CN109988839B

CN109988839B - Application of MINDY1 in cervical lesion diseases

Info

Publication number: CN109988839B
Application number: CN201910254339.7A
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: 2021-11-12
Anticipated expiration: 2039-03-31
Also published as: CN109988839A

Abstract

The invention discloses application of MINDY1 in cervical lesion diseases, discovers that MINDY1 is expressed and reduced in cervical squamous cell carcinoma tissues and cervical intraepithelial neoplasia tissues for the first time by combining a high-throughput sequencing technology and bioinformatics analysis, and proves that MINDY1 is related to occurrence and development of cervical squamous cell carcinoma through further QPCR and cell experiments, so that the invention provides a method and a product for diagnosing and treating the cervical lesion diseases.

Description

Application of MINDY1 in cervical lesion diseases

Technical Field

The invention belongs to the field of biological medicines, and relates to application of MINDY1 in cervical lesion diseases, in particular to cervical intraepithelial neoplasia and cervical cancer.

Background

Cervical cancer is one of the most common malignancies in gynecology, and is the 4 th leading cause of death in female cancer patients. The proportion of the cervical squamous carcinoma in the cervical cancer is 75-80%. The progression of cervical cancer is the result of a multifactorial co-action, with persistent infection of HPV, particularly of high-risk types, being the major etiological factor in the development and progression of cervical cancer (Li N, France Schi, Howell-Jones R, snivers PJ, Clifford GM. human papulomyvir type distribution in 30,848 innovative scientific cancers region, pathological by genetic geographic region, pathological type and layer of publication. International Journal of cancer.2011, 128(4): 927-935.). With the popularization of cervical cancer screening and the application of Human Papilloma Virus (HPV) vaccines, the incidence and mortality of cervical cancer are reduced (Seoud M, Tjalma Rosse V.Cervical adonnarcinoma: moving towards Vaccine-2011.291491: 9148-one 9158), but the occurrence and development of tumors are the result of multi-factor interaction. In most cases, HPV infection can be cleared by the immune system and only a small proportion of HPV persistent infectors progress to cervical cancer (Wheeler CM, Hunt WC, Cuzick J, Langsfeld E, Robertson M, Castle PE. the infection of type-specific human papillomavirus infections on the detection of cellular precancer: A position-based study of cellular nuclear expression in the underlying stages.and Int J cancer.2013, doi:10.1002.) suggesting that, in addition to HPV infection, abnormal expression of host cell growth differentiation-related genes is also important in the pathogenesis of cervical cancer.

The normal cervical epithelium develops into cervical intraepithelial neoplasia, progresses to cervical cancer and needs to go through a plurality of different pathological stages, wherein a plurality of molecular events are involved, and the occurrence and development of cervical cancer should be the result of the combined action of host genetic factors and HPV. Therefore, the research on the gene and the signal path related to the growth and the differentiation of the cervical squamous cell carcinoma can provide a new strategy for the screening, the diagnosis and the prognosis of the cervical carcinoma.

The basis and starting point of gene function and structure studies is transcriptome studies, and RNA sequencing (RNA-Seq) is transcriptome sequencing of cells OR tissues using deep sequencing techniques (Ramskold D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Danies GA, Khrebtukova I Loring JF, Laurent LC, Schroth GP, Sandberg R.Full-length mRNA-Seq from single-cell levels of RNA and induced viral circulating tumor cells. Nat Biotechnol.2012,30(8): 777-782). Compared with the traditional chip hybridization platform, the RNA-Seq has obvious advantages in the aspects of detecting new genes, analyzing transcripts of tumor tissues, detecting differential genes of the tumor tissues and normal tissues and the like. The invention searches differentially expressed genes by carrying out high-throughput sequencing on a sample of a disease patient, thereby providing a molecular target and a theoretical basis for early diagnosis and treatment of cervical cancer.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, the present invention aims to provide a cervical treatment device which is compatible with cervical diseases: cervical intraepithelial neoplasia and cervical squamous carcinoma development related gene markers.

