CN108624694B

CN108624694B - Application of CMC2 as cervical cancer diagnosis and treatment marker

Info

Publication number: CN108624694B
Application number: CN201810868349.5A
Authority: CN
Inventors: 杨承刚; 孙耀兰
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2021-03-16
Anticipated expiration: 2038-08-02
Also published as: CN108624694A

Abstract

The invention discloses CMC2 as a diagnosis and treatment target of cervical cancer. Therefore, the CMC2 can be used for developing products for diagnosing cervical cancer and medicaments for treating the cervical cancer. The research result of the invention provides a theoretical basis for the clinician to formulate a personalized treatment scheme and can provide a new drug target for the development of cervical cancer drugs.

Description

Application of CMC2 as cervical cancer diagnosis and treatment marker

Technical Field

The invention relates to the field of biomedicine, in particular to application of a CMC2 gene in preparing a product for diagnosing and treating cervical cancer.

Background

Cervical cancer is the most common gynecological malignancy, and is the first among female genital tumors. According to the 2014 statistical data of the world health organization, the number of new annual cases of cervical cancer is about 52.8 ten thousand, the number of annual deaths is about 26.6 ten thousand, 85% of the patients occur in developing countries, and rural areas are higher than cities. According to the latest Cancer statistical data published by the national tumor center, the number of new cases of cervical Cancer in China is about 9.89 ten thousand in 2015 years and the number of annual deaths is about 3.05 ten thousand in 2015 years, and the data shows a trend of rising year by year and the onset of disease younger [ Chen W, Zheng R, Baade P D, et al. The occurrence of cervical cancer is closely related to various factors, such as early marriage, early childbirth, prolificacy and sexual disturbance, but Human Papilloma Virus (HPV) infection is considered as a necessary condition for the onset of cervical cancer, HPV screening is positive in 99.7% of cervical cancer patients, and the risk of HPV persistent infection of patients suffering from cervical cancer is more than 250 times that of HPV-negative [ Wentzensen N, Arbyn M.HPV-based clinical cancer screening-factors, diagnosis, and missierceptions [ J ]. Prev Med,2017,98:33-35 l; torre L A, Islami F, Siegel R L, et al, Global Cancer in who: burden and tresds [ J ]. Cancer epidemic biomekers Prev,2017 ]. After the fact that HPV infection is the important cause of cervical cancer is firstly determined in the seven eighties of the last century, students carry out deep research on the mechanism of inducing the cervical cancer by HPV infection, and therefore, diagnosis methods and treatment strategies aiming at the cervical cancer are developed, including sensitive and specific precancerous diagnosis methods, targeted immunoprophylaxis measures and treatment methods, and the survival and life quality of cervical cancer patients are greatly improved.

With the development of modern biological detection technology and the continuous and deep research on the pathogenesis of tumors, various molecules in various biological processes related to tumorigenesis, such as nucleic acid, protein, carbohydrate, lipid, small molecule metabolites and even free tumor cells in blood, can be used as important tumor markers, and provide definite bases for clinical prevention, diagnosis and treatment. In cervical cancer, several tumor markers have been found to be used for clinical prevention of cervical cancer.

Ki-67 and p16INK4a are two common molecular markers for analyzing the proliferation state of tumor cells and the malignancy degree of tumors. Wherein Ki-67 is not expressed in silent GO phase cells but is highly expressed in G1, S, G2 and M phase cells, so it can be widely used to analyze the proliferative activity of cells to assess tumor progression [ Endl E, Gerdes J.the Ki-67protein: stimulating for and an unknown function [ J ] Exp Cell Res,2000,257(2): 231-; meanwhile, because the expression pedigree is wide, the tumor marker serving as a specific tumor marker still has certain defects, and other molecular markers are needed for auxiliary diagnosis in clinic. p16INK4a, a cyclin-regulating protein, specifically binds CDK4 and CDK6 to inhibit its activity and phosphorylation of downstream pRb, regulate cell cycle progression and cell differentiation processes; it has been found that p16INK4a expression is positively correlated with HR-HPV persistent infection and cervical tumor pathological grading, and has certain guiding value for patient post-operative HPV clearance and persistent infection [ Koh J, Enders G H, Dynlash B D, et al. Tumour-derived p16 allols encoding proteins deletion in cell-cycle inhibition [ J ] Nature,1995,375(6531): 506-.

