CN109811059B

CN109811059B - Application of biomarker UGGT1 in cervical diseases

Info

Publication number: CN109811059B
Application number: CN201910254347.1A
Authority: CN
Inventors: 杨承刚; 孙耀兰; 黄露宁
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2019-03-31
Filing date: 2019-03-31
Publication date: 2021-10-19
Anticipated expiration: 2039-03-31
Also published as: CN109811059A

Abstract

The invention provides application of a biomarker UGGT1 in cervical diseases, wherein the cervical diseases comprise cervical intraepithelial neoplasia and cervical squamous carcinoma. The invention provides application of UGGT1 in diagnosis and treatment of cervical intraepithelial neoplasia and cervical squamous cell carcinoma. The invention also provides application of UGGT1 in screening candidate drugs for treating cervical squamous cell carcinoma.

Description

Application of biomarker UGGT1 in cervical diseases

Technical Field

The invention belongs to the field of biological medicines, and relates to application of a biomarker UGGT1 in cervical diseases.

Background

Cervical squamous carcinoma is one of the most common gynecological malignancies currently in use and which greatly threatens the health of women in general (Wu C, KraR P, Zhai K, Chang J, Wang Z, Li Y Hu Z, He Z, Jia W, Abnet CC, et. oncogene-side association analysis of biological tissue in Chinese identity multiple surgery location and gene environment interaction. Nat. Gene t 2015.44: 1090-1097.). Cervical squamous carcinoma mortality is statistically secondary to female tumors, with a proportion of cervical squamous carcinoma third among female malignancies worldwide (Chen M, Huang J, Zhu Z, Zhang J, Li K: Systematic review and meta-analysis of tumor in diagnosis in cervical Cancer. BMC 2014,13: 539.). According to global data statistics in 2015, 531,800 new cervical squamous carcinoma cases are estimated each year, and 272,700 cases are death cases. More than 130,000 new patients with cervical squamous carcinoma are present in China every year, accounting for about one third of the global morbidity, and becoming a heavy medical burden affecting social development [4 ]. Since the 20 th century and the 50 th century, the cytological screening, prevention and treatment of cervical cancer has advanced greatly, and the method is widely applied to the global range, so that cervical squamous carcinoma can be discovered earlier, and the treatment of cervical squamous carcinoma has a remarkable good treatment effect. However, in recent 50 years, the number of Human Papillomavirus (HPV) infections has increased significantly from now on due to changes and advances in social life, and the onset of cervical cancer has tended to increase in some areas, and the onset of cervical squamous cell carcinoma has become younger in recent years. According to the 2013 International Federation of Gynecology and Obstructrics, FIGO survey report, the age of onset of cervical squamous cell carcinoma averages 60 years in the last 50 th century, and the average age of onset of cervical squamous cell carcinoma drops to 50 years in the last 90 th century, and cervical squamous cell carcinoma becomes the leading killer of women's health again (Ghadban T, Schmidt-Yang M, Uzunoglu F q Perez DR 2015ui TY E1Gamma Ar, ErbesPJ, Zilbennins V, Wellner U, Patent K, a1: An A/C germ line single-nuclear surgery polymerized in the TNAIRFP 3gene associated with varied disease and gene J107).

The pathological type of cervical cancer is simpler than other malignant tumors, and is mainly divided into two types of squamous carcinoma and adenocarcinoma (Ren Z, Zhu J, Gu H, Liu R, Chen S, Rong G Sun B: Decoy receptor 3 polyrrphism now associated with the roof of esophageal cancer in a cervical cancer biological-markers 2014,19:340-344.), wherein the squamous carcinoma accounts for 80 percent, the adenocarcinoma accounts for less than 20 percent, and the rest pathological types are rare and comprise adenosquamous carcinoma, small cell carcinoma, melanoma, sarcoma and the like. The occurrence and development of cervical squamous carcinoma undergo a complex process. Such complex systems include: the abnormal regulation of partial genes in terms of expression, the inhibition of signal channels of partial genes and the activation of signal channels of partial genes further cause the over-proliferation of tumor cells and the abnormal differentiation of tumor cells, further progress to precancerous lesion and then show malignant biological behaviors such as the specific invasion and metastasis of tumors.

