge tubes.
The absolute ethyl alcohol of l/3 volumes is added, is added to after mixing completely in the centrifugal column in collecting pipe (every time at most
700 μ l), room temperature centrifuges lmin with 10000rpm, collects efflux.
The absolute ethyl alcohol of its 2/3 volume is added in the efflux of collection, is then added to after mixing completely mounted in collection
In another new centrifugal column in pipe, room temperature centrifuges lmin with l0000rpm, discards efflux.
700 μ lmiRNA washing lotions 1, centrifugation 5-10s rinsings are added in centrifugal column.
Rinsed twice with 500 μ l washing lotions 2/3 as stated above again.Lmin is centrifuged again to remove ethanol completely.
Add the eluent of 100 μ l room temperatures, l0000rpm centrifugation lmin, reclaim RNA.
After RNA dilutes 25 times, ultraviolet specrophotometer surveys its OD260 and OD280 value, and calculates OD260/D280, than
When value is more than 1.8, show that RNA purity is preferable.RNA concentration is calculated according to OD260 simultaneously.
4th, mimic or inhibitor transient transfection
1. mimic or inhibitor is prepared：250 μ 1 × Generic buffers of l are added in 5nmol double-strand siRNA, are obtained
To mimic the or inhibitor mother liquors that concentration is 20 μM, -20 degrees Celsius of preservations are put.
2. taking the cell that growth conditions are good, cell is inoculated in 60mm culture dishes in the day before transfection and (is not added with antibiosis
Element), cell density reaches 30% or so during transfection.
3. prepare following complex：A liquid：The mimic of debita spissitudo or inhibitor are diluted in the training of 500 μ l serum-frees
Support in base, gently mix；B liquid：10 μ l Lipofectamine 2000 (softly being mixed before) are taken to be diluted in 500 μ l serum-frees
In culture medium, mix.It is incubated at room temperature 5min.
4. the liposome of dilution is mixed with the mimic or inhibitor diluted, gently mix, be incubated at room temperature 20 minutes
(complex can keep stable in 6 hours in room temperature).
5. the mixed μ l of compound 1000 are added in Tissue Culture Dish, serum free medium is added gently to be mixed to 5ml
It is even.Former culture medium is discarded after 6 hours, changes the culture medium containing 10% serum.
6. cell is collected after 48 hours carries out Westernblot, RT-PCR, MTS etc. are accordingly tested.
5th, MTS methods detection cell growth curve
When cell growth to exponential phase, carry out cell transfecting or carry out respective handling, collect cell, it is suitable to be diluted to
The cell suspension of concentration, cell suspension is added in 96 porocyte culture plates, per 5000, hole cell/100 μ l, in incubator
Continue culture to appropriate time.Each experimental group does 6 parallel holes, only to add the hole of culture medium to make blank control.If at four
Between point：0h (was both surveyed) after adherent after plating cells, 24h, 48h, 72h, 96h.Using MTS colorimetric method for determining cytoactives, per hole
15 μ l MTS reagents (500 μ g/ml) are added, continue to determine light suction at wavelength 570nm with enzyme mark spectrophotometer after cultivating 2h
Receipts value (OD), is returned to zero with blank well, and OD values are higher, and cell quantity is more.
6th, Colony forming experiment is cloned
1. carry out cell transfecting or processing cell according to requirement of experiment.
2. preparing big ware, 10ml training bases, and 800 cells are added per hole.
3. cell is cultivated -14 days 10 days at the standard conditions, observation Clone formation situation
4. terminating culture when cell forms macroscopic clone, culture medium is discarded, cell is carefully cleaned with PBS 2 times,
Add 4% formaldehyde to fix, 1ml/ holes, fixed 15min, discard fixer, slowly rinsed well with flowing water, add 1ml crystal violets to contaminate
Liquid dyes 3min.Dyeing liquor is slowly washed away with flowing water, dried in fume hood, photograph.
7th, chemotactic cell experiment (Transwell)
1. cell count, 5 × 10 are added in cell5Individual cell, in upper chamber plus 500ul serum free mediums, it is lower indoor plus
800ul contains 10% blood serum medium.