The proExC antibody (BD Co.) specifically recognizes the nuclear protein MCM2 and the topoisomerase TOP2A complex induced by HPV infection. In cervical gland and squamous cell dysplasia, the cellular S-phase gene induced by the E7 oncogene promotes high expression of TOP2A and MCM2 in the nucleus, while TOP2A can combine with 6 MCM2 molecules to form a stable structure retained in the nucleus [ Santin A D, Zhan F, Bignoti E, et al. Gene expression profiles of primary HPV16-and HPV 18-induced initial stage domains and nuclear molecules: identification of non-catalytic molecular markers for nuclear catalytic nuclear differentiation and thermal differentiation [ J. Virology,2005,331 (291) ]. Because it is not expressed in normal cervical epithelial cells, but is significantly highly expressed in HPV-induced squamous cells with active proliferation, it can be clinically used to distinguish atypical hyperplasia from similar changes such as incomplete squamous cell development or atrophy.

HPV DNA integrity and stability are mainly maintained by the capsid protein L1 and L2 together forming a stable protective shell. Among them, the L1 protein can also promote the invasion of virus particles into mucosal basement membrane zone cells or cervical epithelial cells by recognizing corresponding receptors on host cells. Expression of the L1 protein continues to be detected during mild to moderate dysplasia with HPV infection, but L1 protein gradually disappears as cervical carcinogenesis progresses [ McMurray H R, Mccance D J.human papillomavir type 16E6 activities TERT gene transcription prevention of c-Myc and release of USF-mediated expression [ J ]. J Virol,2003,77(18): 9852-). Therefore, the disappearance of the expression of the HPV-L1 indicates that the viral genome is integrated in the host genome, and the diagnosis of the CIN3 stage of cervical cancer can be realized.

Lanminin-5 is a tumor marker closely related to tumor invasion, and the expression of the Lanminin-5 gene is closely related to the tumor progression in various malignant tumors. In the process of cervical cancer, the Lamin-5 is mainly expressed in the early stage of tumor, especially in the skin lesion of micro-infiltration, so that the method can be used for detecting the early stage of cervical squamous cell infiltration. MIB-I has an expression pattern similar to that of Ki-67, i.e., tumor cells that are active in a proliferative state with significantly high expression in stages of GINS, and detection in combination with Ki-67 can be used to assist in the analysis of the periodic state in which the tumor cells are located. Therefore, MIB-1 is another important indicator for detecting the proliferative activity of cells in the course of atypical hyperplasia.

In summary, although many tumor markers are currently used for clinical diagnosis of cervical cancer, tumor markers are constitutively expressed under normal conditions or increased in non-malignant tumor diseases, and thus lack tumor specificity. Therefore, the search of tumor specific antigens as biomarkers for the early diagnosis and prognosis determination of clinical cervical cancer is urgently needed.

Disclosure of Invention

One of the purposes of the invention is to provide a method for diagnosing cervical cancer by detecting the expression difference of CMC2 gene.

It is another object of the present invention to provide a method for treating cervical cancer by inhibiting the expression of CMC2 gene.

The invention also aims to provide a method for screening a cervical cancer treatment drug.

The fourth purpose of the invention is to provide a medicine for treating cervical cancer.

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

the invention provides an application of a reagent for detecting CMC2 in preparing a tool for diagnosing cervical cancer.

Further, the reagent for detecting CMC2 includes a reagent for detecting the expression level of CMC2 gene.

Further, the reagent for detecting CMC2 includes a reagent capable of quantifying CMC2 gene mRNA and/or a reagent capable of quantifying CMC2 protein.

