CN113265468B

CN113265468B - MiRNA related to cervical cancer diagnosis, treatment and prognosis

Info

Publication number: CN113265468B
Application number: CN202110824037.6A
Authority: CN
Inventors: 尚春亮; 乔杰; 郭红燕
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2021-10-12
Anticipated expiration: 2041-07-21
Also published as: CN113265468A

Abstract

The invention relates to the technical field of biology, in particular to diagnosis, treatment and prognosis application of miRNA related to cervical cancer, and specifically relates to a method for indicating the prognosis of cervical cancer, especially cervical cancer lymph node metastasis patients by detecting the miRNA level, and treatment of cervical cancer by combining the miRNA with obesity-reducing drug orlistat.

Description

MiRNA related to cervical cancer diagnosis, treatment and prognosis

Technical Field

The invention relates to the field of biotechnology, in particular to diagnosis, treatment and prognosis application of miRNA related to cervical cancer.

Background

Cervical Cancer (CC) is the second most common malignancy that severely affects female health, with new cases of Cervical Cancer accounting for approximately 1/4 of the total number of worldwide occurrences each year in our country. The tumor metastasis is the main reason for the postoperative recurrence and death of cervical cancer patients, lymph node metastasis can occur in the early stage of cervical cancer, and researches show that the 5-year survival rate of early-stage cervical cancer patients without lymph node metastasis is as high as 80-90%, while the 5-year survival rate of patients with lymph node metastasis is only 50-65%. Therefore, lymph node metastasis is the main cause of death of cervical cancer patients, and the research on new diagnosis and prognosis prediction markers of cervical cancer has important significance.

The incidence of cervical cancer has decreased significantly in developed countries, largely due to early diagnosis and treatment of precancerous lesions. However, cervical cancer is often asymptomatic in its early stages, and may be already advanced when it is found. Cervical cancer diagnosis mainly depends on vaginal cytology examination, local smear rapid diagnosis, biopsy examination and the like. The auxiliary detection means also comprises immunological reaction, in vivo measurement of special chemical components, enzyme reaction and the like. The detection of squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA), cytokeratin fragment antigen (CYFRA21-1), cancer antigen 125(CA-125) in blood can provide reference for the diagnosis of cervical cancer, but the detection has high false positive and false negative and low specificity.

MicroRNAs (i.e., miRNAs) are a hotspot in the field of molecular biology research in recent years, and the mature state of the MicroRNAs is a small single-stranded RNA molecule with the length of about 19-23 nucleotides and has high conservation in evolution. The main function of miRNA is to regulate the expression of the gene related to the growth, development and disease process of organism. Since the discovery of lin-4 and let-7, which are involved in the regulation of nematode timing development, mirnas were introduced in the Science journal of the ten-year major scientific breakthrough in 2002 and 2003, respectively. miRNAs were predicted to regulate 5300 human genes, at least 30% of all genes, in 2005. As research progresses, more and more miRNAs are continuously discovered. In recent years, the relation between miRNA and diseases has become a focus and a focus of research, and it has been found that the expression of miRNA through negative regulatory genes is highly related to the onset of various human diseases such as tumor and diabetes.

Studies have demonstrated the presence of hundreds of miRNAs in tissues, and these small molecular RNAs are stable in nature, abundant in content, easy to quantitatively detect, and have significant disease specificity. The existing mature technologies, including qualitative and quantitative miRNA molecular technologies, show that the method using miRNAs as molecular biomarkers is more effective than the traditional specific protein molecular marking method, and develops a new visual angle for the biomarkers.

