CN109811059A

CN109811059A - Application of the biomarker UGGT1 in cervical disease

Info

Publication number: CN109811059A
Application number: CN201910254347.1A
Authority: CN
Inventors: 杨承刚; 孙耀兰; 黄露宁
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2019-03-31
Filing date: 2019-03-31
Publication date: 2019-05-28
Anticipated expiration: 2039-03-31
Also published as: CN109811059B

Abstract

The present invention provides application of the biomarker UGGT1 in cervical disease, the cervical disease includes cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma.The present invention provides application of the UGGT1 in cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma diagnosing and treating.Invention also provides application of the UGGT1 in the drug candidate of screening treatment cervical squamous cell carcinoma.

Description

Application of the biomarker UGGT1 in cervical disease

Technical field

The invention belongs to biomedicine fields, are related to application of the biomarker UGGT1 in cervical disease.

Background technique

Cervical squamous cell carcinoma is presently the most common and high degree threatens the gynaecology of vast female body health pernicious swollen One of tumor (Wu C, KraR P, Zhai K, Chang J, Wang Z, Li Y Hu Z, He Z, Jia W, Abnet CC, et.algenome-wide association analyses of cervical squamous cell carcinoma in Chinese identify multiple susceptibility loci and gene environment interactions.Nat Genet 2015.44:1090-1097.).The cervical squamous cell carcinoma death rate occupies female tumor second according to statistics Position, for ratio in women worldwide malignant tumour, the disease incidence of cervical squamous cell carcinoma occupies third position (Chen M, Huang J, Zhu Z,Zhang J,Li K:Systematic review and meta-analysis of tumor bio-markers in predicting prognosis in cervical cancer.BMC Cancer 2014,13:539.).It is complete according to 2015 Ball data statistics, it is contemplated that about have 531,800 new hair cervical squamous cell carcinoma cases every year, wherein having 272,700 are death. 130,000 is alreadyd exceed in the patient of China's cervical squamous cell carcinoma newly-increased every year, accounts for about the one third of whole world morbidity quantity, The heavy medical burden [4] of social development is influenced through becoming.From after the 1950s, the cytology correlation of cervical carcinoma is sieved The work looked into, prevent and treated also makes significant progress, and extends to global range and is widely applied, so that uterine neck squama Cancer can be found earlier, so that the treatment of cervical squamous cell carcinoma achieves good therapeutic effect highly visible.However, Since nearly 50 years, due to the change and progress of social life, Subclinical papillomavirus infection (Human Papillomavirus, HPV) Number of the infected increased significantly earlier above, and the morbidity of cervical carcinoma has the tendency that rising in some areas, and occurs cervical squamous cell carcinoma in recent years The phenomenon that morbidity rejuvenation.According to International Association for Obstetrics (International Federation of in 2013 Gynecology and Obstetrics, FIGO) survey report statistics, the age of onset of cervical squamous cell carcinoma is from the fifties in last century Average out to 60 years old, and the nineties in last century that arrives, the average age of onset of cervical squamous cell carcinoma have dropped down to 50 years old, cervical squamous cell carcinoma is again Primary killers (Ghadban T, Schmidt-Yang M, Uzunoglu F q Perez DR, TSui TY as WomanHealth E1Gammal Ar,ErbesPJ,Zilbennints V,Wellner U,Pantel K,et a1:An A/C germ line single-nucleotide polymorphism in the TNRFAIP3gene is associated with advanced disease stage and survival in only surgically treated esophageal cancer.J HumGenet 2015,60:107.)。

It is fairly simple for the relatively other malignant tumours of the pathological of cervical carcinoma, it is broadly divided into squamous carcinoma and two type of gland cancer Type (Ren Z, Zhu J, Gu H, Liu R, Chen S, Rong G Sun B:Decoy receptor 3polymorphisms are not associated with the risk of esophageal cancer in a Chinese population Bio-markers 2014,19:340-344.), wherein specific gravity shared by squamous carcinoma be 80%, the specific gravity of gland cancer less than 20%, Remaining histological type is more rare, including adenosquamous carcinoma, small cell carcinoma, melanoma, sarcoma etc..The generation and development of cervical squamous cell carcinoma Undergo a kind of process of complexity.The system of this complexity includes Abnormal regulation, the part base for including: portion gene in terms of expression Because further causing the hyper-proliferative of tumour cell, swelling by inhibiting its signal path, portion gene then to activate its signal path The exception of the differentiation of oncocyte, further progresses to precancerous lesion, and it is pernicious then to show as the distinctive invasion of tumour and transfer etc. Biological behaviour.