The reagent for quantifying CMC2 gene mRNA of the present invention can exert its function based on a known method using a nucleic acid molecule: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The assay can be performed qualitatively, quantitatively, or semi-quantitatively using the reagent.

Further, the PCR method is a known method, for example, ARMS (Amplification Mutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.

The reagent capable of quantifying the mRNA of the CMC2 gene can be a specific primer of the CMC2 gene or transcript, can also be a specific recognition probe of the CMC2 gene or transcript, or comprises the primer and the probe.

The above-mentioned primers specific to the CMC2 gene or transcript include primers for the specific amplification of the CMC2 gene used in real-time quantitative PCR. In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The primer can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.

The probe may be prepared by chemical synthesis, by appropriately designing with reference to known information using a method known to those skilled in the art, and by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.

The reagent for quantifying CMC2 protein of the present invention can exert its function based on a known method using an antibody: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.

The reagent for quantifying the CMC2 protein comprises an antibody or a fragment thereof which specifically binds to the CMC2 protein. Antibodies of any structure, size, immunoglobulin class, origin, etc. may be used orA fragment thereof, as long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')₂Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The reagent for quantifying CMC2 protein of the present invention may include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding a fragment of an antibody, a vector containing the nucleic acid, and a cell carrying the vector.

Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against CMC2 protein can be obtained by subjecting the obtained antibody to antigen-specific purification using CMC2 protein or a part thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.

Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.

The obtaining of the sample for detecting the expression level of the CMC2 gene is routine in the art, and preferably can be achieved by a method which is non-invasive or minimally invasive.

The sample may be (but is not limited to): tissue, peripheral blood, bone marrow, lymph nodes, peritoneal cavity wash, urine, sweat. In a specific embodiment of the invention, the sample is from a tissue of a subject.

The invention also provides a tool for diagnosing cervical cancer, which can detect the expression level of the CMC2 gene.

Further, the means comprise an agent capable of quantifying CMC2 gene mRNA, and/or an agent capable of quantifying CMC2 protein.

Typically, the reagents are present in suitable containers. Each of the primers or probes can be adjusted to at least one desired amount of concentration using a diluent such as deionized water and dispensed into a container.

Further, the reagent capable of quantifying the mRNA of the CMC2 gene comprises a primer for specifically amplifying the CMC2 gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Further, the tool for diagnosing cervical cancer includes, but is not limited to, a chip, a kit, a strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool, and with the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of the CMC2 gene is related to the occurrence of cervical cancer in high-throughput sequencing also belongs to the novel application of the CMC2, and is also within the protection scope of the invention.

The kit of the present invention may further comprise a reagent for extracting nucleic acid, a reagent for PCR, a reagent for staining or developing color, and the like. For example, such agents include, but are not limited to: an extraction solution, an amplification solution, a hybridization solution, a color development solution, a washing solution, and the like.

In addition, the kit can also comprise instructions and the like for describing a method for detecting the expression of the CMC2 gene.

The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagents are capable of diagnosing cervical cancer based on the detection of CMC2 gene or transcript.

The present invention also provides a method for diagnosing cervical cancer, comprising the steps of:

(1) obtaining a sample from a subject;

(2) detecting the expression level of CMC2 gene in the sample of the subject;

(3) correlating the measured CMC2 gene expression level with a disease association of the subject.

(4) A statistically elevated level of CMC2 gene expression compared to a normal control indicates that the subject is judged to have cervical cancer or that the subject is judged to be at high risk of having cervical cancer.

The invention also provides a method for treating cervical cancer, which comprises inhibiting the expression of the CMC2 gene or inhibiting the activity of the expression product of the CMC2 gene.

The invention also provides a screening method of a candidate drug for treating cervical cancer, which can measure the effect of the candidate drug on improving prognosis by measuring the expression level of the CMC2 gene or the CMC2 protein at a certain period after adding a test drug to a model cell. More specifically, when the expression level of CMC2 gene or CMC2 protein is decreased or restored to a normal level after the addition or administration of a test drug, the drug can be selected as a therapeutic drug for improving cervical cancer.

