Investigation Of The Effect And Mechanism Of MiR-145on Regulating The Radiosensitivity Of Cervical Cancer

BackgroundCervical cancer is one of the worldwide most common malignant tumors amongwomen, and its morbidity ranks only second to breast cancer. According to statistics,China’s annual newly increased number of the infected reaches up to130,000, and the ageof those diseased women presents younger trend. It is definite now that the persistentinfection of high-risk human papilloma virus is the primary cause of cervical cancer. In theaspect of treatment, the continued perfection of radical hysterectomy and adjuvantradiotherapy and chemotherapy makes the5-year survival rate raised to about90%forearly patients of cervical cancer. But this cannot still bring obvious effects on thoseadvanced stages patients. Radiotherapy is the main therapy method of locally advancedcervical, but it usually generates resistance effect of tumor radiation, then tumor recurrenceand metastasis will be induced, which has a serious impact on radiation effect.miRNAs is a kind of small non-coding RNA with the length equal to20to22nucleotides. In recent years, studies found that abnormal expression of miRNAs plays animportant role in tumor occurrence and development, and it affects tumor cell proliferation,apoptosis, differentiation and metastasis etc. Besides, miRNAs is also closely related to thetumor treatment response, and a growing number of studies confirm multiple miRNAs canadjust the radiation sensitivity of tumor cells. Sift miRNAs which can predict radiationsensitivity, and study its function and mechanism in the process of radiation sensitivityadjustment, and it’s able to explain better the reason for the radiation resistance effect, tohelp to develop more efficient and less harmful individualized treatment plan for cancerpatients, and to provide a new thought for enhancing radiotherapy sensitization, it hasimportant clinical significance.Previous researches have verified that Hsa-miR-145-5p (miR-145) is an importanttumor suppressors which is down-regulated in many kinds of cancers. Simultaneously,miR-145is also a star molecule with high research value in miRNAs related toradiotherapy. Some studies have reported that the expression of miR-145is regulated byp53which as a guard genes plays tumor suppressor role of promoting cell cycle arrest andapoptosis when cell stress. What’s more, studies have found that the expression ofmiR-145increases obviously in both thymus tumor tissue of the radiation induced cancermouse model and prostate cancer cell after the radiostimulation.However, there’s no relevant research reports on correlation between miR-145andcervical cancer clinical pathological factors, and whether it can participate in cervical cancer radiotherapy sensitivity adjustment. In this study, combined with clinical samples,we firstly further studied the clinical significance of down-regulated miR-145expressionin cervical cancer. Then, the role of miR-145in radiation sensitivity was explored incervical cancer cell lines and mouse models.MethodsFirstly we collected cervical cancer tissues and adjacent non-tumor tissues of42clinical patients with cervical cancer. Then we used of RNA Isolation, inverse transcriptionand quantitative PCR technology to test the level miR-145presents in the tissues, andanalyzed the association between the expression level and cervical cancer clinicalpathological factors. Then, we selected the Me180cell lines which is sensitive toradiotherapy, and Hela&SiHa cell lines which are prone to be resistant to radiotherapy, inorder to examine the present differences of miR-145in cervical carcinoma cell lines ofdifferent radiotherapy sensitivity and the situation after radiation treatment. Besides, weused miRNA mimic to up-regulate miR-145in cervical cancer cells, and Cell CountingKit-8reagent, microplate reader to examine the cell viability of cervical cancer cells andchanges of multiplication capacity after treatment. Annexin V-FITC combined withPropidium Iodide(PI) double marking flow cytometry was used to test the apoptosis afterradiation treatment. We also built HeLa cell line nude mice transplanted tumor model, andinjected intratumorally miRNA agomir, exogenous overexpression of miR-145in tumors,combined with the radiation treatment, studied the effects of the radiation sensitivity ofmiR-145cervical cancer cells in vitro level. Finally, we used bioinformation prediction andmicroarray analysis filtering the most likely target genes of miR-145in cervical cancer,and do RT-qPCR and Western blot in the overexpression of miR-145cells, detectedchanges of mRNA and protein in HLTF, also maked immunohistochemical detection intransplanted tumor, analyzed the expression level of HLTF.ResultsmiR-145was significantly decreased in cervical cancer tissues, and down-regulatedexpression level was closely related to the advanced stages (ⅡA2-ⅣA), large size tumors,and moderate/poor differentiation of cervical cancer. miR-145expression level in theradiation sensitive cell of Me180was higher than that of radiotherapy resistance cellsHeLa, SiHa, and after radiotherapy of radiation resistance cells, the levels of miR-145alsoincreases accordingly. Over expressing miR-145could increase the cell apoptosis level,reduce cell survival rate and inhibit cell proliferation after radiotherapy. Injecting miR-145-agomir in the tumor could inhibit growth rate of tumor and enhance thesensibility of radiotherapy. According to bioinformation prediction and microarray analysis,we identified HLTF as the uppermost candidate target gene of miR-145. Then,experimental results showed that miR-145could inhibit the expression of HLTF.Conclusion1、miR-145was significantly decreased in cervical cancer tissues, anddown-regulated expression level was closely related to the advanced stages, large sizetumors, and moderate/poor differentiation of cervical cancer.2、miR-145could adjust and control the radiotherapy sensibility of cervical cancer inforward direction through promoting cell apoptosis and reduce the survival rate of cell.3、miR-145could inhibit the expression of HLTF, mir-145might play the function ofreducing the radiotherapy resistance of cancer cell through regulating HLTF.