It can be found that clinical pathology and by stages approximate advanced squamous carcinoma of uterine cervix patient in clinical position, although with The Role of Concurrent Chemoradiotherapy scheme of identical standard, therapeutic effect are also to have biggish difference, and Partial controll rate patient occurs sometimes DISTANT METASTASES IN situation, or even there are some patientss to show apparent chemicotherapy during radiotherapy and resist, therapeutic effect is very poor (Maruyama R,Suzuki H:Long noncoding RNA involvement in cancer.BMB Rep2012,45: 604-611.).The insensitive patient of this part of radiotherapy just becomes the problem in clinical treatment, that is, needs clinician to find quick Feel special prediction technique.Therefore, in order to further change cervical carcinoma rapid growth high incidence, improve facing for cervical carcinoma Bed magnetic target therapy scheme, the identification for finding the cervical carcinoma related neoplasms gene of tissue specificity become it in theoretical and technology The pre- new tool examined, treated.

Summary of the invention

In order to make up for the deficiencies of the prior art, it the purpose of the present invention is to provide a kind of clinical diagnosis of cervical disease and controls The molecular target for the treatment of.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides the reagents of detection UGGT1 to early diagnose cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma in preparation Product in application.

Further, the product includes: chip, preparation, kit or nucleic acid film item.

The present invention provides the product of a kind of diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma, the product includes inspection Survey the reagent of UGGT1 level.

Further, the reagent is selected from probe, primer or the protein binding agent that specificity is directed to UGGT1.

Further, for the primer sequence of UGGT1 as shown in NO.1~2 SEQ ID.

The present invention provides UGGT1 in the drug candidate that cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma are treated in screening Using.

The present invention provides a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, step includes:

The cultivating system of expression or the albumen containing UGGT1 gene or its coding is handled with substance to be screened；With

Detect the expression or activity of the albumen of UGGT1 gene or its coding in the system；

Wherein, if the substance to be screened can inhibit the level or expression activity of UGGT1 gene, show that this is to be screened Substance is the drug candidate for treating cervical squamous cell carcinoma.

The present invention provides the inhibitor of UGGT1 preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in answer With.

Further, the inhibitor includes reducing high UGGT1 gene or its expression product stability, lowering UGGT1 gene Or expression, reduction UGGT1 gene or the substance of its expression product effective acting time of its expression product；It is preferred described Inhibitor is siRNA.

The present invention provides a kind of pharmaceutical composition for treating cervical squamous cell carcinoma, described pharmaceutical composition includes the suppression of UGGT1 Preparation, the inhibitor of preferred UGGT1 are the nucleic acid inhibitor of UGGT1, and more preferably, the nucleic acid inhibitor is siRNA.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection UGGT1 gene in cervical squamous cell carcinoma patient；

Fig. 2 is UGGT1 gene expression dose figure；

Fig. 3 is the CCK-8 method detection active influence diagram of UGGT1 cell proliferation；

Fig. 4 is using the influence diagram of the cell Transwell detection UGGT1 gene pairs cell migration invasion, wherein figure A is pair The influence diagram of cell migration；Figure B is the influence diagram to cell invasion.

Specific embodiment

The present invention is by high throughput sequencing technologies and carries out high-flux sequence analysis, detection cervical intraepithelial neoplasia sample disease Become, the gene expression dose in cervical squamous cell carcinoma patient tissue, discovery wherein expresses the gene with notable difference, inquires into itself and palace Neck intraepithelial neoplasia, cervical squamous cell carcinoma generation between relationship, thus be cervical squamous cell carcinoma early detection and targeted therapy Find better approaches and methods.By screening present invention firstly discovers that UGGT1 expresses up-regulation in cervical squamous cell carcinoma patient, and UGGT1 is further demonstrated by gene silent technology and participates in the proliferation of cervical cancer cell, invasive procedure, prompts UGGT1 that can make It, can also be with other gene marker use in conjunction for the independentpredictor of cervical squamous cell carcinoma.