The invention also provides a medicament for treating cervical cancer, which comprises an agent for inhibiting CMC 2.

The agent for inhibiting CMC2 of the present invention is not limited as long as the agent is capable of inhibiting the expression or activity of CMC2 or a substance involved in the upstream or downstream pathway of CMC2, and is a drug effective for the treatment of cervical cancer.

The invention also provides application of the CMC2 gene or its expression product in preparing a medicament for treating cervical cancer.

Further, the medicament comprises an interfering RNA aiming at the expression of the CMC2 gene, or a negative regulation miRNA, a negative regulation type transcription regulation factor or a repression type targeting small molecule compound.

The medicaments of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the agents of the present invention may be administered alone, in various combinations, and in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. When using pharmaceutical compositions, a safe and effective amount of the drug of the present invention is administered to a human, and the specific dosage will depend on factors such as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.

The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The sequence of "CMC 2 gene" (Chromosome 16, NC _000016.10(80975795..81007036, completion)) of the present invention can be queried in the NCBI database.

In the context of the present invention, "diagnosis of cervical cancer" includes judging whether a subject has suffered from cervical cancer, judging whether a subject is at risk of suffering from cervical cancer, or judging that a patient suffering from cervical cancer has relapsed.

As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:

(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;

(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or

(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".

The invention has the advantages and beneficial effects that:

the invention discovers and confirms the close correlation between the CMC2 gene expression and the generation of cervical cancer for the first time, and has a large number of verified samples and accurate results. The correlation provides a new way for diagnosing and treating the cervical cancer.

Drawings

FIG. 1 shows a statistical graph for detecting differential expression of CMC2 gene in cervical cancer tissues and normal control tissues using QPCR;

FIG. 2 shows a statistical graph of the differential expression of CMC2 protein in cervical cancer tissues and normal control tissues detected by Western blot experiment;

FIG. 3 shows a statistical chart of the inhibition rate of CMC2 gene expression detected by Western blot experiment.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of cancer tissues of cervical cancer patients for genes abnormally expressed

1. Tissue collection

4 cases of cervical cancer tissues provided in obstetrics and gynecology department of hospitals were collected from patients who were pathologically diagnosed as cervical cancer after hysterectomy (conization or total section), with the surrounding normal tissues as a control group.

2. Tissue RNA extraction

Taking about 50mg of cervical cancer patient tissues cryopreserved at minus 80 ℃ and peripheral normal cervical tissues, putting the tissues into liquid nitrogen for grinding, transferring the tissues into a 1.5mL EP tube when no large granular tissues exist, adding 1m L Trizol for total RNA extraction and real-time quantitative PCR analysis, and specifically carrying out the following steps:

1) adding 200 mu L of chloroform into the tissue suspension containing 1mL of Trizol, manually fully shaking and uniformly mixing, and standing at room temperature for 10 min;

2) centrifuging at 12000rpm and 4 deg.C for 15 min;

3) after the centrifugation is finished, the upper aqueous phase of the EP tube is gently sucked to a new 1.5mL EP tube, 600 muL isopropanol is added, the mixture is fully mixed by turning upside down, and the mixture is placed at room temperature for 20min (or placed at minus 20 ℃ for 2h, RNA precipitation can be increased);

4) centrifuging at 12000rpm and 4 deg.C for 15 min;

5) discarding the supernatant, adding 75% ethanol diluted by DEPC water, and slightly blowing and sucking the suspension precipitate by a gun head;

6) centrifuging at 12000rpm and 4 deg.C for 15 min;

7) discarding the supernatant, sucking the residual liquid by using a gun head, and airing at room temperature for 5 min;

8) dissolving 20 μ L of RNAase-free water in the precipitate, and using or freezing at-80 deg.C for use.

