The Role And Mechanism Of LncRNA KCNQ1OT1 In Promoting Autophagy Mediated Radiation Resistance In Lung Cancer Cells

In recent years,the number of lung cancer cases caused by various reasons has gradually increased.At present,radiotherapy is the most important treatment for lung cancer.However,there is evidence that about 16%of patients still fail due to local recurrence[1].Therefore,the treatment failure caused by radiotherapy resistance becomes the bottleneck of improving the curative effect of lung cancer.Studies have shown that radiation therapy can directly or indirectly induce cell stress to lead to the death of cancer cells,but a small number of cancer cells may survive due to activation of intracellular damage repair signal.After radiotherapy,the surviving cancer cells show radiation resistance and can promote tumor recurrence[2-3].Therefore,it is an urgent problem to understand the molecular mechanism of radiotherapy resistance of lung cancer and to find the potential target of radiotherapy sensitization.With the discovery of long-chain non coding RNA(lncRNA),we have realized that lncRNA is a kind of non coding transcripts with a length of more than 200nt,which is abnormally expressed in a variety of cancer types,and directly or indirectly participates in the regulation of target genes from the level of transcription and translation in various ways[4].Previous studies have confirmed that lncRNA KCNQ10T1 is highly expressed in lung cancer,colorectal cancer,breast cancer and ovarian cancer,and that it is closely related to the treatment resistance and poor prognosis of tumor[5].Autophagy is a highly conservative metabolic cycle process in cells.It has been proved that protective autophagy in cancer cells can recover the damaged organelles by decomposition,limit the killing effect of radiotherapy on cancer cells,and play an important role in the radiotherapy resistance and recurrence of tumor[6].Therefore,further understanding of the mechanism of IncRNA KCNQ1OT1 and autophagy in the radiotherapy resistance of lung cancer is expected to provide a new idea for the radiotherapy sensitization in the clinical treatment of lung cancer.Based on the above research background,we plan to carry out the following research to further explore.[Objective]:to analyze the relationship and mechanism between IncRNA KCNQ1OT1 and autophagy and radiation resistance of lung cancer cells,and screen downstream factors regulated by KCNQ1OT1[Method]:(1)A549 cells of lung cancer were subcultured,and A549 cells were screened by radiotherapy.A549 cells with resistance to radiotherapy were isolated and cultured,named A549/IR.The radiosensitivity of A549/IR cells was evaluated by clone formation experiment.The expression level of IncRNA KCNQ1OT1 in A549/IR cells and their parents A549 cells was detected by qPCR.LncRNA was located by fluorescence in situ hybridization Expression of KCNQ1OT1 in A549/IR.(2)The expression of lncRNA KCNQ1OT1 in A549/IR cells was interfered by shRNA technology.The control group(A549/IR-NC)and the down-regulation group(A549/IR-KCNQ10T1-shRNA)were set up.The effect of down-regulation of KCNQ1OT1 expression on the radiosensitivity of a549ir cells was detected by in vitro and subcutaneous tumorigenesis experiments in nude mice The relationship between the expression of KCNQ1OT1 and radiotherapy resistance of lung cancer A549/IR cells;the effect of down-regulation of KCNQ1OT1 on the expression of autophagy related proteins lc3b and p62 was detected by Western blotting;the number of autophagy bodies in the cells after down-regulation of KCNQIOT1 was observed by transmission electron microscopy,and the relationship between the radiosensitivity mediated by KCNQ1OT1 and the level of autophagy was analyzed.(3)The second generation sequencing technology was used to detect the expression of IncRNA KCNQ1OT1 in A549/IR cells.Compared with the control group,the changes of miRNA expression profile in A549/IR cells were analyzed.The possible signal pathways and biological functions of the differentially expressed miRNA in the radiosensitivity regulation were analyzed.The possible miRNA targets involved in the regulation were screened and the IncRNA was predicted KCNQ1OT1 may mediate the downstream molecular mechanism of radiosensitivity of lung cancer cells.