The Long Non-Coding RNA KCNQ1OT1 Promotes The Progression And Chemoresistance Of Small Cell Lung Cancer Via Regulating TGF-β1-Mediated Epithelial-Mesenchymal Transition

Background and purpose:The latest statistics show that the number of new lung cancer cases in China is as high as 828,100,and the number of deaths is 657,000,which is the type of cancer with the largest number of deaths in China.Small cell lung cancer(SCLC)accounts for about 15%of all lung cancers and is the most malignant subtype of lung cancer,with a five-year survival rate of less than 2%.SCLC is poorly differentiated and progresses rapidly,and more than 70%of patients are in extensive stage when diagnosed.Therefore,chemotherapy is the first-line clinical treatment for SCLC.Although SCLC is extremely sensitive to chemotherapy drugs at the time of initial treatment,most patients achieve objective response after 2-4 cycles of chemotherapy,and the objective response rate(Objective Response Rate,ORR)can reach 70-80%,SCLC patients are prone to recurrence and metastasis due to chemotherapy resistance in a short period of time,which eventually leads to the patient’s mortality.Therefore,it is quite urgent to clarify the underlying mechanisms that induce SCLC to acquire chemoresistance and find effective therapeutic targets,expecting reverse the chemosensitivity of patients with drug-resistant SCLC to prolong the overall survival time.KCNQ1OT1 is a long non-coding RNA(lncRNA)with a base sequence length of 9.1kb,which has been confirmed to be abnormally highly expressed in a variety of tumors,such as colon cancer,breast cancer,and cervical cancer,and participate in the regulation of malignant progression of those tumors,including tumor proliferation,apoptosis,migration,invasion and chemotherapy resistance.However,the regulatory function and molecular mechanism of KCNQ1OT1 in SCLC are still unclear.Our previous study found that LncRNA KCNQ10T1 was significantly highly expressed in acquired chemotherapy-resistant SCLC cell lines through microarray screening.In our recent study,we aim to explore the expression level of lncRNA KCNQ1OT1 in SCLC tissues and cell lines,the correlation between KCNQ10T1 and clinicopathological characteristics of patients with SCLC,the impact of KCNQ1OT1 on SCLC chemotherapy resistance and the possible underlying mechanisms,and the value of KCNQ1OT1 as a prognostic biomarker for SCLC and the possibility as a therapeutic target for SCLC.Methods:The lncRNA KCNQ1OT1,which is highly expressed in chemotherapy-resistant SCLC,was selected as the follow-up research molecule by analyzing the lncRNA microarray in our previous research;clinical tumor samples and normal lung tissue samples of patients with SCLC were collected,and RT-qPCR method was used to analyze the differential expression of KCNQ1OT1 in SCLC and normal lung tissue,then analyze the association between KCNQ1OT1 expression and clinical characteristics,as well as survival prognosis of patients;we then use RNAi technology and lentiviral vector to transiently and stably knock down the expression of KCNQ1OT1 in SCLC cells,the role of KCNQ1OT1 in proliferation and apoptosis of SCLC was assessed by CCK8,flow cytometry,clonogenicity and mice subcutaneous tumor models;on the basis of knockdown of KCNQ10T1 expression,wound healing and transwell assays were used to evaluated the effect of KCNQ10T1 on the migration and invasion ability of SCLC cells;functionally,the effect of KCNQ1OT1 on the chemotherapeutic responsiveness of SCLC was evaluated by CCK8,flow cytometry and mice subcutaneous tumor model.In order to explore the potential mechanism of KCNQ1OT1 regulating the malignant progression of SCLC,next-generation sequencing technology was used to analyze the changes of transcriptome profile after knocking down the expression of KCNQ1OT1 in SCLC cells,and the differentially expressed genes were enriched by function and signal transduction pathway.Finally,RT-qPCR and Western blotting assays were used to explore the regulatory effect of KCNQ1OT1 on TGF-β/EMT signaling axis.Results:Microarray results showed that the expression of KCNQ1OT1 was up-regulated in chemotherapy-resistant SCLC cell lines;the expression of KCNQ1OT1 in SCLC tissues was significantly upregulated than that in normal lung tissue.High KCNQ1OT1 expression predicts poor survival prognosis in patients with SCLC;both in vitro and in vivo experiments show that knocking down KCNQ1OT1 expression can inhibit the proliferation of SCLC,promote cell apoptosis,inhibit the migration and invasion ability of SCLC cells,and sensitize SCLC cells to chemotherapy drugs.Mechanistically,knockdown of KCNQ1OT1 was found to inhibit the expression of TGFB1,SMAD2,SMAD3,TWIST1,ZEB2,and VIM,while promote the expression of epithelial cell marker CDH1.However,after the intervention of exogenous TGF-β1 in SCLC,the expressions of SMAD2,SMAD3,TWIST1,ZEB2,and VIM were up-regulated,and the expression of epithelial cell marker CDH1 was decreased.Conclusion:The expression of KCNQ10T1 in SCLC tissue is significantly higher than that in normal lung tissue,and the expression KCNQ10T1 in chemotherapy-resistant cell lines is higher than that in chemotherapy-sensitive cell lines.The high expression of KCNQ10T1 indicates poor prognosis in patients with SCLC;KCNQ1OT1 might affect the TGF-β/EMT signaling axis to regulate the malignant progression of SCLC,including the regulation of proliferation,apoptosis,migration,invasion and chemotherapy resistance of SCLC;Inhibition of KCNQ1OT1/TGF-β/EMT signaling axis may act as a new therapeutic target for SCLC.