Terms used herein " differential expression " indicates to mark with one or more present invention biologies identical in second of sample The expression of will object compares, after measured the amount or level of mRNA, one or more biology marks of the invention in a sample The difference of one or more splice variant expressions of the RNA of the will object and/or biomarker mRNA." differential expression " it can also include compared with protein expression quantity in second of sample or sample group or level, to this in sample or sample group The measurement of the protein of invention biomarker coding.Differential expression can it is as described herein and those skilled in the art understand that Method determines.Given biomarker can in term " differential expression " or " variation of expression " expression and second of sample Measurement expression compares, after measured the amount of RNA and/or the amount of protein, and expression can be measured by giving biomarker in sample Horizontal increases or decreases.Term " differential expression " or " variation of expression " also may indicate that and life in second sample group The expression that measures of object marker compares, and biomarker is given in sample group can measure the increase or drop of expression It is low." differential expression " used herein can be with the expression of given biomarker relative to biomarker given in control The ratio of Average expression level be measured, wherein ratio is not equal to 1.0.Differential expression can also be measured with p value.Work as use When p value, when p value is less than 0.1, biomarker is accredited as the differential expression between the first and second groups.More preferable p value Less than 0.05.Even more preferably p value is less than 0.01.Even more preferably from p value less than 0.005.Most preferably p value is less than 0.001.When being based on When ratio determines differential expression, if the ratio of expression is more than or less than 1.0, RNA in the first and second of sample Or protein is differential expression.For example, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1 Rate, such as 0.8,0.6,0.4,0.2,0.1,0.05.In another embodiment of the present invention, if the first group is averaged The ratio of expression and the Average expression level of the second group is more than or less than 1.0, then transcribed nucleic acid is originally differential expression. For example, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1, for example, 0.8,0.6,0.4,0.2, 0.1,0.05.In another embodiment of the present invention, if put down in expression and second colony in first sample The ratio of equal expression is more than or less than 1.0, is greater than 1.2,1.5,1.7,2,3,4,10,20 or ratio for example including ratio Less than 1, such as 0.8,0.6,0.4,0.2,0.1,0.05, then transcribed nucleic acid is originally differential expression.

" differential expression increase " or " up-regulation " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display increase at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more.

" differential expression reduction " or " downward " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display reduce at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0 times, 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times or less gene.For example, up-regulation gene include with The expression of the mRNA or protein that separate from normal individual are compared, from the individual separation characterized by with cervical lesions disease MRNA or the increased gene of protein expression level in tissue.For example, down-regulated gene includes the tissue separated with from normal individual It compares, the base that mRNA or protein expression level reduce from the tissue of the individual separation characterized by with cervical lesions disease Cause.

UGGT1 gene

Term " UGGT1 " (gene I/D: 56886) refers to UDP-glucose 1 gene of glycoproteinglucosyltransferase and albumen and cover its homologue, mutation and isoform.The art Language covers overall length, unprocessed UGGT1, and from any type of UGGT1 processed in cell.The term covers UGGT1 Natural generation variant (such as splice variant or allelic variant).The term covers such as UGGT1 gene, the mRNA of hCCSP GT1 Amino acid sequence (the GenBank accession number NP_ of sequence (such as GenBank accession number NM_020120.3) and hCCSP GT1 064505.1) and from any other vertebrate origin, including mammal, such as Primate and rodent (example Such as mouse and rat) UGGT1DNA, mRNA and amino acid sequence.In a specific embodiment of the invention, UGGT1 is people UGGT1 gene and its expression product.

UGGT1 nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, recombination method or artificial conjunction At method obtain.

The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage Solution, the means for measuring gene expression are not importances of the invention.Biology can be detected on transcriptional level or translation skill The expression of marker.

Gene and albumen of the invention is detected using multiple technologies known to persons of ordinary skill in the art, these skills Art includes but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.