2. after migrating 12-18 hours, the nutrient solution of the upper and lower room of filter membrane is discarded, with the PBS of preheating, gently blows and beats PBS
Have the function that cleaning filter membranes lower surface, be repeated 1 times；
3. moving into the μ l of 4% paraformaldehyde 600 in lower room, filter membrane lower surface is set to be immersed in wherein fixed cell, 15min；
4. discarding fixer, transwell cells are inverted, make filter membrane following table face-up, natural air drying；
5. few drops of Giemsa stains are directly dripped after air-drying in the filter membrane lower surface of inverted transwell cells,
10min；
6. distilled water cleans, the cell that cell face do not migrate is wiped with cotton balls, observes and counts under inverted microscope.
8th, small amount plasmid is extracted
1. by 1-5ml (plasmid of high copy number) or 10ml (plasmid of low copy number) bacterial cultures with desk-top centrifugation
Machine 10,000g is centrifuged 5 minutes.Test tube is simultaneously inverted on paper handkerchief and sucks remaining nutrient solution by supernatant discarding.
2. add 250 μ l cell suspending liquids and vortex concussion or piping and druming are with abundant suspension cell.Abundant suspension cell is very
It is crucial.If not in centrifuge tube, the cell of suspension is transferred in 1.5ml sterilization centrifuge tubes.Add 250 μ l cell pyrolysis liquids
And reverse centrifuge tube 4 times is to be sufficiently mixed (should not vortex shake).It is incubated to cell suspending liquid and clarifies, takes around 1-5 minutes.
Pay attention to：It is extremely important that the clarification of lysate part is observed before adding alkaline protease solution (the 4th step)；But incubation time should not
More than 5 minutes.
3. add 10 μ l alkaline protease solutions and overturn centrifuge tube 4 times to be sufficiently mixed.In incubation at room temperature 5 minutes.
Alkali protease can inactivate nuclease and it is other discharged during bacteria lysis can influence separate matter
The albumen of grain quality.Add 350 μ lIn Plus SV and solution and rapidly reverse centrifuge tube 4 times to be sufficiently mixed (no
Vortex is wanted to shake).Cell pyrolysis liquid (about 14,000g) is centrifuged 10 minutes in room temperature.Cleared lysate (850 μ l) is transferred to
In ready centrifugal column.It not stir or be transferred along any white precipitate with supernatant.
4. in room temperature centrifuge supernatant 1 minute.Centrifuge tube is taken out from collecting pipe and is discarded in collecting pipe
Liquid.Centrifugal column is reinserted in collecting pipe.Add the post cleaning fluid that 750 μ l were previously diluted with 95% alcohol.In room temperature
With centrifuge with maximum velocity centrifugation 1 minute.Centrifuge tube is taken out from collecting pipe and discards the liquid in collecting pipe.By centrifugal column
Reinsert in collecting pipe.
5. with 250 μ l post cleaning fluid repeated washing steps.In room temperature centrifuge with maximum velocity centrifugation 2 minutes.
Centrifugal column is transferred in a new 1.5ml sterilizations centrifuge tube, need during operation it is careful, should not be by any point post
Cleaning fluid is transferred along with centrifugal column.If being stained with post cleaning fluid on centrifugal column, again with maximum velocity centrifugation 1 minute.Will
Centrifugal column is transferred in a new 1.5ml sterilization centrifuge tube.The water of 100 μ l nuclease frees is added into centrifugal column to elute plasmid
DNA.In room temperature centrifuge 1 minute.
6. after eluting DNA, centrifugal column is sterilized from 1.5ml and is taken out in centrifuge tube and discards centrifugal column.It is not added with buffering
The aqueous dna of liquid at -20 DEG C and it is following be stable.(TE buffer solutions, are broadly divided into three (methylol) aminomethane buffer solutions
0.1mol/L trishydroxymethylaminomethanes, 0.1mol/LEDTA) in DNA at 4 degree be stable.If necessary to which DNA is deposited
In TE buffer solutions, 11 μ 10 × TE of l buffer solutions are added in the DNA of 100 μ l elutions.Centrifuge tube is covered and by after purification
DNA deposits in -20 DEG C or less.