The miRNA screened by the invention, such as miR-532-5p is located on Xp11.23 of human chromosome, has a complementary sequence of 3' untranslated region, and can down-regulate a plurality of gene products by inhibiting transcription. miR-532-5p was first found to have a carcinogenic effect in cutaneous melanomas (KitagOM, MartinezSR, NakamuraT, et. Regulation of RUNX3 turmor suppresor gene Expression in Cancer melanoma [ J ]. Clin Cancer Res,2009,15(9):2988-2994), but it was also subsequently found to be down-regulated in a variety of solid tumors, such as ovarian Cancer, lung Cancer and liver Cancer, and cell function tests suggested that it has an effect of inhibiting tumor cell proliferation (Wang F, ChangJT, KaoCJ, et. high Expression of miR-532-5p, a turmor suppresor, lead to beta promoter in ovo toxin cell and in vitro [ J ]. Mol, The 2016 (2016) (1133): 2016 1). Further, patent document WO2009155455a1 provides a method for detecting cancer. The method comprises providing a test biological sample from the subject and determining the expression level of miR-532-5p in the test sample. If the expression level of miR-532-5p in the test sample is higher than the expression level in the normal sample, the subject may have cancer. The cancer may be melanoma, breast, stomach, pancreas, colon, or esophagus cancer. The examples demonstrate the expression of miR-532-5p in melanoma, but the effect and mechanism of miR-532-5p in other cancers are not verified in detail. However, as is well known, various mirnas play important roles in various cell biological behaviors such as cell proliferation, aging, apoptosis, metastasis and the like, are closely related to the occurrence and development of tumors, and different mirnas have different cancer promotion or cancer inhibition effects in various tumors, so that the determination of the up-regulation or down-regulation of a specific miRNA in a specific cancer is particularly important.

Disclosure of Invention

The invention provides a miRNA for diagnosing, treating and prognosing cervical cancer aiming at the problem of poor diagnosis and treatment effects of metastatic cervical cancer clinically, in particular to a miRNA level which can indicate the prognosis of cervical cancer, especially cervical cancer patients with lymph node metastasis sources, and relates to the treatment of cervical cancer by the miRNA.

In a first aspect of the present invention, a miRNA marker for detecting, diagnosing, and determining a prognosis of cervical cancer is provided, wherein the miRNA marker includes any one or more of the following mirnas that are stably present and detectable in a human tissue: miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3 p.

Preferably, detection of the miRNA levels is indicative of a prognosis for cervical cancer, particularly cervical cancer lymph node metastasis.

In a second aspect of the invention, the application of the miRNA marker in the preparation of a kit for detection, diagnosis and prognosis judgment is also provided. The kit contains a primer for detecting the miRNA marker. The diagnostic kit comprises enzymes and reagents commonly used for PCR reactions.

The third aspect of the invention also provides a kit for preparing cervical cancer detection, diagnosis and prognosis judgment, which is characterized in that the kit contains a primer for detecting the miRNA marker.

In the fourth aspect of the invention, the pharmaceutical composition for treating cervical cancer is further provided, and the pharmaceutical composition comprises a reagent for over-expressing miRNA, wherein the miRNA is selected from one or more of miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3 p.

The reagent for over-expressing miRNA is an over-expression vector containing the miRNA,

in the fifth aspect of the invention, the application of the miRNA in preparing a reagent for down-regulating vimentin and N-cadherin is also provided.

In the sixth aspect of the invention, the application of the miRNA in preparing a reagent for up-regulating E-cadherin is also provided.

The miRNA related to cervical cancer is screened to obtain the miRNA for detection diagnosis, prognosis judgment and tumor treatment, and the miRNA can be used as a molecular marker or a target point for cervical cancer diagnosis and applied to clinical early diagnosis, prognosis judgment or targeted therapy of cervical cancer.

Specifically, the application provides a miRNA marker combination for cervical cancer detection, diagnosis and prognosis, wherein the marker combination comprises any 2 or more than 2 of the following miRNAs which are stably present and detectable in human tissues: miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3p, and the detection of the level of the combination of the miRNAs can indicate the prognosis condition of the cervical cancer, particularly the cervical cancer lymph node metastasis patient.

The application also provides application of the miRNA marker combination in preparation of a kit for cervical cancer detection, diagnosis and prognosis, and is characterized in that the kit contains a primer for detecting the miRNA marker, and enzymes and reagents commonly used in PCR reaction.

The application also provides a kit for detecting, diagnosing and judging the prognosis condition of cervical cancer, which is characterized by comprising a primer for detecting the combination of the miRNA markers, and enzyme and reagent which are commonly used in PCR reaction.