3. Quantification of RNA

Diluting the extracted total RNA 2 μ L in 98 μ L DEPC water, measuring absorbance values at wavelengths of 260nm and 280nm by using an ultraviolet spectrophotometer, judging the purity of the RNA according to the A260/A280 ratio, and calculating the concentration of the sample RNA according to the A260 value.

4. High throughput transcriptome sequencing

(1) RNA-seq read mapping

The clean reads were first removed from the low quality reads and then matched to the UCSC h. sapiens reference genome (hg19) using TopHat v1.3.1, a pre-constructed index of h.sapiens UCSC hg19 version was downloaded from the TopHat homepage and used as the reference genome, allowing multiple matching sites per read (default to 20) with up to 2 mismatches when matched to the genome using TopHat. TopHat builds a pool of possible cleavage sites based on the exon regions and GT-AG cleavage signals, from which reads that are not mapped to the genome are mapped to the genome. We use the system default parameters of the TopHat method.

(2) Transcript abundance assessment

The matched read files are processed by Cufflinks v1.0.3, and the Cufflinks v1.0.3 standardizes the number of RNA-seq segments to calculate the relative abundance of the transcript. The FPKM value refers to the number of fragments that match to a region of the exon 1kb long for a particular gene per million sequenced fragments. And calculating a confidence interval of the FPKM estimated value by a Bayesian inference method. The referenced GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo _ sapiens. grch37.63. GTF).

(3) Detection of differentially expressed genes

The downloaded Ensembl GTF file and the original file matched by TopHat are transmitted to Cuffdiff, and the Cuffdiff uses the original matched file to re-estimate the expression abundance of the transcripts listed in the GTF file and detect differential expression. Only q values < 0.01 in the Cuffidff output, tests showed that successful comparisons were considered differential expression.

4. Results

The RNA-seq result shows that compared with the normal control tissue, 351 genes with high expression and 294 genes with low expression are in the cervical cancer tissue, and the difference has statistical significance (P < 0.05).

Example 2 Large sample validation of expression of differentially expressed genes

Detection at the transcriptional level

1. Tissue collection

Cervical cancer tissues and corresponding normal control tissues were collected in 40 cases according to the criteria of example 1.

2. Tissue RNA extraction and identification

The procedure is as in example 1.

3. Design and preparation of primers

The real-time quantitative PCR primer sequences used in the present application were as follows (designed and synthesized by Competition Biotechnology (Shanghai) Ltd.):

CMC2 gene primer:

an upstream primer: 5'-TGACTTATCTCCACACTTG-3' (SEQ ID NO. 1);

downstream primer 5'-TACTCATTCTTCAGGCATT-3' (SEQ ID NO.2),

GAPDH gene primers:

an upstream primer: 5'-GACCTGACCTGCCGTCTA-3' (SEQ ID NO. 3);

a downstream primer: 5'-AGGAGTGGGTGTCGCTGT-3' (SEQ ID NO. 4).

4. Reverse transcription PCR

Reverse transcription was performed using a Takara reverse transcription kit (containing genomic DNA-removing enzyme) in the following reaction system:

reaction system for removing genomic DNA:

TABLE 1 reaction System for removing genomic DNA

Reagent	Volume of
		Total RNA	1.0μg
gDNA Eraser	1.0μL
		5*gDNA Eraser B buffer	2.0μL
RNase Free water	Make up to 10 mu L

The reaction was carried out at 42 ℃ for 5 min.

Reverse transcription PCR reaction System:

TABLE 2 reverse transcription PCR reaction System

Reagent	Volume of
		RT Primer Mix	1.0μL
PrimeScript RT Enzyme Mix I	1.0μL
		5*Prime Script Buffer	4.0μL
Reaction system for removing genomic DNA	10.0μL
		RNase Free water	4.0μL

The reaction was carried out at 37 ℃ for 15min and at 85 ℃ for 5 s.