In clinical work, it can be found that the treatment effect of the patients with intermediate and advanced cervical squamous carcinoma with similar clinical pathology and stage is greatly different despite the same standard synchronous radiotherapy and chemotherapy scheme, and the local control rate patients sometimes have distant metastasis, even some patients show obvious radiotherapy and chemotherapy resistance during radiotherapy, and the treatment effect is very poor (Maruyama R, Suzuki H: Long non-coding RNA inhalation in cancer. BMB Rep2012,45:604 and 611.). This fraction of patients who are not sensitive to radiation therapy becomes a problem in clinical treatment, requiring clinicians to find sensitivity-specific predictive methods. Therefore, in order to further change the rapidly-increased high incidence rate of the cervical cancer, perfect the clinical targeted treatment scheme of the cervical cancer, and find the identification of the tissue-specific cervical cancer-related tumor gene to become a new means for the theoretical and technical pre-diagnosis and treatment of the cervical cancer.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a molecular target for clinical diagnosis and treatment of cervical diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent for detecting UGGT1 in preparing a product for early diagnosis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

Further, the product comprises: a chip, a preparation, a kit or a nucleic acid membrane strip.

The invention provides a product for diagnosing cervical intraepithelial neoplasia and cervical squamous carcinoma, which comprises a reagent for detecting UGGT1 level.

Further, the agent is selected from a probe, primer or protein binding agent specific for UGGT 1.

Furthermore, the primer sequence aiming at UGGT1 is shown in SEQ ID NO. 1-2.

The invention provides application of UGGT1 in screening candidate drugs for treating cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

The invention provides a method for screening candidate drugs for treating cervical squamous carcinoma, which comprises the following steps:

treating a culture system expressing or containing the UGGT1 gene or a protein encoded thereby with a substance to be screened; and

detecting expression or activity of UGGT1 gene or its encoded protein in said system;

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

The invention provides application of an inhibitor of UGGT1 in preparing a medicament for treating cervical squamous cell carcinoma and metastasis and invasion thereof.

Further, the inhibitor comprises a substance that decreases the stability of the high UGGT1 gene or its expression product, down-regulates the expression level of the UGGT1 gene or its expression product, decreases the effective acting time of the UGGT1 gene or its expression product; preferably the inhibitor is an siRNA.

The invention provides a pharmaceutical composition for treating cervical squamous carcinoma, which comprises an inhibitor of UGGT1, preferably the inhibitor of UGGT1 is a nucleic acid inhibitor of UGGT1, more preferably the nucleic acid inhibitor is siRNA.

Drawings

FIG. 1 is a graph of the detection of UGGT1 gene expression in cervical squamous cell carcinoma patients using QPCR;

FIG. 2 is a graph of UGGT1 gene expression level;

FIG. 3 is a graph showing the effect of the CCK-8 method on UGGT1 on cell proliferation activity;

FIG. 4 is a graph showing the effect of UGGT1 gene on invasion of cell migration by Transwell chamber detection, in which graph A is a graph showing the effect on cell migration; panel B is a graph of the effect on cell invasion.

Detailed Description

The invention detects the gene expression level in the tissues of cervical intraepithelial neoplasia and cervical squamous cell carcinoma patients by high-throughput sequencing technology and high-throughput sequencing analysis, finds genes with obvious difference in expression, and discusses the relationship between the genes and the occurrence of the cervical intraepithelial neoplasia and cervical squamous cell carcinoma, thereby finding a better way and method for early detection and targeted therapy of the cervical squamous cell carcinoma. The invention discovers that UGGT1 is up-regulated in cervical squamous cell carcinoma patients for the first time through screening, and further verifies that UGGT1 participates in the proliferation and invasion process of cervical cancer cells through a gene silencing technology, which indicates that UGGT1 can be used as an independent prediction factor of cervical squamous cell carcinoma and can also be applied in combination with other gene markers.