9th, plasmid extraction is identified
The measure of plasmid concentration is carried out with Amersham Bioscience Gene Quant concentration mensurations instrument.DNA exists
There is the absworption peak of maximum at 260nm, protein has the absworption peak of maximum at 280nm, and salt and small molecule then concentrate on 230nm
Place.Therefore, spectrophotometry DNA concentration can be carried out with 260nm wavelength, OD values are 1.0 double equivalent to about 50 μ g/ml
Chain DNA.1cm optical paths are such as used, with 100 times of deionized water dilution DNA sample and using water as blank control, according to what is now read
OD260 values can calculate the concentration before sample dilution：
DNA (mg/ml)=50 × OD260 readings × extension rate/1000；
The OD260/OD280 of DNA sterlings is 1.8, therefore DNA purity can be estimated according to OD260/OD280 value.If than
The higher explanation of value contains RNA, and the low explanation of ratio is with the presence of residual protein.OD230/OD260 ratio should 0.4-0.5 it
Between, if the higher explanation of ratio is with the presence of remnants salt.
Tenth, luciferase reporter gene is built
Utilize PCR method, the 3'UTR sequences using 293T cell genomic dnas as template PCR amplifications MMP19 genes, by it
It is cloned into pmiR-RB-REPORTTMIn Dual-Luciferase report carrier, the reporter fluorescence of used carrier is hRluc, corrects fluorescence
For hluc (doing internal reference correction).
1. gene sequencing
Gene order and carrier sequence are analyzed, can utilize Xhol, two restriction enzymes of Not1 are by target gene
Fragment is cloned into carrier, and Vector map is as follows：
②PCR
Reaction system is designed as 30 μ l total systems, μ l, 2.5mM the deoxyribose cores of specifically 5 × DNA polymerase buffer liquid 6
The μ l of guanosine triphosphate 2, each μ l, DNA moulds of 1 μ l, PrimeSTAR HS archaeal dna polymerases (2.5U/ μ l) 0.3 of upstream and downstream primer (10uM)
The μ l (about 100ng) of plate 1,30 μ l systems are supplied with aqua sterilisa.Reaction condition is (touchdown PCR)：98 DEG C of 2min pre-degenerations, in circulation
98 DEG C of 10s denaturation, from 68 DEG C of each 1 DEG C of annealing of cycle down, 72 DEG C of extension 45s；10 circulations；In 62 DEG C of annealing, 15 are carried out
Circulation；72 DEG C are continued to extend 3min after PCR reaction cycles, then 4 DEG C of preservations.
3. digestion
Double digestion is carried out to above-mentioned PCR primer and carrier with XhoI, NotI, endonuclease reaction system is as follows:10X H buffer solutions
3 μ l, DNA about 2 μ g, restriction endonuclease (10U/ μ l) each 1 μ l, sterile deionized water are supplied to 30 μ l.37 DEG C of reaction condition, 3hr.
4. connect
The μ l (about 150ng) of purpose fragment DNA 15, carrier 1 μ l (about 50ng), 10X T4 connection buffer solution 2ul, T4 connection
Enzyme 1ul, sterile deionized water are supplied to 20 μ l, 22 DEG C of connection 3hr.
5. convert
Take connection product to be added in 100 μ lDH5 α competent cells, mix, ice bath 30 minutes；Above-mentioned conversion fluid is placed in
42 DEG C of water-baths 90 seconds, placement 2-3 minutes in ice bath are immediately placed on after taking-up；The LB of 900 μ l, 37 DEG C of preheatings is added thereto (no
Containing antibiotic) culture medium, 150rpm, 37 DEG C of shaken cultivations 45 minutes；2500rpm centrifuges 5min, and supernatant is siphoned away, stays 100 μ
L mixes bacterium solution, (the μ g/ml of antibiotic concentration 100) is added on the LB solid agar mediums of antibiotic containing Amp, with sterile elbow
Glass rod with gentle cell is uniformly spreadable.After planar surface drying, flat board, 37 DEG C of culture 12-16 hours are inverted.