The invention also aims to provide the application of the biomarker in the clinical diagnosis and treatment of cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

The invention also aims to provide application of the biomarker in clinical treatment of cervical squamous cell carcinoma.

The fourth purpose of the invention is to provide the application of the biomarker of cervical squamous cell carcinoma in screening drugs for treating cervical squamous cell carcinoma.

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

in a first aspect of the invention, there is provided an agent capable of detecting the level of MINDY1 gene or its expression product.

Further, the reagent comprises:

a probe that specifically recognizes MINDY1 gene; or

Primers for specifically amplifying MINDY1 gene; or

An antibody or ligand that specifically binds to a protein encoded by MINDY1 gene.

Furthermore, the primer sequence of the specific amplification MINDY1 is shown in SEQ ID NO. 1-2.

In a second aspect the invention provides a kit comprising the reagents of the first aspect of the invention.

In a third aspect, the invention provides a chip comprising a reagent according to the first aspect of the invention.

A fourth aspect of the present invention provides a nucleic acid membrane strip comprising the reagent according to the first aspect of the present invention.

In a fifth aspect, the invention provides a composition comprising an enhancer of MINDY1, and/or a pharmaceutically acceptable carrier. The promoter comprises substances for improving the stability of MINDY1 gene or expression products thereof, up-regulating the expression level of MINDY1 gene or expression products thereof, and prolonging the effective action time of MINDY1 gene or expression products thereof; the pharmaceutically acceptable carrier and/or adjuvant includes (but is not limited to) diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant; the pharmaceutical composition may further include additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

Further, the promoter is a carrier containing MINDY 1.

A sixth aspect of the present invention provides a method for screening a candidate drug for treating cervical squamous carcinoma, the method comprising:

treating a culture system expressing or containing MINDY1 gene or its encoded protein with a substance to be screened; and

detecting the expression or activity of MINDY1 gene or its encoded protein in said system;

wherein, if the substance to be screened can promote the level or the expression activity of MINDY1 gene, the substance to be screened is a candidate drug for treating cervical squamous cell carcinoma.

In the present invention, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

In the present invention, the steps further include: and carrying out further cell experiments and/or animal experiments on the obtained candidate drugs so as to further select the drug capable of treating the cervical squamous carcinoma from the candidate drugs.

A seventh aspect of the invention provides a use as claimed in any one of:

a. the reagent of the first aspect of the invention is applied to the preparation of products for early diagnosis of cervical intraepithelial neoplasia and cervical squamous carcinoma;

b. the kit of the second aspect of the invention is applied to the preparation of products for diagnosing cervical intraepithelial neoplasia and cervical squamous cell carcinoma;

c. the chip of the third aspect of the invention is applied to the preparation of products for diagnosing cervical intraepithelial neoplasia and cervical squamous carcinoma;

d. the nucleic acid membrane strip of the fourth aspect of the invention is applied to the preparation of products for diagnosing cervical intraepithelial neoplasia and cervical squamous cell carcinoma;

e. the use of a composition according to the fifth aspect of the invention for the manufacture of a medicament for the treatment of squamous cell carcinoma of the cervix;

f. the method of the sixth aspect of the invention is applied to screening of candidate drugs for treating cervical squamous carcinoma;

the application of MINDY1 in screening candidate drugs for treating cervical squamous cell carcinoma;

application of MINDY1 in preparing medicines for treating cervical squamous cell carcinoma.

Drawings

FIG. 1 is a diagram showing the QPCR assay of the expression of MINDY1 gene in patients with cervical squamous cell carcinoma;

FIG. 2 is a graph showing the expression level of MINDY1 gene;

FIG. 3 is a graph showing the effect of MINDY1 on cell proliferation activity measured by the CCK-8 method;

FIG. 4 is a graph showing the effect of MINDY1 gene on invasion of cell migration measured using a transwell chamber, in which Panel 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 screening shows that MINDY1 is expressed and down-regulated in cervical squamous cell carcinoma patients for the first time, and the gene overexpression technology further verifies that MINDY1 participates in the proliferation and invasion processes of cervical cancer cells, and prompts that MINDY1 can be used as an independent prediction factor of cervical squamous cell carcinoma and can also be combined with other gene markers for application.