The term "differential expression" as used herein means the difference in the level of expression of the RNA of one or more biomarkers of the invention and/or one or more splice variants of the mRNA of said biomarker in one sample as compared to the level of expression of the same one or more biomarkers of the invention in a second sample, as measured by the amount or level of mRNA. "differentially expressed" may also include the determination of a protein encoded by a biomarker of the invention in a sample or sample population as compared to the amount or level of protein expression in a second sample or sample population. Differential expression can be determined as described herein and understood by those skilled in the art. The term "differential expression" or "change in expression level" means an increase or decrease in the measurable expression level of a given biomarker in a sample as compared to the measurable expression level of the given biomarker in a second sample, as measured by the amount of RNA and/or the amount of protein. The term "differential expression" or "change in expression level" may also mean an increase or decrease in the measurable expression level of a given biomarker in a sample population as compared to the measurable expression level of the biomarker in a second sample population. As used herein, "differential expression" can be determined as the ratio of the expression level of a given biomarker relative to the average expression level of the given biomarker in a control, wherein the ratio is not equal to 1.0. Differential expression can also be measured using p-values. When using a p-value, biomarkers are identified as differentially expressed between the first and second populations when the p-value is less than 0.1. More preferably, the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Still more preferably, the p-value is less than 0.005. Most preferably, the p value is less than 0.001. When differential expression is determined based on the ratio, the RNA or protein is differentially expressed if the ratio of the expression levels in the first and second samples is greater than or less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, the nucleic acid transcript is differentially expressed if the ratio of the average expression level of the first population to the average expression level of the second population is greater than or less than 1.0. For example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05. In another embodiment of the invention, a nucleic acid transcript is differentially expressed if the ratio of the expression level in the first sample to the average expression level in the second population is greater than or less than 1.0, for example including ratios greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or ratios less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.

By "differential expression increase" or "upregulation" is meant that gene expression (as measured by RNA expression or protein expression) exhibits an increase of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold or more, of the gene relative to a control.

By "differential expression reduction" or "down-regulation" is meant a gene whose expression (as measured by RNA expression or protein expression) exhibits a reduction in gene expression relative to a control of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less. For example, an up-regulated gene includes a gene that has an increased level of expression of mRNA or protein in tissue isolated from an individual characterized as having a cervical disorder as compared to the expression of mRNA or protein isolated from a normal individual. For example, a down-regulated gene includes a gene that has a reduced level of mRNA or protein expression in a tissue isolated from an individual characterized as having a cervical disorder as compared to a tissue isolated from a normal individual.

UGGT1 gene

The term "UGGT 1" (Gene ID: 56886) refers to the UDP-glucose binding lucosylsyltransferase 1 gene and protein and encompasses homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed UGGT1, as well as any form of UGGT1 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of UGGT 1. The term encompasses, for example, the UGGT1 gene, the mRNA sequence of human UGGT1 (e.g., GenBank accession No. NM _020120.3), and the amino acid sequence of human UGGT1 (GenBank accession No. NP _064505.1) as well as UGGT1DNA, mRNA, and amino acid sequences from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats). In a particular embodiment of the invention UGGT1 is the human UGGT1 gene and its expression products.

The full-length nucleotide sequence of UGGT1 or its fragment of the present invention can be obtained by PCR amplification, recombination or artificial synthesis.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level or the translational level.

The genes and proteins of the invention are detected using a variety of techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

The nucleic acid amplification technique is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

Nucleic acid hybridization techniques of the invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH).

Protein immunoassays of the invention include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

The reagent for detecting the UGGT1 protein is a specific binding agent of the UGGT1 protein. Specific binding agents are for example receptors for protein UGGT1, lectins binding protein UGGT1, antibodies against protein UGGT1, peptide antibodies (peptidebody) against protein UGGT1, bispecific dual binding agents or bispecific antibody formats.

Examples of specific binding agents are peptides, peptidomimetics, aptamers, spiegelmers, dappin, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments. In a specific embodiment of the invention, the specific binding agent is an antibody specific for UGGT 1.

The invention provides products, including but not limited to chips, kits, for detecting expression levels of UGGT 1. Wherein the chip includes: a solid support; and an oligonucleotide probe or antibody immobilized on the solid support in an ordered manner, the oligonucleotide probe specifically corresponding to part or all of the sequence indicated by UGGT1, and the antibody specifically binding to UGGT1 protein.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The invention provides a kit, which can be used as a reagent for detecting UGGT1 gene or protein. One or more selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the present invention may further comprise instructions for use of the kit, which describe how to use the kit for detection.

In certain embodiments, provided herein are kits for detecting the protein level of one or more biomarkers. In certain embodiments, the kit comprises a test strip coated with an antibody that recognizes a protein biomarker, a wash solution, reagents for performing the assay, protein isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit may be customized for home use, clinical use, or research use.

Inhibitors and pharmaceutical compositions

The invention provides a medicament (composition) which contains an effective amount of the UGGT1 inhibitor and a pharmaceutically acceptable carrier. The inhibitor comprises a substance which reduces the stability of the UGGT1 gene or an expression product thereof, down regulates the expression level of the UGGT1 gene or an expression product thereof, and reduces the effective acting time of the UGGT1 gene or the expression product thereof. For example, the inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules.