The nucleic acid amplification technologies be selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification based on nucleic acid sequence of transcriptive intermediate (NASBA).Wherein, PCR is needed RNA reverse transcription before amplification into DNA (RT-PCR), TMA and the direct cloning RNA of NASBA.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.

Protein immunization technology of the invention includes sandwich immunoassay, such as sandwich ELISA, wherein using identification biology mark Two kinds of antibody of different epitopes carry out the detection of the biomarker on will object；Radiommunoassay (RIA), directly, indirectly or Comparison enzyme linked immunosorbent assay (ELISA) (ELISA), fluorescence immunoassay (FIA), immunoblotting, is exempted from enzyme immunoassay (EIA) (EIA) The epidemic disease precipitation method and immunoassays based on any particle are (as used gold particle, Argent grain or latex particle, magnetic-particle or quantum Point).Immunization for example can be implemented in the form of microtiter plate or item.

The reagent for detecting UGGT1 albumen is the specific binding agent of UGGT1 albumen.Specific binding agent is such as protein The receptor of UGGT1, the agglutinin of conjugated protein UGGT1, for the antibody of protein UGGT1, for the peptide of protein UGGT1 Antibody (peptidebody), the agent of bispecific dual combination or bispecific antibody form.

The example of specific binding agent is peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repetition Albumen, Kunitz type domain, antibody, single domain antibody and monovalent antibody fragments.In a specific embodiment of the present invention, the specificity Bonding agent is UGGT1 specific antibody.

The present invention provides the products of the expression of detection UGGT1, and the product includes but is not limited to chip, reagent Box.Wherein chip includes: solid phase carrier；And orderly it is fixed on oligonucleotide probe or antibody on the solid phase carrier, institute The oligonucleotide probe stated some or all of specifically corresponds to shown in UGGT1 sequence, the combination of the antibody specificity UGGT1 albumen.

The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but is not limited to have silicon carrier, Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

The present invention provides a kind of kit, the kit can be used for detecting the reagent of UGGT1 gene or albumen.It is selected from One or more substances of the following group: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or solvent.

In kit of the invention can also have kit operation instructions, be described how using kit into Row detection.

In certain embodiments, it provided herein is the protein levels for detecting one or more biomarkers Kit.In certain embodiments, the kit includes the coated examination of antibody with identification of protein biomarker Paper slip, washing solution, the reagent for carrying out the test, Separation of Proteins or tools for purification, detection instrument and the positive and feminine gender are right According to.In certain embodiments, the kit also includes the specification using the kit.The kit can customize confession Use at home, clinical use or research use.

Inhibitor and pharmaceutical composition

The present invention provides a kind of drug (compositions), it contains the inhibitor and medicine of a effective amount of UGGT1 Acceptable carrier on.The inhibitor includes reducing UGGT1 gene or its expression product stability, lowering UGGT1 gene Or expression, reduction UGGT1 gene or the substance of its expression product effective acting time of its expression product.Such as the suppression Preparation includes nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule.

As a kind of selection mode of the invention, the inhibitor of the UGGT1 is a species specificity in conjunction with UGGT1 Antibody.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody molecule, Any segment or modification including antibody, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the segment can Retain the binding ability with UGGT1 albumen.It is well known to those skilled in the art when preparation for the antibody of protein level , and the present invention may use any method to prepare the antibody

As a kind of preferred embodiment of the invention, the inhibitor of the UGGT1 is a kind of small interference of UGGT1 specificity RNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous complementary The mRNA of sequence is the target specific mRNA of degradation, this process is exactly RNA interference (RNA interference) process.It is small RNA interfering can be prepared into the form of double-strandednucleic acid, it contains a positive-sense strand and an antisense strand, this two chains are only hybridizing Under conditions of form double-strand.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Therefore, For example, complementary positive-sense strand and antisense strand are chemical synthesis, and can generate the double-strand of synthesis by anneal thereafter RNA compound.

When screening effective siRNA sequence, the present inventor is by largely comparing analysis, to find out optimal effective Segment.The present inventor's design has synthesized a variety of siRNA sequences, and by they respectively by transfection reagent transfect relevant cell system into Row verifying, selects the optimal siRNA of interference effect, and further tests in cellular level, to prove that gene pairs is related thin The influence of born of the same parents.