The application also provides a molecular model for assessing prognosis of a cervical cancer patient, which is characterized by comprising five target miRNAs: miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3p, detecting to obtain an expression value RE of miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3p in a cervical cancer tissue,

calculating a prognostic risk score using the formula:

risk Score = [ RE value of miR-142-3p (-0.00002696198) ] + [ RE value of miR-532-5p (-0.0001861752) ] + [ RE value of miR-378a-5p (-0.0003280755) ] + [ RE value of miR-150-3p (-0.03612045) ] + [ RE value of miR-361-3p (-0.0000827455) ], when Risk Score > -0.261 value, the prognosis high Risk group can be judged, and when Risk Score is less than or equal to-0.261 value, the prognosis low Risk group can be judged.

The application also provides a pharmaceutical composition for treating cervical cancer, which is characterized in that the pharmaceutical composition comprises a reagent for over-expressing miRNA, and the miRNA is selected from one or more of miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3 p. The reagent for over-expressing miRNA is an over-expression vector containing miRNA.

Preferably, the pharmaceutical composition further comprises a FASN inhibitor.

The application also provides application of an agent for over-expressing miRNA and a FASN inhibitor in preparation of medicines for treating cancer or inhibiting cancer metastasis, wherein the miRNA is selected from one or more of miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3 p.

Preferably, the FASN inhibitor is orlistat.

The application also provides application of the miRNA marker in preparing a reagent for down-regulating the wave-shaped protein and the N-cadherin or a reagent for up-regulating the E-cadherin.

Drawings

Figure 1 a workflow diagram for selecting mirnas.

Figure 2 relative expression of 5 candidate prognostic mirnas detected by qRT-PCR in cervical cancer tissues and normal tissues.

FIG. 3qRT-PCR results show that miR-532-5p is significantly and lowly expressed in cervical cancer in-situ focal tissues with positive lymph node metastasis.

FIG. 4 expression of miR-532-5p in three cervical cancer cell lines with different metastatic potential.

FIG. 5 uses qRT-PCR to confirm the overexpression and interference efficiency of miR-532-5P.

FIG. 6 shows that, compared with miR-Ctrl control cells, miR-532-5p overexpression can significantly reduce cell proliferation capacity as shown in MTT assay.

FIG. 7 Transwell experiment demonstrates the effect of miR-532-5p overexpression on the invasive potential of cervical cancer cells (scale bar, 500 μm).

FIG. 8 Transwell experiment demonstrates the effect of inhibiting the expression of miR-532-5p on the invasion capacity of cervical cancer cells (graduated bar, 500 um).

FIG. 9 Effect of miR-532-5p on human lymphatic endothelial cell tubulogenesis ability (bar, 100 μm).

FIG. 10 Effect of miR-532-5p overexpression on changes in epithelial-mesenchymal transition (EMT) marker protein expression.

FIG. 11 is a representative image of the tumorigenesis in a nude mouse model of stably over-expressing miR-532-5p or miR-Ctrl cells by xenografting.

Figure 12 subcutaneous tumor growth curves and mean weights of tumors in mice at the same time points in different treatment groups.

FIG. 13 construction of lymph node metastasis model of cervical cancer in mice.

FIG. 14 effect of different treatment groups on lymph node metastasis ratio in cervical cancer mouse model.

Figure 15 subcutaneous tumor growth curves and mean weights of tumors in mice in different treatment groups.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 experimental method:

1. RNA extraction, qRT-PCR:

total RNA was extracted using TRIzol reagent (TaKara, Japan). QRT-PCR was performed on ABI7500 real-time PCR system (Thermo Scientific) using SYBR Premix Ex Taq (TaKaRa). To detect mirnas and U6, RNA samples were Reverse Transcribed (RT) to complementary DNA by specific stem-loop RT primers using the PrimeScrip RT kit (TaKaRa, japan). The results were calculated by the 2- Δ Δ CT method.

2. Western blot

Cell samples were collected and lysed with cold RIPA lysis buffer containing protease inhibitors. Total proteins were separated by SDS-polyacrylamide gel and then electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Mass.). Membranes were blocked with 5% skim milk and then incubated with appropriate antibodies. Antigen-antibody complexes on the membrane were detected with Pierce ECL Western blot substrate (Thermo Scientific, Waltham, MA).