In an alternative embodiment of the invention, the inhibitor of UGGT1 is an antibody that specifically binds to UGGT 1. The specific antibody comprises a monoclonal antibody and a polyclonal antibody; the invention encompasses not only intact antibody molecules, but also any fragment or modification of an antibody, e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc. As long as the fragment retains the ability to bind to UGGT1 protein. The preparation of antibodies for use at the protein level is well known to those skilled in the art and any method may be used in the present invention to prepare such antibodies

In a preferred embodiment of the invention, the inhibitor of UGGT1 is a small interfering RNA molecule specific for UGGT 1. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting related cell lines with transfection reagents respectively, selects siRNA with the best interference effect, and further performs experiments at a cell level, thereby proving the influence of genes on related cells.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

In an alternative embodiment of the present invention, the inhibitor of UGGT1 can be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, wherein the Small hairpin RNA is capable of inhibiting gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

In the present invention, these inhibitors may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

In the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, diluents, binders, surfactants, humectants, adsorptive carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may further include any one of glycine, cysteine and glutamic acid. Saccharides include monosaccharides such as glucose, mannose, galactose, fructose, and the like; sugar alcohols such as mannitol, cellosolve, xylitol, and the like; disaccharides such as sucrose, maltose, lactose, and the like; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, etc. and their derivatives. The cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium hydroxymethylcellulose. Surfactants include ionic or non-ionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and the corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium, potassium, calcium, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylene diamine tetracetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The pharmaceutical composition of the invention can also be used in combination with other drugs for the treatment of cervical squamous carcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient.

Preferably, it can be carried out by means of gene therapy. For example, an inhibitor of UGGT1 can be administered directly to a subject by a method such as injection; alternatively, the modulator-promoting expression unit carrying UGGT1 (e.g., an expression vector or virus) can be delivered to the target site by a route that depends on the type of inhibitor, as is well known to those skilled in the art.

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with cervical squamous cell carcinoma

1. Sample collection

32 cervical squamous carcinoma (CESC) tissues and 18 normal tissues (N), 24 Cervical Intraepithelial Neoplasia (CIN) tissues were collected, no immunosuppressant treatment, radiation therapy and chemotherapy was done prior to all cases, and all subjects enrolled were signed an informed consent prior to specimen collection. All the specimens were obtained with the consent of the tissue ethics committee. 4 samples of each group are taken for detection and analysis of gene expression profiles, differential expression genes are screened, and verification experiments are carried out on all case samples of each group.

2. Preparation of RNA samples

The total RNA in each group of tissues is extracted by using a tissue RNA extraction kit of QIAGEN, and the specific steps refer to the instruction.

3. Mass analysis of RNA samples

The RNA extracted above was subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN value was determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

4. construction of cDNA library

The construction of cDNA library was carried out using Illumina Truseq RNA sample Prep Kit, the specific procedures were as described in the specification.

5. Sequencing

And (3) sequencing the cDNA library by using an Illumina X-Ten sequencing platform, wherein the specific operation is carried out according to the instruction.

6. High throughput transcriptome sequencing data analysis

Bioinformatics analysis was performed on the sequencing results using metaMA package analysis, the method used for p-value combination in meta analysis was invert normal method, and the screening criteria for differentially expressed genes was FDR < 0.05.

7. Results

The RNA-seq result shows that compared with a normal control, the expression level of the UGGT1 gene is obviously up-regulated in cervical squamous carcinoma tissues and cervical intraepithelial neoplasia tissues, wherein the expression level of the cervical squamous carcinoma tissues is also obviously up-regulated compared with the cervical intraepithelial neoplasia tissues, and the difference has statistical significance, so that the UGGT1 is further subjected to large sample verification.

Example 2QPCR sequencing validation of differential expression of UGGT1 Gene

1. Large sample QPCR validation was performed on differential UGGT1 gene expression.

2. RNA extraction

3、QPCR

1) Reverse transcription reaction

Using FastQ μ ant cDNA first strand synthesis kit (cat # KR106) to carry out IncRNA reverse transcription, genomic DNA reaction was first removed, 5 XgDNA B μ ffer 2.0 μ l, total RNA1 μ g, RNase Free ddH were added to the test tube₂O to make the total volume to 10 μ l, heating in water bath at 42 deg.C for 3min.