[Results]:(1)The results of qPCR showed that IncRNA in A549/IR cells could be used as the model of radiotherapy resistance The expression of KCNQ10T1 in A549/IR cells was significantly up-regulated compared with that in the control group.The results of clonogenesis showed that the surviving A549/IR cells had stronger clonogenesis ability(P<0.05)than that of their parent A549 cell lines.The results of fish showed that IncRNA KCNQ1OT1 in A549/IR cells was located in the cytoplasm.(2)After stable transformation of A549/IR cells by lentivirus,the control group(A549/IR-NC)and KCNQ10T1 down-regulated group(A549/IR-KCNQ10T1-shRNA)cells were constructed.The results of qPCR showed that KCNQ10T1 in A549/IR cells was significantly down-regulated.The results of clonogenesis experiment showed that after down regulating the expression of KCNQ10T1,the proliferation and tumorigenicity of A549/IR cells decreased and radiosensitivity increased The results of blotting showed that the expression of lc3b in A549/IR-KCNQ10T1-shRNA group was lower than that in A549/IR-NC group,and the expression of p62 in A549/IR-KCNQ10T1-shRNA group was higher than that in A549/IR-NC group;the number of autophagic bodies in A549/IR-KCNQ10T1-shRNA group was less than that in A549/IR-NC group;in the subcutaneous tumorigenesis experiment of nude mice,the ability of tumor formation and proliferation decreased significantly,and the sensitivity to radiotherapy increased in A549/IR-KCNQ10T1-shRNA group(P<0.05).The above results showed that the expression of KCNQ10T1 was positively correlated with the autophagy level of A549/IR cells.(3)The results of miRNA sequencing showed that there were 347 differentially expressed miRN As in A549/IR-KCNQ1OT1-shRNA group compared with A549/IR-NC group,with 1.5-fold difference as the threshold,including 157 up-regulated miRNAs,190 down regulated miRNAs,and KEGG pathway enrichment analysis showed that the differentially expressed miRNAs were mainly involved in autophagy regulation,signal transmission in tumor,non-small cell lung cancer and other related signal pathways.[Conclusion]:1.A549/IR cells with resistance to radiotherapy can be isolated after high-dose irradiation of parent A549.The proliferation ability of these cells is stronger than that of normal A549 cells.2.The expression of lncRNA KCNQ1OT1 in A549/IR cells was higher than that in A549 cells,suggesting that KCNQ1OT1 might mediate the radiosensitivity of A549/IR cells.3.Down regulating the expression of lncRNA KCNQ1OT1 in A549/IR cells can inhibit the level of protective autophagy and proliferation of A549/IR cells,enhance the effect of radiation-induced cell death,and improve the radiosensitivity.It shows that lncRNA KCNQ1OT1 is a key factor in the regulation of radiotherapy resistance in A549/IR cells,and it is positively correlated with the level of autophagy to mediate the radiosensitivity of A549/IR cells.4.LncRNA KCNQIOT1 is located in the cytoplasm of A549/IR cells,which may play the role of miRNA molecular sponge to competitively inhibit miRNA and thus play a regulatory role in mRNA expression.In A549/IR cells,the miRNA profile of differential expression,especially the miRNA with significant multiple of differential expression,was down regulated by lncRNA KCNQIOTl,which may be the key molecule involved in the process of radiotherapy resistance of lung cancer A549/IR cells mediated by autophagy.In this study,we investigated the possible molecular mechanism of radiation resistance in lung cancer cells from the perspective of lncRNA mediated autophagy regulation KCNQIOT1 is an important regulatory factor of radiotherapy resistance in lung cancer.KCNQ1OT1,which is highly expressed in A549/IR,is resistant to radiotherapy by enhancing the level of autophagy in cells,and the miRNAs that may be involved in the regulation are preliminarily screened out by miRNA sequencing technology.The above results provide some new clues to improve the sensitivity of radiotherapy of lung cancer cells with lncRNA KCNQ1OT1 as the entry point.