Nucleic acid inhibitor of the invention such as siRNA can be with chemical synthesis, can also be by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using transfection reagent quilt appropriate It is transported into the cell, or multiple technologies known in the art also can be used and be transported into the cell.

As a kind of optional way of the invention, the inhibitor of the UGGT1 is also possible to a kind of " children purpura nephritis (Small hairpin RNA, shRNA) " is the non-coding small RNA molecular for being capable of forming hairpin structure, children purpura nephritis energy Enough by RNA interference channel come the expression of suppressor.As above-mentioned, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA Template is inserted into a carrier, such as plasmid or viral vectors, is then connected to a promoter carry out table in vitro or in vivo It reaches.ShRNA under the action of DICER enzyme, can be cut into siRNA molecule in eukaryocyte, hence into RNAi approach. " shRNA expression vector " refers to plasmid of some this fields conventionally used for constructing shRNA structure, exist on the usual plasmid " Every sequence " and be located at " intervening sequence " both sides multiple cloning sites or for replace sequence, thus people can by shRNA (or Analog) corresponding DNA sequence dna be inserted by way of forward and reverse multiple cloning sites or replacement thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (Short Hairpin) structure." the shRNA expression vector " is current It can be bought and be obtained by commercially available approach completely, such as some viral vectors.

In the present invention, these inhibitor can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier In medium, wherein pH is usually about 5-8, and preferably pH is about 6-8, although pH value can be with the property for being formulated substance and wait control The illness for the treatment of and be varied.Prepared pharmaceutical composition can be administered by conventional route, including (but not It is limited to): tumor is interior, intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or local administration.

In the present invention, pharmaceutically acceptable carrier, including but not limited to diluent, adhesive, surfactant, Humectant, absorption carrier, lubricant, filler, disintegrating agent.

Wherein, diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinated form sediment Powder, dextrin, maltodextrin, sucrose, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, poly- second Enol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methyl Cellulose etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, lauryl sodium sulfate, stearic acid list glycerol Ester, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap Clay etc.；Lubricant such as zinc stearate, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol, Boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, Dan Yuegui sucrose acid ester, laruyl alcohol sulfuric acid Sodium, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, wheat Bud sugar, erythrose, microcrystalline cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp Polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate etc.；Disintegrating agent such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low Replace hydroxypropyl methyl, croscarmellose sodium, soybean polyoses etc..

Pharmaceutical composition in the present invention can also include stabilizer, fungicide, buffer, isotonic agent, chelating agent, pH control The additives such as preparation and surfactant.

Wherein, stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can be with Including any one in glycine, cysteine and glutamic acid.Carbohydrate includes monosaccharide, such as glucose, mannose, gala Sugar, fructose etc.；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, Such as glucan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.Cellulose derivative includes first Base cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose and sodium cellulose glycolate. Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, rouge Fatty acid glyceride.Additive buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (they Alkali metal or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent include potassium chloride, sodium chloride, sugar and Glycerol.Chelating agent includes sodium ethylene diamine tetracetate and citric acid.

Pharmaceutical composition Orally-administrable of the present invention, parenteral administration are given by sucking spray delivery, part Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral administration or injection Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.

Pharmaceutical composition of the invention can also be with the drug combination of other treatment cervical squamous cell carcinoma, other therapeutic compound It can be administered simultaneously with main active constituent, or even be administered simultaneously in same composition.It can also be with individual composition Or the dosage form different from main active constituent individually gives other therapeutic compounds.

Preferably, the means that gene therapy can be used carry out.For example, can be directly by the inhibitor of UGGT1 by such as infusing It the methods of penetrates and to deliver medicine to subject；Alternatively, the ceneme that the promotion for carrying UGGT1 is adjusted (can be compared by certain approach Such as expression vector or virus) it is delivered on target spot, concrete condition need to be depending on the type of the inhibitor, these are these Known to the technical staff of field.

Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention, It is not intended to limit protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1 screens gene marker relevant to cervical squamous cell carcinoma

1, sample collection

Collect 32 cervical squamous cell carcinoma (CESC) tissues and 18 normal tissues (N), 24 cervical intraepithelial neoplasia (CIN)s (CIN) Tissue, all cases are preoperative not to do any immunosuppressant treatment, radiotherapy and chemotherapy, and all research objects being included in are in receipts Informed consent form is signed before collection sample.The acquirement of above-mentioned all samples passes through the agreement of the committee, organizational ethics.Every group takes 4 Example sample carries out the detection and analysis of gene expression profile, carries out the screening of difference expression gene, and in each group all cases sample Carry out confirmatory experiment.

2, the preparation of RNA sample

The total serum IgE in each group tissue, specific steps reference are extracted using the tissue RNA extracts kit of QIAGEN Book.

3, the quality analysis of RNA sample

The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total 5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, the building of cDNA library

The building of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit By specification carries out.

5, it is sequenced

CDNA library is sequenced using Illumina X-Ten microarray dataset, concrete operations by specification carries out.

6, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, is analyzed using metaMA packet, p value merges side used in meta analysis Method is inverse normal method, and the screening criteria of difference expression gene is FDR < 0.05.

7, result

RNA-seq is the results show that compared with normal control, and UGGT1 gene is in cervical squamous cell cancer and in epithelium of cervix uteri Expression quantity in interior tumor-like lesion tissue significantly raises, wherein the expression quantity in cervical squamous cell cancer and cervical intraepithelial neoplasia sample disease Become tissue to compare, expression quantity also significantly raises, and difference has statistical significance, therefore carries out further large sample to UGGT1 Verifying.

The differential expression of embodiment 2QPCR sequence verification UGGT1 gene

1, large sample QPCR verifying is carried out to UGGT1 gene differential expression.

2, RNA is extracted

3、QPCR

1) reverse transcription reaction

LncRNA reverse transcription is carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106), is gone first Except genomic DNA reacts, 5 × gDNA B μ ffer, 2.0 μ l is added in test tube, 1 μ g of total serum IgE adds RNase Free ddH₂O Make total volume to 10 μ l, 42 DEG C of heating 3min. in water-bath

By 10 × Fast RT B μ, 2.0 μ l, RT Enzyme Mix of ffer, 1.0 μ l, FQ-RT Primer Mix, 2.0 μ L, RNase Free ddH₂5.0 μ l of O is added in above-mentioned test tube after mixing and is mixed together totally 20 μ l, 42 DEG C of heating in water-bath 15min, 95 DEG C of heating 3min.

2) design of primers

QPCR amplimer is designed according to the coded sequence of UGGT1 gene and GAPDH gene in Genebank, by Bo Maide Biotech firm's synthesis.Specific primer sequence is as follows:

UGGT1 gene:

Forward primer is 5 '-TCTCCACTGTTCACTCTG-3 ' (SEQ ID NO.1)；

Reverse primer is 5 '-CACTGTCCACCTCTTCTAA-3 ' (SEQ ID NO.2).

GAPDH gene:

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).

3) QPCR amplification is examined

It with SuperReal PreMix Plus (SYBR Green) (article No.: FP205), is expanded, experimental implementation is by production Product specification carries out.

Using 20 μ l reaction systems: 2 × SuperReal PreMix Plus 10 μ l, each 0.6 μ of forward and reverse primer (10 μM) L, 5 × ROX Reference Dye^△2 μ l, 2 μ l of DNA profiling, 4.8 μ l of sterile purified water.3 parallel pipes are arranged in each sample, All amplified reactions are repeated three times the above reliability to guarantee result.

Amplification program are as follows: 95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C 60s, 95 DEG C of 15s).

4) screening of cDNA template concentrations

After each sample cDNA is mixed, 10 times of gradients (10,100,1000,10000,100000 times) are carried out as template Dilution, sample respectively takes 2 μ l to make template after dilution, is expanded respectively with target gene primer and reference gene primer, while 60-95 DEG C of progress melt curve analysis analysis carries out the screening of template concentrations according to amplification efficiency height and the unimodal principle of solubility curve.