6. bacterium colony PCR is identified
Single bacterium colony on the above-mentioned flat board of picking, in the molten water to 3ul, take 0.5ul to do template, enter performing PCR.Reaction system designs
For 10 μ l total systems, specifically 10X PCR amplification buffers (predominantly 100mM Tris-HCl buffer solutions, 15mM Mg2+,
500mM KCl) 1 μ l, 25mM MgCl2The μ l of 1 μ l, 2.5mM deoxyribonucleoside triphosphate 0.8, carrier upstream and downstream primer (10uM)
Each μ l of 0.5 μ l, Taq archaeal dna polymerase (2.5U/ μ l) 0.1, the μ l (about 100ng) of DNA profiling 0.5,10 μ l bodies are supplied with aqua sterilisa
System.Reaction condition is 95 DEG C of 3min pre-degenerations, 95 DEG C of 30s denaturation in circulation, 57 DEG C of annealing, 72 DEG C of extension 90s；25 circulations；
72 DEG C are continued to extend 5min after PCR reaction cycles, then 4 DEG C of preservations.
7. send the clone of the positive to sequencing company sequencing further identification.
8. mutational vector is built
A, it is mutated PCR
Reaction system is designed as 30 μ l total systems, μ l, the 2.5mM dNTPmix 2 of specifically 5 × PrimeSTARBuffer 6
μ l, upstream and downstream primer (10uM) each 1 μ l, PrimeSTARHS archaeal dna polymerase (2.5U/ μ l) 0.5 μ l, the μ of wild plasmid template 1
L (about 20ng), 30 μ l systems are supplied with aqua sterilisa.Reaction condition is (touchdown PCR)：98 DEG C of 2min pre-degenerations, 98 DEG C in circulation
10s is denatured, from 66 DEG C of each 1 DEG C of annealing of cycle down, 72 DEG C of extension 45s；10 circulations；62 DEG C of annealing again, carry out 20 circulations；
72 DEG C are continued to extend 5min after PCR reaction cycles, then 4 DEG C of preservations.
B, the wild plasmid template of DnpI endonuclease digestions residual
State directly up and 1.5 μ l DnpI restriction endonucleases, 37 DEG C of digestion 1hr are added in PCR reaction products.
C, convert
A, take the μ l of connection product 10 to be added in 100 μ lDH5 α competent cells, mix, ice bath 30 minutes.
B, above-mentioned conversion fluid is placed in 42 DEG C of water-baths 90 seconds, placement 2-3 minutes in ice bath is immediately placed on after taking-up.
C, LB (being free of antibiotic) culture medium of 900 μ l, 37 DEG C of preheatings, 150rpm, 37 DEG C of shaken cultivations are added thereto
45 minutes.
D, 2500rpm centrifuges 5min, and supernatant is siphoned away, and stays 100 μ l to mix bacterium solution, is added to the LB solids of antibiotic containing Amp
It is with sterile elbow glass rod with gentle that cell is uniformly spreadable on agar medium (the μ g/ml of antibiotic concentration 100).Treat flat board
After dry tack free, flat board, 37 DEG C of culture 12-16 hours are inverted.
D, fall to expand culture extraction plasmid
Single bacterium colony on the above-mentioned flat board of picking, it is inoculated into antibiotic containing Amp (the μ g/ml of antibiotic concentration 100) 4ml LB
In fluid nutrient medium, in 220rpm shaking table cultures 12h；Then plasmid is extracted.
9. the cell in exponential phase is taken to be inoculated in 12 orifice plates, using as the sea pansy of internal reference and MMP
19th, P21 wild types or mutant plasmids are with 1:500 ratio is mixed in the RPMI1640 culture mediums of serum-free
Close, then miR-X mimic or control miRNA are added in mixed liquor.After transfecting 48h, according to Promaga Dual-Luciferase reports
Accuse the Activity determination that kit gene specification carries out luciferase.
11, transplanted tumor in nude mice and pulmonary metastases formation and Experiment on therapy
SPF level BABIC nude mices (5-6 week old, male and female half and half, 10/group/cell line), respectively by 1 × 106Non-small cell
Lung cancer cell line is inoculated in dorsal sc and tail vein on the right side of nude mice, to form transplanted tumor in nude mice and metastatic tumor model, after 7 days,
Dorsal sc and tail vein injection miR-769-5p upper adjustment antagomir on the right side of nude mice, 5 days once, totally 7 times.After 42 days
It is used uniformly and draws cervical approach to put to death mouse, transplanted tumor in nude mice and pulmonary metastases tissue, formalin fixing process is peeled off in dissection.Specifically
Pulmonary metastases are formed and Experiment on therapy is referring to Figure 12 and Figure 13, are shown in Figure 12, after miR-769-5p lowers expression, hence it is evident that are promoted
The growth of transplanted tumor in nude mice.Shown in Figure 13, after miR-769-5p lowers expression, hence it is evident that promote the generation of pulmonary metastases.