3. Cell transfection

For transient transfection assays, miRNA mimics synthesized by geneCreate (Wuhan, China) and miRNA inhibitors were transfected into SiHa and HeLa cells using Lipofectamine RNAIMAX reagent (Invitrogen, USA). Supplementary methods and supplementary table 1 provides a detailed description.

4. Cell proliferation assay, transwell invasion assay and endothelial cell tube formation assay

For cell proliferation assays, MTT (Sigma-Aldrich) solution was added to each well containing the appropriate number of cells after transfection. The absorbance values were measured at 490 nm to assess cell viability. For Transwell analysis, cells were seeded into the upper chamber, while the lower chamber was filled with medium containing 10% bovine serum albumin. Cells that migrated to the lower surface were fixed, stained and counted. For tube formation assays, HLEC were treated with conditioned media from cervical cancer cells and incubated for 6 hours. The tube structure was quantified in terms of total tube length or number of branching nodes using ImageJ software (NIH, Boston, MA, USA).

5. Xenograft model

All animal experiments were approved by the animal ethics and welfare committee of the university of beijing. A xenograft model of female BALB/c nude mice (4-5 weeks old) was used. For the subcutaneous xenograft tumor model, 1X 10 transfected with an overexpression vector or control vector containing miR-532-5p⁷Cells/150. mu.l were injected subcutaneously into nude mice. Tumor growth in mice was measured three times a week for 30 days after injection. Tumor volume was calculated by the following formula: length x width²×0.52。

6. Bioinformatics analysis

OncomiR or OncoLnc was used to retrieve a list of mirnas that are closely related to CC patient survival, and the R package TCGABiolinks provided a bioinformatics solution to allow users to download TCGA data using a guided workflow.

7. Statistical analysis

All statistical analyses were performed using SPSS software (version 13.0) and prism5.0 (GraphPad software). Comparisons were made using Student's t-test or one-way ANOVA.

The primers used, siRNAs, miRNA mics and inhibitors are shown in Table 1, and the antibodies used are shown in Table 2.

TABLE 1 primers, siRNAs, miRNA mics and inhibitors for qRT-PCR

Antibodies used in Table 2

Example 2 screening of MiRNAs correlated with survival in cervical cancer patients

To identify mirnas that are associated with survival in cervical cancer patients, expression profiles of mirnas were obtained based on their correlation with pathological nodal or distant metastasis status, clinical stage, differences in expression in different survival status and significance thresholds (P < 0.01) using the OncomiR and OncoLnc databases (the overall scheme is shown in figure 1).

These miRNAs were analyzed in a Lasso regression model, and finally 5 candidate prognostic miRNA signatures were identified, namely miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3 p. And obtaining expression values RE detected by miR-142-3p, miR-532-5p, miR-378A-5p, miR-150-3p and miR-361-3p in cervical cancer tissues and normal tissues.

Calculating a prognostic risk score using the formula:

risk Score = [ RE value of miR-142-3p (-0.00002696198) ] + [ RE value of miR-532-5p (-0.0001861752) ] + [ RE value of miR-378a-5p (-0.0003280755) ] + [ RE value of miR-150-3p (-0.03612045) ] + [ RE value of miR-361-3p (-0.0000827455) ].

The patients are divided into two groups of high-risk and low-risk according to the median value, and data analysis shows that the miRNAs prognostic model can be used as an effective biomarker for predicting the prognosis of the cervical cancer patients.

When the Risk Score > is-0.261 value, the prognosis is judged as high-Risk group, and when the Risk Score is less than or equal to-0.261 value, the prognosis is judged as low-Risk group.

Example 3 analysis of miRNA expression

To validate the analysis of miRNA expression described above, the expression levels of 5 candidate mirnas were measured using the method of example 1.

Results of the experiment

As shown in figure 2, expression of 5 mirnas in candidate prognostic targets was significantly reduced in cervical cancer tissues compared to normal controls. The miR-532-5P has the lowest P value in the expression (P < 0.001) and prognosis prediction (P < 0.0001) of RNA level, the miR-532-5P is taken as an example for further research, and similar situations exist in other miRs, and the details are not repeated for the sake of brevity.