According to solubility curve, it can be seen that when 10 times of dilutions of carry out of cDNA, the amplification efficiency of PCR is higher, and dissolution is bent Line is unimodal relatively good.

5) sample RealTime PCR is detected

2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progresss solubility curve analysis, purpose band is determined by melt curve analysis analysis and electrophoresis, 2^-ΔΔCT Method carries out relative quantification.

4, result

As a result as shown in Figure 1, compared with normal tissue, in cervical squamous cell cancer and cervical intraepithelial neoplasia (CIN) tissue UGGT1 expression quantity significantly raises, and wherein conspicuousness up-regulation is presented compared with normal tissue in cervical intraepithelial neoplasia (CIN) tissue, Conspicuousness up-regulation is presented compared with cervical intraepithelial neoplasia (CIN) tissue in cervical squamous cell cancer, and difference all has statistical significance (P<0.05)。

The overexpression of embodiment 3UGGT1 gene

1, cell culture

Cervical squamous cell carcinoma cell (Hela) is with the RPIM-1640 culture medium containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5% CO₂Incubator in cultivate, when cell it is long to 80%~90% when, trypsase conventional digestion of the use 0.25% containing EDTA passes Generation.

Before transfection, the cell dissociation of logarithmic growth phase is gently blown and beaten into single cell suspension, the not antibiotic culture of use Base is inoculated in 6 orifice plates, and the cell densities of 6 orifice plates is up to 2 × 10⁵A/hole, when cell confluency carries out transfection examination up to 40%-60% It tests.

2, the design of UGGT1 gene siRNA

The sequence of negative control siRNA-NC:

Positive-sense strand: 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.5),

Antisense strand: 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.6)；

SiRNA1:

Positive-sense strand: 5 '-AACCAUUUUGUUGUAAGAGAG-3 ' (SEQ ID NO.7),

Antisense strand: 5 '-CUCUUACAACAAAAUGGUUUU-3 ' (SEQ ID NO.8)；

SiRNA2:

Positive-sense strand: 5 '-AAUUCCAAAAUUUCUCUUGAC-3 ' (SEQ ID NO.9),

Antisense strand: 5 '-CAAGAGAAAUUUUGGAAUUUU-3 ' (SEQ ID NO.10)；

SiRNA3:

Positive-sense strand is 5 '-AUCAGAUUCACAAGUCUUCUU-3 ' (SEQ ID NO.11),

Antisense strand is 5 '-GAAGACUUGUGAAUCUGAUAC-3 ' (SEQ ID NO.12)

3, it transfects

Experiment is divided into 3 groups, respectively control group (Hela), negative control group (siRNA-NC), experimental group (transfection SiRNA1~3), it is transfected according to the 2000 transfection reagent specification of lipofectamine of invitrogen company.

4, QPCR detects the transcriptional level of UGGT1 gene

1) extraction of cell total rna

The extraction of cell total rna is carried out using the cell RNA extracts kit of QIAGEN, specific steps, which are detailed in kit, to be said Bright book

3.2 reverse transcription steps are the same as embodiment 2.

3.3QPCR amplification step is the same as embodiment 2.

5, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, the difference between silencing UGGT1 gene expression panel and control group is adopted It is examined with t, it is believed that there is statistical significance as P < 0.05.

6, result

As a result such as Fig. 2 is shown, compared with the control group, the level of negative control group UGGT1 is without significant changes, compared to the control group And negative control group, the horizontal of experimental group UGGT1 significantly reduces, and the silencing efficiency of siRNA1 is the most significant, therefore selects SiRNA1 carries out subsequent experiment.

Embodiment 4CCK-8 method detects cervical squamous cell carcinoma cell-proliferation activity

1, with 0.25% trypsin digestion and cell after transfecting 6 hours, list is made into the culture solution containing 10% fetal calf serum A cell suspension (1 × 10⁴/ hole), (100 hole μ l/) is inoculated in 96 well culture plates, and every group sets 6 multiple holes, while setting up nothing The culture solution blank control of cell.

