| BackgroundCervical cancer is the most common gynecological cancer in China. With the changing of people’s sexual attitudes and sexual behavior, the onset age of cervical cancer showed younger trend. There are a large number of new cases and deaths from cervical cancer each year, even though cervical cancer screening program are implemented in the growing popularity. It is clear that persistent high risk human papilloma virus (HPV) infection is the main cause of cervical cancer, but HPV vaccine have not yet approved to prevent cervical cancer in mainland China. Even if HPV vaccine were approved, in order to effectively reduce the incidence of cervical cancer through mass HPV vaccination, it is estimated to need at least 20 years, during which new high-risk HPV infection and cervical cancer cases will appear. With the young tendency of cervical cancer and delayed child-bearing age in Chinese women, a growing number of women are diagnosed with cervical cancer before completing child-bearing, leading more patients calling for fertility-preserving treatment. Current fertility preserving treatment is surgery, which may cause cervical stenosis, pregnancy complications such as miscarriage or premature birth. In addition, there are still a lot of patients are diagnosed with advanced cervical cancer every year, and some are less sensitive to chemotherapy, although the majority of cervical cancer patients are diagnosed at early stage. The reasons for ineffective treatment of cervical cancer is invasive uterine cervical cancer and adjacent tissueinvolved or even distant metastasis. Therefore, there is need of new ideas and methods for treatment of cervical cancer.MicroRNA is non-coding RNA of about 20 nucleotides, and could combine with 3’ untranslated region (UTR) of the target gene, causing degradation of mRNA or inhibitory protein translation. A variety of miRNA regulate expression of human genes and involve in the development and metastasis of cancer. The studies found that miRNA-146 is not only closely related to the inflammatory and immune diseases, and is closely related to various cancer such as prostate cancer, lung cancer. MiRNA-146a and miRNA-146b are highly homologous, belonging to the miRNA-146 family. MiR-146b-5p comes from miRNA-146b precursor by processing the arm of 5’end. The expression of miR-146b-5p were abnormally down-regulated in many malignant tumors, and overexpression of miR-146b-5p could inhibit tumor growth, invasion, and metastasis in vitro, suggesting that miR-146b-5p is a tumor suppressor gene. Studies have shown that transfection of miR-146a in cervical cancer cell CaSki could promote CaSki cell growth, suggesting that miR-146a is an oncogene. However, the role of miR-146b-5p in cervical cancer cell remains unclear. Therefore, this project was designed to explore the biological effects of miRNA-146b-5p on cervical carcinoma cell proliferation, cell cycle, invasion, telomerase activity and secretion of cytokines, and explore their mechanism preliminarily, providing insights and laboratory evidence for target therapy of cervical cancer.Part I:THE EFFEECTS OF MiR-146b-5p ON BIOLOGICAL BEHAVIORS IN HUMAN CERVICAL CANCER CELL LINE CASKIObjective:To explore the effects of miRNA-146b-5p in cervical cancer cell line Caski on biological behaviors such as cell proliferation, cell cycle, invasion, adhesion, telomerase activity and secretion of cytokines.Methods:(1) Select and confirm cells with stable overexpression of miRNA-146b-5p: Transfect recombinant plasmid pGPU6/Neo-miR-146b-5p into Caski cells using liposomes and select cells with stable overexpression of miRNA-146b-5p by G418, using real-time quantitative PCR to detect the relative expression of cells transfected with miRNA-146b-5p. (2) MTS were used to detect cell proliferation activity. (3) Transwell invasion model were used to detect the effect of miR-146b-5p on cell invasion. (4) Cell adhesion test was used to detect the effect of miR-146b-5p on cell adhesion. (5) Flow cytometry was used to detect the effect of miR-146b-5p on cell cycle. (6)ELISA was used to detect the effect of miR-146b-5p on secretion of cytokines TGF-β1, MCP-1, TNF-a. (7)TRAP-PCR-ELISA was used to detect the effect of miR-146b-5p on telomerase activity.Results:(1) Caski cells with stable expression of miR-146b-5p were selected, and expression of miR-146b-5p in transfected group was significantly increased. (2) Overexpression of miR-146b-5p inhibited cell proliferation activity of Caski. (3) Overexpression of miR-146b-5p reduced invasion of Caski cell. (4) Overexpression of miR-146b-5p reduced adhesion of Caski cell. (5) Overexpression of miR-146b-5p increased the proportion of Caski in GO/G1 cell cycle. (6) Overexpression of miR-146b-5p 5p inhibited the secretion of cytokines TGF-β1, MCP-1, TNF-a. (7) Overexpression of miR-146b-5p reduced telomerase activity of Caski cell.Conclusions:MiR-146b-5p could inhibited cell proliferation, invasion and adhesion, made cell cycle arrest in G0/G1 phase, inhibited the secretion of cytokines TGF-pi, MCP-1 and TNF-α, and also reduced telomerase activity of Caski cell, thus inhibiting cell growth of cervical cancer.Part Ⅱ:THE MECHANISM OF MiR-146b-5p ON BIOLOGICAL BEHAVIORS IN HUMAN CERVICAL CANCER CELL LINE CASKIObjective:To explore the effects of miRNA-146b-5p in cervical cancer cell line Caski on expression of CXCR4, MMP-2, MMP-9, c-Myc, cyclin D1, p27, p53 and HPV16 E6 as well as signal transduction pathway key factors such as phosphorylation of JNK, Akt and NF-κB/STAT pathway, thus preliminarily exploring the mechanism of that miR-146b-5p effected biological behaviors.Methods:(1) The mRNA and protein expression of CXCR4, MMP-2, MMP-9, c-Myc, cyclin D1, p27, p53 and HPV16 E6 were detected using real-time PCR and Western blot. (2) Phosphorylation of JNK and Akt were detected using Western blot. (3) Dual Luciferase Reporter Gene Assay Kit were used to detect transcriptional activity of NF-κB, STAT3and STAT5.Results:(1) Overexpression of miR-146b-5p reduced the expression of mRNA and protein of CXCR4, MMP-2, MMP-9, c-Myc, cyclinDl and HPV16 E6, increased the expression of mRNA and protein of p27and p53. (2) Overexpression of miR-146b-5p reduced phosphorylation of JNK and Akt in Caski cell. (3) Overexpression of miR-146b-5p reduced transcriptional activity of NF-κB, STAT3and STAT5 in Caski cell.Conclusions:MiR-146b-5p downregulated the expression of CXCR4, MMP-2, MMP-9, c-Myc, cyclinD1 and of HPV16 E6 and upregulated p27and p53 via JNK, Akt and NF-κB cell signaling transduction pathway, thus affecting biological behaviors of Caski such as cell proliferation, cell cycle, invasion, adhesion, telomerase activity and secretion of cytokines. |
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