10 XFast RT B. mu.ffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3min.

2) Primer design

QPCR amplification primers were designed based on the coding sequences of UGGT1 gene and GAPDH gene in Genebank and were synthesized by Bomeide Biometrics. The specific primer sequences are as follows:

UGGT1 gene:

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

the reverse primer was 5'-CACTGTCCACCTCTTCTAA-3' (SEQ ID NO. 2).

GAPDH gene:

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

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 4).

3) QPCR amplification assay

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

A20. mu.l reaction was used: 2 XSuperReal PreMix Plus 10. mu.l, forward and reverse primers (10. mu.M) 0.6. mu.l each, 5 XROX Reference Dye ^△2. mu.l, DNA template 2. mu.l, sterilized distilled water 4.8. mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was: 95 ℃ 15min, (95 ℃ 10s, 55 ℃ 30s, 72 ℃ 32s) x 40 cycles, 95 ℃ 15s, 60 ℃ 60s, 95 ℃ 15 s).

4) Screening for cDNA template concentration

Mixing cDNA of each sample, diluting the cDNA by 10 times gradient (10 times, 100 times, 1000 times, 10000 times and 100000 times) by taking the cDNA as a template, taking 2 mu l of each diluted sample as the template, respectively amplifying by using a target gene primer and an internal reference gene primer, simultaneously carrying out melting curve analysis at 60-95 ℃, and screening the concentration of the template according to the principle of high amplification efficiency and single peak of the melting curve.

From the dissolution curve, it can be seen that when 10-fold dilution of cDNA was performed, the amplification efficiency of PCR was high and the single peak of the dissolution curve was good.

5) Sample RealTime PCR detection

After 10-fold dilution of cDNA of each sample, 2 μ l of cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. Simultaneously performing dissolution curve analysis at 60-95 deg.C, and determining purpose by dissolution curve analysis and electrophoresisStrip of (2)^-ΔΔCTThe method is used for relative quantification.

4. Results

As shown in fig. 1, the expression level of UGGT1 in cervical squamous carcinoma tissue and cervical intraepithelial neoplasia tissue was significantly increased compared to normal tissue, wherein cervical intraepithelial neoplasia tissue showed significant up-regulation compared to normal tissue, and cervical squamous carcinoma tissue showed significant up-regulation compared to cervical intraepithelial neoplasia tissue, and the difference was statistically significant (P < 0.05).

Example 3 overexpression of UGGT1 Gene

1. Cell culture

Cervical squamous carcinoma cell (Hela) was cultured in RPIM-1640 medium containing 10% fetal bovine serum and 1% P/S at 37 deg.C and 5% CO₂When the cells grow to 80% -90%, the cells are subjected to conventional digestion and passage by using 0.25% of trypsin containing EDTA.

Before transfection, cells in logarithmic growth phase were digested and gently blown into single cell suspensions, which were inoculated into 6-well plates with a 6-well plate cell density of 2X 10⁵One/well, transfection assays were performed when cell fusion rates reached 40% -60%.

2. Design of UGGT1 gene siRNA

Sequence of negative control siRNA-NC:

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

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

siRNA1：

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

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

siRNA2：

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

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

siRNA3：

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

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

3. Transfection

Experiments were divided into 3 groups, namely a control group (Hela), a negative control group (siRNA-NC) and an experimental group (transfection siRNA 1-3), and transfection was performed according to the instructions of lipofectamine 2000 transfection reagent of Invitrogen corporation.

4. QPCR detection of transcription level of UGGT1 gene

1) Extraction of Total RNA from cells

Extracting total RNA of cells by using QIAGEN cell RNA extraction kit, wherein the detailed steps are described in the kit specification

3.2 reverse transcription procedure as in example 2.

3.3QPCR amplification step as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, the results were expressed as mean ± sd, and the statistical analysis was performed using SPSS18.0 statistical software, and the differences between the silent UGGT1 gene expression group and the control group were considered statistically significant when P <0.05 using t-test.

6. Results

As a result, as shown in fig. 2, the level of UGGT1 in the negative control group was not significantly changed compared to the control group, and the level of UGGT1 in the experimental group was significantly decreased compared to the control group and the negative control group, while the silencing effect of siRNA1 was most significant, so siRNA1 was selected for subsequent experiments.