5. Real-time quantitative PCR

Reaction system:

TABLE 3 reverse transcription PCR reaction System

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s;

15s at 95 ℃, 15s at 58 ℃ and 20s at 72 ℃, and the total cycle time is 42 times;

extension at 72 ℃ for 5min (GAPDH as internal control).

By using 2^-△△CtThe expression level of the CMC2 is analyzed by a relative quantification method, and Ct is the intensity value of a fluorescence signal detected in a reaction system by a thermal cycler. The calculation method comprises the following steps: Δ Δ Ct ═ (Ct target gene-Ct reference gene) cervical cancer tissue experimental group- (Ct target gene-Ct reference gene) normal control tissue group, 2^-△△CtThe expression of the target gene in the experimental group is shown as the fold change relative to the control group, and the analysis of the experimental data is performed by the Bio-RAD analysis software.

6. Statistical analysis statistical software SPSS19.0 is used for data analysis, and a paired T test is used for judging whether the expression of CMC2 in the cervical cancer tissue and the normal control tissue sample has a statistical difference. The statistical tests are bilateral tests, and the difference is statistically significant when P is less than 0.05.

7. Results

Compared with the normal control tissue, the expression of the CMC2 gene of 39 of 40 cervical cancer tissues is obviously increased. As shown in FIG. 1, the CMC2 gene was significantly increased in the cervical cancer tissue compared to the normal control tissue, and the difference was statistically significant (P < 0.05).

Secondly, detecting at protein level

1. Extracting collected cervical cancer tissue total protein

(1) Taking out the preserved cervical cancer patient tissues and the peripheral normal cervical tissues from the liquid nitrogen, cutting the tissues to be about 100mg after the tissues are thawed at room temperature, transferring the tissues to a tissue homogenizer and cutting the tissues into pieces by using clean scissors;

(2) about 300. mu.L of RIPA lysate containing 1% PMSF was added, followed by homogenization on ice;

(3) transferring the tissue homogenate to a 1.5mL EP tube, and standing on ice for 15 min;

(4) centrifuging at 12000rpm and 4 deg.C for 15 min;

(5) the supernatant was transferred to a new 1.5mL EP tube, 10. mu.L of the supernatant was quantitated for BCA protein, and the remainder was stored frozen at-20 ℃.

2. Electrophoresis

Beta-actin is used as an internal reference. After 50. mu.g of total protein was fractionated by SDS-PAGE, it was electrically transferred to a PVDF membrane and blocked by gentle shaking at room temperature for 1 hour using 1 XTSST containing 5% skimmed milk powder; adding CMC2 monoclonal antibody and beta-actin monoclonal antibody separately, and staying overnight at 4 ℃; washing the membrane with 1 × TBST for 4 times, adding a secondary antibody, and incubating at room temperature for 1 h; washing the film for 4 times with 1 × TBST, reacting in Super Signal chemiluminescence reagent for 2min, exposing with X-ray film in dark room, developing and fixing by conventional method, and selecting exposed picture for gray level analysis with Image Pro software.

3. Statistical treatment

The grey scale values of the protein bands were analyzed by using Image J software, and the grey scale values of the CMC2 protein bands were normalized by using beta-actin as an internal reference. The results were expressed as mean ± sd, statistically analyzed using SPSS13.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

4. Results

Compared with the normal control tissue, the protein level of the CMC2 in 39 of 40 cervical cancer tissues is obviously increased, and the difference has statistical significance. The statistical results are shown in fig. 2, the protein level of CMC2 in the cervical cancer tissue is significantly increased compared to the normal control tissue, and the difference is statistically significant (P < 0.05).