2, it is separately added into cell Proliferation detection reagent CCK-8 in detection time point (0h, for 24 hours, 48h, 72h, 96h, 120h), Working concentration is 1:10, i.e. 10 μ l CCK-8 are added in 100 μ l culture solutions；

3, at 37 DEG C, 5%CO₂It after being incubated for 1h in incubator, is detected, microplate reader reads OD 450nm.

4, result judges: 0h after transfection, for 24 hours, 48h, 72h, 96h, 120h observe the proliferation of Hela cell using microplate reader Activity, absorption photometric value (OD value) of the cell at wavelength 450nm indicates the cell quantity for being in vegetative state, with cell-free Culture solution group OD value is control.

5, result

As a result as shown in figure 3, the cell Proliferation of the experimental group of transfection siRNA1 substantially reduces, the table for changing UGGT1 is prompted The proliferative capacity that can change cervical squamous cell carcinoma cell up to level, illustrates that UGGT1 is related to the proliferation of cervical squamous cell carcinoma cell.

Embodiment 5Transwell method detects cervical squamous cell carcinoma cell migration and invasion

1, cell migration ability detects

1) migration the previous day, complete medium was added in orifice plate, is put into cell and is placed in incubator overnight；

2) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA It is made 1 × 10⁵Suspension.

3) take 200 μ l cell suspension inoculations in the small indoor culture of Transwell.

4) cell is taken out, remaining liq is blotted and is placed on the fixed 30min of 70% methanol, wipe the thin of upper chamber face with cotton swab Born of the same parents.

5) cell immerses 0.4% violet staining 10min, PBS twice of cleaning, randomly selects the visual field under microscope and counts film The cell number of bottom surface.

2, cell invasion ability detects

1) matrigel, each 24 hole suspension type cell bottom are diluted with 1640 1:8 of RPIM on ice on the day before Matrigel Portion film upper chamber face adds the 60 μ l dried for standby of matrigel after dilution.

2) aquation basilar memebrane: 50 serum-free mediums of the μ l containing 10g/LBSA are added in every hole, and 37 DEG C, 30min, culture is sucked out Residual liquid in plate.

3) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA It is made 1 × 10⁵Suspension.

3, data processing

Statistical analysis is carried out to data with SPSS18.0 software.Measurement data is indicated with mean ± standard deviation.Multiple samples This mean compares using one-way analysis of variance, and P < 0.05 is that difference is statistically significant.

4, result

As a result as shown in figure 4, the cell migration of experimental group and invasion number relatively transfect lacking for empty plasmid respectively, illustrate to reduce The expression of UGGT1 gene can reduce migration and the invasive ability of cervical squamous cell carcinoma cell, and UGGT1 is prompted to can be used as molecular target Mark is applied to the treatment of cervical squamous cell carcinoma.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

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Claims

1. detecting application of the reagent of UGGT1 in the product that preparation early diagnoses cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma.

2. application according to claim 1, which is characterized in that the product includes: chip, preparation, kit or nucleic acid Film item.

3. the product of a kind of diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma, which is characterized in that the product includes detection The reagent of UGGT1 level.

4. product according to claim 3, which is characterized in that the reagent is selected from specificity and is directed to the probe of UGGT1, draws Object or protein binding agent.

5. product according to claim 4, which is characterized in that for primer sequence such as NO.1~2 SEQ ID of UGGT1 It is shown.

Application of the 6.UGGT1 in the drug candidate that cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma are treated in screening.

7. a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, which is characterized in that step includes:

Wherein, if the substance to be screened can inhibit the level or expression activity of UGGT1 gene, show the substance to be screened It is the drug candidate for treating cervical squamous cell carcinoma.

The inhibitor of 8.UGGT1 preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application.

9. application according to claim 8, which is characterized in that the inhibitor includes reducing high UGGT1 gene or its table Have up to product stability, the expression of downward UGGT1 gene or its expression product, reduction UGGT1 gene or its expression product Imitate the substance of action time；The preferred inhibitor is siRNA.

10. a kind of pharmaceutical composition for treating cervical squamous cell carcinoma, which is characterized in that described pharmaceutical composition includes the inhibition of UGGT1 Agent, the inhibitor of preferred UGGT1 are the nucleic acid inhibitor of UGGT1, and more preferably, the nucleic acid inhibitor is siRNA.