Example 4CCK-8 assay of the proliferative Activity of cervical squamous carcinoma cells

1. After 6 hours of transfection, cells were digested with 0.25% trypsin and single cell suspensions (1X 10) were prepared using culture medium containing 10% fetal bovine serum⁴And/well) were inoculated into 96-well culture plates (100. mu.l/well), each set was 6 duplicate wells, and cell-free culture medium blanks were set.

2. Adding cell proliferation detection reagent CCK-8 at detection time points (0h, 24h, 48h, 72h, 96h and 120h) respectively, wherein the working concentration is 1:10, namely adding 10 mul CCK-8 into 100 mul culture solution;

3. at 37 ℃ 5% CO₂After incubation for 1h in the incubator, detection was performed, and the microplate reader read OD 450 nm.

4. And (5) judging a result: proliferation activity of Hela cells was observed with a microplate reader at 0h, 24h, 48h, 72h, 96h, and 120h after transfection, and absorbance luminosity value (OD value) of the cells at a wavelength of 450nm indicates the number of the cells in a proliferation state, and the OD value of the cell-free culture solution group was used as a control.

5. Results

The results are shown in fig. 3, the cell proliferation of the experimental group transfected with siRNA1 is significantly reduced, suggesting that altering the expression level of UGGT1 may alter the proliferative capacity of cervical squamous carcinoma cells, suggesting that UGGT1 is associated with the proliferation of cervical squamous carcinoma cells.

Example 5Transwell method for detecting migration and invasion of cervical squamous cell carcinoma cells

1. Cell migration ability assay

1) Adding the complete culture medium into the pore plate one day before the migration, placing the pore plate into a small chamber, and placing the small chamber in an incubator overnight;

2) cells after 48h transfection were starved, collected for counting, prepared to 1X 10 in 0.2% BSA in serum-free medium⁵A suspension of (a).

3) 200. mu.l of the cell suspension was inoculated into a Transwell chamber and cultured.

4) The chamber was removed, the remaining liquid was blotted dry and fixed in 70% methanol for 30min, and the cells on the upper chamber were wiped off with a cotton swab.

5) The chamber was immersed in 0.4% crystal violet and stained for 10min, washed twice with PBS, and the number of cells on the bottom of the membrane was counted under a microscope at random.

2. Cell invasion capacity assay

1) The day before the invasion experiment matrigel was diluted with RPIM 16401: 8 on ice, and 60. mu.l of the diluted matrigel was added to the membrane upper surface of the bottom of each 24-well suspension chamber and dried for use.

2) Hydrated basement membrane: mu.l of serum-free medium containing 10g/LBSA was added to each well at 37 ℃ for 30min, and the residual liquid in the plate was aspirated.

3) Cells after 48h of transfectionStarvation was performed, cells were collected for counting, and 1X 10 cells were prepared in 0.2% BSA in serum-free medium⁵A suspension of (a).

3. Data processing

Statistical analysis of the data was performed using SPSS18.0 software. The metrology data is expressed as mean ± standard deviation. The average number of a plurality of samples is compared by adopting one-factor variance analysis, and the difference with P <0.05 has statistical significance.

4. Results

The results are shown in fig. 4, the cell migration and invasion numbers of the experimental group are respectively less than those of the transfection blank plasmid, which indicates that the reduction of the expression level of UGGT1 gene can reduce the migration and invasion capacity of cervical squamous cell carcinoma cells, and suggests that UGGT1 can be used as a molecular target for the treatment of cervical squamous cell carcinoma.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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Claims

The application of UGGT1 gene in screening candidate drugs for treating cervical cancer is realized by detecting the expression level of UGGT1 gene mRNA.
2. A method of screening for a candidate drug for the treatment of cervical cancer, comprising the steps of:

treating the culture system expressing or containing UGGT1 gene mRNA with the substance to be screened; and

detecting the expression level of UGGT1 gene mRNA in said system;

wherein, if the substance to be screened can inhibit the expression level of UGGT1 gene mRNA, the substance to be screened is indicated to be a candidate drug for treating cervical cancer.
Application of UGGT1 gene mRNA inhibitor in preparing medicine for treating cervical cancer.
4. The use according to claim 3, wherein the inhibitor comprises an agent that down-regulates the expression level of UGGT1 gene mRNA.
5. The use according to claim 3 wherein the inhibitor is a nucleic acid inhibitor of UGGT1 gene mRNA.
6. The use of claim 5, wherein the nucleic acid inhibitor is an siRNA.