12, statistical analysis
All data of this experiment be more than 3 times repeat experiment draw.Data processing software be Prism5 and
WordExcel.Data are through SPSS16.0 statistical analyses, P<0.05 thinks statistically significant.
The present embodiment additionally provides finds the transfer of non-small cell lung carcinogenesis, prognosis and targeted therapy using the above method
Mark is being prepared with the application in suppressing tumour medicine：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
Wherein, microRNA-769-5p sequences are：UGAGACCUCUGGGUUCUGAGCU.
The present embodiment finds the mark of the transfer of non-small cell lung carcinogenesis, prognosis and targeted therapy using the above method
MicroRNA-769-5p has following experiment effect：
Referring to Fig. 1, expressions of the microRNA-769-5p in non-small cell tissue is significantly lower than normal lung group by cancer
Knit.
Referring to Fig. 2, miR-769-5p expressions are with clinical stages in obvious negatively correlated figure, and miR-769-5p is in III+IV
Expression in phase Non-Small Cell Lung Carcinoma is less than the expression in I+II phase Non-Small Cell Lung Carcinomas.
Referring to Fig. 3, miR-769-5p expressions are with lymphatic metastasis in obvious negative correlation, and miR-769-5p is in lymph node
The expression in Non-Small Cell Lung Carcinoma is shifted less than the expression water in without lymphatic metastasis Non-Small Cell Lung Carcinoma
It is flat.
Referring to Fig. 4, survival analysis shows that miR-769-5p low expression group patient's (n=70) life cycles are substantially less than high expression
Group.
Referring to Fig. 5, survival analysis shows that miR-769-5p low expression group patient's (n=93) life cycles are substantially less than high expression
Group.
Referring to Fig. 6, the high expression of miR-769-5p can suppress non-small cell lung cancer cell strain cell propagation.
Referring to Fig. 7, miR-769-5p is high, and expression can suppress non-small cell lung cancer cell strain cell plates clonality.
Referring to Fig. 8, the high expression of miR-769-5p can suppress the transfer of non-small cell lung cancer cell strain cell invasion.
Referring to Fig. 9, miR-769-5p low expressions can promote the multiplication capacity of non-small cell lung cancer cell strain cell.
Referring to Figure 10, miR-769-5p low expressions can promote the plate clone of non-small cell lung cancer cell strain cell to form energy
Power.
Referring to Figure 11, miR-769-5p low expressions can promote the invasion and attack transfer ability of non-small cell lung cancer cell strain cell.
Wherein Fig. 2, Fig. 3 and Fig. 8 show that miR-769-5p labels have and suppress the transfer of non-small cell lung carcinogenesis, diffusion
Design sketch.
Wherein Fig. 4 and Fig. 5 shows that miR-769-5p labels have the design sketch for suppressing non-small cell lung cancer prognosis.
The expression conditions that the present embodiment additionally provides miR-769-5p are being prepared with suppression tumor prognosis or targeting
Application in medicine：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
Tumour in the present embodiment is non-small cell lung cancer.
The molecular marked compound for the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy is present embodiments provided, should
Molecular marked compound is microRNA-769-5p.
In the present embodiment molecular marked compound microRNA-769-5p prepare non-small cell lung carcinogenesis transfer, prognosis or
Application in targeted therapy diagnostic reagent or kit.
Diagnostic reagent or kit in this implementation are conventionally prepared.
Prognosis monitors for detection, curative effect evaluation or transfer and relapse in the present embodiment.
Molecular marked compound microRNA-769-5p kit monitoring non-small cell lung cancer transfer and relapse in the present embodiment.