Example 2 interference with CMC2 Gene expression

1. Interfering RNA design Synthesis

The siRNA was designed and synthesized by Shanghai Jima pharmaceutical technology, Inc. according to the CMC2 gene sequence. The Shanghai Jima pharmaceutical technology Co., Ltd simultaneously provided a negative control siRNA (siRNA-NC) having no sequence homology with the CMC2 gene.

siRNA-CMC2：

The sense strand is 5'-UCAACAUCAUUACAAUAACCA-3' (SEQ ID NO. 5);

the antisense strand is 5'-GUUAUUGUAAUGAUGUUGAUC-3' (SEQ ID NO.6),

2. culture of Hela cells

The cells were incubated at 37 ℃ in 1640 medium containing polyclonal antibody and 10% fetal Bovine Serum (BSA) in 5% CO₂The culture medium is changed for 1 time every 24 hours, and the culture medium is subcultured for 1 time in 48 hours. Cells in logarithmic growth phase were taken for subsequent experiments.

3. Transient transfection of cells

Cells were digested the day before transfection and seeded into 6-well plates at approximately 2 × 10 per well⁵The cells are cultured overnight, and the transient transfection of siRNA is carried out by observing the fusion degree of the cells (about 50-70%), which comprises the following steps:

1) the siRNA was diluted to 20. mu.M with RNase Free water as specified in the siRNA synthesis protocol;

2) marking two sterile EP tubes as A and B respectively, adding 125 mu L/hole serum-free culture medium and 5 mu L siRNA into the tube A, and adding 125 mu L/hole serum-free culture medium and 5 mu L Lipofectamine 3000 into the tube B;

3) mixing the solutions in tubes A and B, and standing at room temperature for 5 min;

4) replacing the culture medium in the 6-well plate with a serum-free culture medium, wherein each well is 2 mL;

5) adding the uniformly mixed solution A and B, and shaking in a cross manner to fully and uniformly mix the cells;

6) is placed in CO₂Performing conventional culture at 37 ℃ in an incubator;

7) after transfection for 48h, 1ml of Trizol or 200 μ L of RIPA was added to lyse cells, RNA and protein were extracted for real-time quantitative PCR and Western blot detection, and subsequent experiments were performed after analysis of interference effects.

4. Results

As shown in FIG. 3, compared with the negative control group (transfected siRNA-NC), the expression level of CMC2 protein in the cells of the experimental group (transfected siRNA-CMC2) is significantly reduced, and the difference is statistically significant (P < 0.05).

Example 4 Effect of expression of CMC2 Gene on Hela cell proliferation

The cell proliferation is detected by CCK-8 method

(1) Transfection was performed according to the method of example 3;

(2) HeLa cells after 24 hours of transfection, the medium was aspirated off, the cells were digested with 0.25% trypsin, the cells were counted with crystal violet stain, the cells were suspended in 1640 medium containing 0.5% FBS and adjusted to a cell concentration of 2X10⁴/ml；

(3) Add 100. mu.l of cell suspension to each well of a new 96-well plate under sterile conditions, i.e., 2X10³Single cell, 37 ℃, 5% CO₂Standing for 24 h;

(4) adding 10 mul of CCK solution into each hole respectively, and culturing for 2 h;

(5) the wavelength of 450nm, and the light absorption value of each experimental group is measured by an ultraviolet spectrophotometer.

As a result:

the results showed that the OD value of the cells of the negative control group (transfected siRNA-NC) was 0.957. + -. 0.116, and the OD value of the cells of the experimental group (transfected siRNA-CMC2) was 0.414. + -. 0.071. The results show that the cervical cancer cell proliferation inhibiting CMC2 expression is inhibited, and the difference has statistical significance (P < 0.05).

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.

Sequence listing

<110> Beijing, the deep biometric information technology GmbH

Application of <120> CMC2 as cervical cancer diagnosis and treatment marker

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgacttatct ccacacttg 19

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tactcattct tcaggcatt 19

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacctgacct gccgtcta 18

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggagtgggt gtcgctgt 18

<210> 5

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ucaacaucau uacaauaacc a 21

<210> 6

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

guuauuguaa ugauguugau c 21

Claims

1. Application of a reagent for inhibiting CMC2 gene expression in preparing a medicament for treating cervical cancer.