MINDY1 gene

The gene ID of MINDY1 in the present invention is 55793, and MINDY1 in the present invention includes a wild type, a mutant type or a fragment thereof. One skilled in the art will appreciate that in performing sequencing analysis, the original sequencing results will be aligned to the human reference genome, and thus MINDY1 in the screening results may contain different transcripts, as long as MINDY1 (Gene ID: 55793) on the reference genome can be aligned. MINDY1 has been disclosed so far as having six transcripts, the sequences are shown as NM _001040217.2, NM _001163258.1, NM _001163259.1, NM _001163260.1, NM _001319998.1, NM _018379.4, respectively. In the present invention, a representative MINDY1 gene sequence is shown in NM-001040217.2.

The MINDY1 nucleotide full-length sequence or its fragment of the present invention can be obtained by PCR amplification method, recombination method or artificial synthesis method.

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 MINDY1 protein is a specific binding agent of MINDY1 protein. Specific binders are for example receptors for the protein MINDY1, lectins binding to the protein MINDY1, antibodies against the protein MINDY1, peptide antibodies (peptidebody) against the protein MINDY1, bispecific dual binders 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 MINDY 1.

The invention provides products for detecting the expression level of MINDY1, and the products include (but are not limited to) chips, kits and nucleic acid membrane strips. Wherein the chip includes: a solid support; and an oligonucleotide probe or an antibody orderly fixed on the solid phase carrier, wherein the oligonucleotide probe specifically corresponds to a part or all of the sequence shown in MINDY1, and the antibody specifically binds to MINDY1 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 MINDY1 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.

The invention provides a nucleic acid membrane strip, which comprises a substrate and oligonucleotide probes fixed on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

Accelerator and pharmaceutical composition

The invention provides a medicament (composition) which contains an effective amount of the MINDY1 promoter and a pharmaceutically acceptable carrier. The composition can be used for treating cervical squamous carcinoma.

As a preferred mode of the invention, the promoter of MINDY1 is an expression vector of MINDY 1. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

The pharmaceutical compositions of the present invention may be in a form suitable for administration by injection, in a form suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs), in the form of an ointment, cream or lotion suitable for topical administration, in a delivery form suitable for use as eye drops, in an aerosol form suitable for administration by inhalation (e.g., by intranasal or oral inhalation), in a form suitable for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection.

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, the promoter of MINDY1 can be administered directly to a subject by a method such as injection; alternatively, the expression unit carrying MINDY1 that facilitates dispensing (e.g., an expression vector or virus, etc.) can be delivered to the target site by any route, depending on the type of promoter, 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 MINDY1 gene in cervical squamous carcinoma tissues and cervical intraepithelial neoplasia tissues is obviously reduced, wherein the expression level of the cervical squamous carcinoma tissues is also obviously reduced compared with the cervical intraepithelial neoplasia tissues, and the difference has statistical significance, so that the large sample verification is further carried out on MINDY 1.

Example 2 QPCR sequencing validation of differential expression of MINDY1 Gene

1. Large sample QPCR validation of differential expression of MINDY1 gene was performed.

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 RNA 1 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 MINDY1 gene and GAPDH gene in Genebank and were synthesized by Bomader Biotech. The specific primer sequences are as follows:

MINDY1 gene:

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

the reverse primer was 5'-CTTGCTCCTCCTGTAGAA-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

Each sample cDNA was diluted 10-fold and 2. mu.l was takenAs a template, a target gene primer and an internal reference gene primer are used for amplification respectively. Simultaneously performing dissolution curve analysis at 60-95 deg.C, and determining target band by dissolution curve analysis and electrophoresis, 2^-ΔΔCTThe method is used for relative quantification.

4. Results

As shown in fig. 1, the expression level of MINDY1 in cervical squamous carcinoma tissue and cervical intraepithelial neoplasia tissue was significantly reduced compared to normal tissue, wherein cervical intraepithelial neoplasia tissue showed significant down-regulation compared to normal tissue, and cervical squamous carcinoma tissue showed significant down-regulation compared to cervical intraepithelial neoplasia tissue, and the difference was statistically significant (P < 0.05).

