The Role Of Hsa＿circ＿0006157 In Promoting Radioresistance Of Cervical Cancer Cells

Objective:Cervical cancer(CC)is the fourth most common cancer among women in the world and is a serious threat to women’s health.Radiotherapy is one of the main treatment modalities for cervical cancer.With the continuous development of radiotherapy techniques in recent years,the clinical outcome of cervical cancer has improved in some extent.However,the overall outcome for patients with advanced disease is still unsatisfactory.This is largely explained by the fact that some of the tumor cells have gradually become resistant to radiation due to mutation during the course of radiotherapy,which then leads to local recurrence or regional lymph node recurrence.Therefore,radioresistance is currently one of the major obstacles to the treatment of cervical cancer.Circumscribed RNA(circ RNA)can be involved in resistance to radiotherapy in many tumours,but there are few studies on whether and how circ RNA acts in resistance to radiotherapy of cervical cancer.In this study,we investigated the function and preliminary mechanism of circ RNA Hsa＿circ＿0006157 in radiotherapy resistance of cervical cancer in the uterus.This may provide a molecular marker for predicting the efficacy of radiotherapy in cervical cancer and a molecular target for radiotherapy sensitization,which is important for improving the outcome and quality of life of cervical cancer patients.Methods:(1)Establishment of cervical cancer radioresistant cell sublines and detection of their radioresistance properties: Hela-RR and Siha-RR were established by repeated and interrupted X-ray irradiation;the DNA damage markers and EMT-related markers were detected by plate clone formation assay,wound healing assay,Western blot and subcutaneous tumour formation assay in nude mice.The radioresistance properties of Hela-RR and Siha-RR were confirmed using plate clone formation assay,trauma healing assay,Western blot for DNA damage markers and EMT-related markers,and subcutaneous tumor formation assay in nude mice.(2)RNA high-throughput sequencing was used to map the differential circ RNA expression profile of hypopharyngeal cancer radioresistance,perform functional analysis of the differential circ RNA,and verify the expression of the differentially expressed circ RNA Hsa＿circ＿0006157 by q PCR,and further use Sanger sequencing and RNase R assay to detect the circ structure of Hsa＿circ＿0006157.0006157 by circular structure(3)Proliferation,migration,invasion,DNA damage repair and EMT-related protein expression in Hela-RR and Siha-RR cells after silencing of Hsa＿circ＿0006157 expression by RNA interference technology(RNAi)Results:1)Radiotherapy-resistant cell sublines Hela-RR and Siha-RR were successfully constructed from cervical cancer cell lines Hela and Siha,and showed stronger radioresistance in vitro and in vivo compared with Hela and Siha.2)RNA-seq revealed that Hsa＿circ＿0006157 was up-regulated in Hela-RR and Siha-RR.q PCR verified that Hsa＿circ＿0006157 was highly expressed in Hela-RR and Siha-RR;Sanger sequencing,RNase digestion assay showed that Hsa＿circ＿0006157 had a circular structure.3)RNA interference technology(RNAi)silenced the expression of Hsa＿circ＿0006157 expression,functional assays showed that knockdown of Hsa＿circ＿0006157 significantly attenuated the proliferation migration invasion ability,DNA damage repair of Hela-RR and Siha-RR,and reduced the expression levels of EMT-related proteins N-cadherin,EPCAM,Snail and Vimentin.Conclusion:1)The established cervical cancer radiotherapy-resistant cell sublines He La-RR and Siha-RR cells have a certain degree of radioresistance;further high-throughput sequencing and bioinformatics analysis methods were used to establish the differential expression of circ RNAs related to cervical cancer radiotherapy resistance spectrum;2)circ RNA Hsa＿circ＿0006157 is up-regulated in cervical cancer radiotherapy-resistant cells,and has the effect of promoting cervical cancer proliferation,migration,invasion and radiotherapy resistance.