The patient that 10 pathological examinations are diagnosed as non-small cell lung cancer is chosen, follow-up is tracked to it.Clinical samples
From the Patients with Non-small-cell Lung of Tangshan City People's Hospital, the patient of all collections is by effective treatment, clinical data
And follow-up resource is complete.The six weeks blood for gathering patient first first after 10 Treatment for Non-small Cell Lung of collection, detects blood
The expression quantity of middle corresponding gene, later every three months check once, track nine months, detect four times altogether.
The relative expression quantity of corresponding gene in 10 patient's Patients with Non-small-cell Lung blood is detected using kit.According to
Patients with Non-small-cell Lung treatment after 6 weeks, 3 months, 6 months, 9 months when blood sample in corresponding gene relative expression quantity and
Judge whether Patients with Non-small-cell Lung occurs transfer and relapse compared to the level of change before treatment.Criterion is set to, treatment
When the relative expression quantity of gene is declined more than or equal to 35% compared with pre-treatment afterwards, it is judged as transfer and relapse；Base after treatment
When the relative expression quantity of cause is declined more than or equal to 35% compared with pre-treatment, it is judged as transfer and relapse；Gene after treatment
When relative expression quantity is dropped by less than 35% compared with pre-treatment, it is judged as Progression free survival.
The detection judged result of kit is as shown in table 1.
Table 1：The detection judged result of kit
| Sequence number | 6 weeks | 3 months | 6 months | 9 months | Clinical effectiveness |
| 1 | Rise 25% | Rise 22% | Rise 20% | Rise 18% | Progression free survival |
| 2 | Rise 20% | Rise 12% | Decline 17% | Decline 45% | Transfer and relapse |
| 3 | Rise 26% | Rise 22% | Rise 23% | Rise 20% | Progression free survival |
| 4 | Rise 17% | Rise 15% | Rise 8% | Decline 42% | Transfer and relapse |
| 5 | Rise 25% | Rise 23% | Rise 20% | Rise 19% | Progression free survival |
| 6 | Rise 23% | Rise 19% | Rise 5% | Decline 38% | Transfer and relapse |
| 7 | Rise 23% | Rise 12% | Rise 4% | Decline 50% | Transfer and relapse |
| 8 | Rise 20% | Rise 18% | Rise 15% | Rise 12% | Progression free survival |
| 9 | Rise 25% | Rise 12% | Decline 22% | Decline 65% | Transfer and relapse |
| 10 | Rise 25% | Rise 8% | Rise 5% | Decline 48% | Transfer and relapse |
Know from table 1, clinical diagnosis result is shown in 10 Patients with Non-small-cell Lung transfer occurs again after there are 6 treatments
Hair, 4 Progression free survivals.Non-small cell lung cancer is monitored using the kit of the present invention, can be found earlier than clinical symptoms and sign,
Carry out intervening in advance for doctor providing reference.
It the above is only the preferred embodiment of the present invention, it is noted that although with reference to preferred embodiment to patent of the present invention
It is explained in detail, for the person of ordinary skill of the art, some improvement can be carried out to technical scheme
And retouching, but the spirit and scope of technical solution of the present invention is not departed from, these improvements and modifications are also considered as the protection model of the present invention
Enclose.
Claims (7)
1. prepared containing material as described below with the application in suppressing tumour medicine：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
2. application as claimed in claim 1, it is characterised in that the suppression tumour turns to suppress non-small cell lung carcinogenesis
Move, diffusion.
3. the expression conditions containing material as described below are being prepared with suppression tumor prognosis or target therapeutic agent
Using：
a)microRNA-769-5p；
B) recombinant vector of the Noncoding gene containing microRNA-769-5p；
C) recombinant viral vector of the Noncoding gene containing microRNA-769-5p.
4. application as claimed in claim 3, it is characterised in that the tumour is non-small cell lung cancer.
5. the molecular marked compound for the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy, it is characterised in that described point
Sub- label is microRNA-769-5p.
6. molecular marked compound as claimed in claim 5 prepare the transfer of non-small cell lung carcinogenesis, prognosis or targeted therapy are examined
Application in disconnected reagent or kit.
7. application as claimed in claim 5, it is characterised in that the prognosis monitors for detection, curative effect evaluation or transfer and relapse.
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| CN112980957A (en) * | 2021-03-19 | 2021-06-18 | 温州医科大学 | Target hsa _ circ _0001326 for inhibiting non-small cell lung cancer metastasis and application thereof |
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