The PCR data showed that miR-532-5p expression was lower in Lymph Node Metastasis (LNM) -positive cervical carcinoma in situ foci than in situ foci tissue without lymph node metastasis (fig. 3). Meanwhile, the expression of miR-532-5p in HeLa and SiHa cells derived from orthotopic foci was significantly higher than that in MS751 cells derived from lymph node metastasis (FIG. 4).

Example 4 MiRNAs inhibit cervical cancer cell metastasis, lymphangiogenesis and epithelial-mesenchymal transition (EMT) in vitro

The miR-532-5P is taken as an example for further research, and other miR have similar conditions and are not repeated for brevity.

In order to further research the potential inhibition effect of miR-532-5p on lymph node transfer, SiHa and HeLa cell lines are selected for a function research experiment. Interference and overexpression efficiency were detected by qRT-PCR using the method of example 1 (figure 5).

First, whether miR-532-5p inhibits CC cell proliferation was investigated, since tumor size is positively correlated with tumor metastasis. Cell proliferation experiments (MTT) revealed that miR-532-5p significantly reduced cell proliferation capacity (FIG. 6).

miR-532-5p overexpression can obviously inhibit the invasion and metastasis capacity of CC cells (figure 7). In contrast, inhibition of miR-532-5p enhances cell invasion and migration ability (FIG. 8).

Furthermore, lymphangiogenesis is a key factor in tumor LNM. Compared with the corresponding control group cells, the CC cell culture supernatant over-expressing miR-532-5p can obviously inhibit HLEC tube forming capability (figure 9). Meanwhile, compared with the tissue with high expression of miR-532-5p, the density of D2-40 labeled lymphatic vessels in the CC tissue with low expression of miR-532-5p is higher.

In addition, it has been demonstrated that cancer cells that develop EMT acquire a greater capacity to spread via the lymphatic system. miR-532-5p overexpression results in significant down-regulation of vimentin and N-cadherin, and up-regulation of E-cadherin (FIG. 10).

Example 5 in vivo targeting of miRNA experiments

The humanized mouse tumor model is used for researching the function of the miR-532-5p in the cervical cancer xenograft. First, miR-532-5p overexpression reduced tumor growth in a subcutaneous xenograft model (fig. 11). In addition, tumor weights and volumes were significantly reduced in the miR-532-5p overexpression group (fig. 12).

Example 6 administration of miRNA in combination with FASN inhibitor (Orlistat )

Further research is carried out by taking miR-532-5p as an example, and similar situations exist in other miR, which is not repeated for brevity.

FASN is an interaction target of miR532-5p, and the expression of FASN can be remarkably inhibited by the over-expression of miR-532-5 p. Orlistat, as a FASN inhibitor, is an FDA approved antiobesity agent acting only on the gastrointestinal tract and inhibits the growth and metastasis of a variety of human cancers. Therefore, we evaluated the efficacy of Orlistat/miR-532-5p combination therapy using the lymph node metastasis nude mouse model (FIG. 13). The results prove that the nude mice receiving miR-532-5p overexpression treatment have low lymph node metastasis rate; moreover, the miR-532-5p overexpression and Orlistat combined treatment of the nude mice has more remarkable lymph node metastasis inhibition effect compared with the singly-used Orlistat or miR-532-5p overexpression treatment (figure 14). In addition, miR-532-5p overexpression combined with Orlistat treatment significantly reduced the rate of growth of subcutaneous tumors in nude mice (fig. 15).

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The pharmaceutical composition for inhibiting the lymph node metastasis of cervical cancer comprises a reagent for over-expressing miRNA and a fatty acid synthetase FASN inhibitor, wherein the miRNA is miR-532-5p, and the fatty acid synthetase FASN inhibitor is orlistat.

The application of the miR-532-5p overexpression reagent in the preparation of the medicine for inhibiting the metastasis of the cervical cancer lymph nodes is characterized in that the medicine further comprises a fatty acid synthase FASN inhibitor, and the fatty acid synthase FASN inhibitor is orlistat.