Example 3 overexpression of MINDY1 Gene

Specific primers are designed according to MINDY1 gene sequence information in NCBI, corresponding genes are obtained through amplification, gene fragment glue is recovered and then is respectively connected with an expression vector pEGFP-C1 and then is transformed into DH5 alpha competent cells, and pEGFP-C1-MINDY1 recombinant plasmids are obtained after positive clones are selected and subjected to PCR and sequencing identification.

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.

2. Transfection

The experiment was divided into 3 groups, control (Hela), blank control (transfection pEGFP-C1) and experimental group (transfection pEGFP-C1-MINDY1), and transfection was performed according to the instructions of lipofectamine 2000 transfection reagent of Invitrogen. The method comprises the following steps:

1) the cells in logarithmic growth phase are digested and lightly blown into single cell suspension, the single cell suspension is inoculated to a 6-well plate by using a culture medium without antibiotics, and the cell density of the 6-well plate reaches 2 multiplied by 10⁵Performing transfection test when the cell fusion rate reaches 40% -60%;

2) the next day of transfection, 10. mu.l Lipofectamine 2000 was diluted in 200. mu.l Opti-MEMI medium without serum and left to stand at room temperature for 5 min;

3) dissolving 4 μ g of plasmid in 200 μ l of serum-free Opti-MEM I medium;

4) mixing the two dilutions uniformly, and incubating for 20 minutes at room temperature; the ratio of plasmid to transfection reagent was 4. mu.g/well to 10. mu.l;

5) the cells in the 6-well plate were replaced with 1.6ml of fresh RPIM-1640 medium. Adding the suspension into 6-well plates respectively, wherein the final volume of culture solution in each well is 2 ml;

6) after 6 hours of transfection, 2ml of fresh complete culture solution is replaced for continuous culture;

7) and collecting cells for 72h, extracting RNA, and performing QPCR detection.

3. QPCR detection of transcript level of MINDY1 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.

3.3 QPCR amplification step as in example 2.

4. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± sd, and statistically analyzed using SPSS18.0 statistical software, and the difference between the expression group of the overexpressed MINDY1 gene and the control group was considered statistically significant when P <0.05 using t-test.

5. Results

The results are shown in FIG. 2, and compared with control group Hela and transfection-unloaded pEGFP-C1, the experimental group pEGFP-C1-MINDY1 can significantly increase the expression level of MINDY1 gene, and the difference has statistical significance.

Example 4 CCK-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 pEGFP-C1-MINDY1 is significantly reduced, which suggests that changing the expression level of MINDY1 can change the proliferation capability of cervical squamous carcinoma cells, and suggests that MINDY1 is related to the proliferation of cervical squamous carcinoma cells.

Example 5 Transwell 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 transfection were starved, collected for counting, prepared to 1X 10 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 over-expression of MINDY1 gene reduces the migration and invasion capacity of cervical squamous carcinoma cells.

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> MINDY1 in cervical lesion diseases

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Claims

1. A method of screening a candidate drug for treatment of cervical squamous carcinoma, the method comprising:

treating a culture system expressing or containing mRNA of MINDY1 gene with a substance to be screened; and

detecting the mRNA expression level of MINDY1 gene in the system;

wherein, if the substance to be screened can promote the expression of mRNA of MINDY1 gene, the substance to be screened is a candidate drug for treating cervical squamous cell carcinoma.

2. Use according to any one of the following:

a. the application of a composition in preparing a medicament for treating cervical squamous carcinoma is characterized in that the composition comprises an accelerant of MINDY1 and a pharmaceutically acceptable carrier;

b. use of the method of claim 1 for screening a candidate agent for the treatment of cervical squamous carcinoma;

c. the application of MINDY1 gene in screening candidate drugs for treating cervical squamous cell carcinoma is realized by detecting mRNA expression level of MINDY1 gene.

3. The use of claim 2, wherein the promoter of MINDY1 is a carrier comprising MINDY 1.