CircESRP1 Modulates The Chemoresistance Of Small Cell Lung Cancer By Affecting TGF-β Signaling Pathway

BackgroundLung cancer is the leading cause of death for cancer patients in China,which accounts for approximately one sixth of the world’s cancer death.Small cell lung cancer(SCLC),taking up 15%to 25%of all lung cancers and chemotherapy,is the main clinical treatment method for SCLC.However,the multidrug resistance of chemotherapy drugs often occurs in the course of treatment for SCLC,which seriously affects the prognosis of cancer patients.Therefore,it is urgent to study the mechanism of drug resistance in SCLC and make certain how to reverse drug resistance.Circular RNAs(circRNAs),a kind of special RNA,are characterized by covalent closed-loop structure.In the past five years,more and more studies on circRNAs have made its role in human diseases become increasingly clear.Many studies have shown that circRNAs are highly conservative in species,It is due to their conservativeness,abundance variability and tissue specificity that circRNA may be used as a molecular marker of some diseases,including cancer.At the same time,circRNAs play an important role in gene regulation,which can regulate the expression of mRNA at multiple levels and thus affect the occurrence and development of diseases.Many evidences show that circRNAs not only affect the proliferation,invasion and metastasis of cancer,but also mediate the resistance to radiotherapy and chemotherapy of cancer.cESRPl is a circRNA derived from ESRP1 gene locus with 287 bp length.Previous gene chips showed that cESRPl was significantly down-regulated in SCLC chemotherapeutic resistant cell line H69AR compared with chemotherapeutic sensitive cell line H69.Our previous studies have confirmed that up-regulation of cESRPl can inhibit the growth of cancer cells and reverse the resistance of cancer cells to chemotherapeutic drugs.TGF-β is a multipotent cytokine,which plays a key role in many physiological processes,including regulating the balance between embryogenesis and tissue development.Abnormal TGF-β signal transduction is also associated with various diseases,including cancer.In normal epithelial cells,TGF-β,as a kind of tumor suppressor,triggers an effective cell inhibition response and then inhibits the growth of tumors.On the contrary,in the course of cancer progression,the tumor suppressive function of TGF-β is lost,and in some advanced cancers,TGF-p becomes a carcinogenic factor which can promote the proliferation,invasion,metastasis and chemotherapy resistance of cancer cells.ObjectivesThrough a series of in vivo and in vitro experiments,we analyzed and explored the possible mechanism of cESRPl involvement in chemotherapy resistance of small cell lung cancer.Furthermore,we constructed a patient-derived xenografts(PDXs)model and successfully induced drug-resistant PDXs model to verify the mechanism of cESRPl in SCLC chemotherapy resistance,enriched the molecular mechanism of circRNAs in SCLC growth and chemotherapy resistance,and provided scientific basis for finding molecular markers and targets of SCLC chemotherapy resistance.Materials and methods(1)RIP experiments showed that cESRPl could bind to the RISC(Ago2)complex,confirming that cESRPl had the sponge function of microRNAs and participated in the mechanism of ceRNA(competitive endogenous RNA).(2)The combination of miR-93-5p and cESRPl was predicted by Gene database.RNA Pull down test,luciferase test and FISH test showed that cESRPl could directly act on miR-93-5p,indicating that the combination of cESRPl and mir-93-5p could play active role.(3)Flow cytometry,CCK8 susceptibility test and nude mouse tumor formation experiment were used to prove that up-regulation of cESRPl could reverse the drug resistance of SCLC by regulating miR-93-5p.(4)It demonstrated that miR-93-5p could target tumor suppressor Smad7 and p21 by predicting on bioinformatics website and referring to the literature.We firstly verified that the transcription and translation levels of Smad7 and p21 in drug-resistant H69AR were significantly down-regulated by fluorescent quantitative PCR and Western blot.Then,Transfection of mimics or inhibitor of miR-93-5p demonstrated that miR-93-5p could negatively regulate smad7 and p21.Further up-and down-regulation of cESRP1 showed that cESRP1 could positively regulate Smad7 and p21 through combining miR-93-5p.(5)The Previous study showed that TGF-(3 might be involved in drug resistance of SCLC through EMT.Western blot showed that TGF-β has a negative feedback regulation effect on Smad7 and p21,and the expression of EMT markers was affected at the same time,which suggested that Smad7 and p21 could affect the EMT pathway induced by TGF-β pathway.The luciferase assay showed that the cESRPl-miR-93-5p-p21/Smad7 axis was involved in the TGF-β signaling pathway.(6)PDX model were used to show that blocking the TGF-β signaling pathway could increase the sensitivity of SCLC to chemotherapeutic drugs.(7)qRT-PCR and immunohistochemical techniques were used to study the relationship between cESRPl and p21 and Smad7 in clinical tissues of SCLC.Fluorescence in situ hybridization(FISH)and immunohistochemical techniques were used to further study the relationship between circESRP1 and the expression of microRNA-93-5p,Smad7 as well as p21 and the effect of chemotherapy and prognosis of patients.Results(l)cESRP1 can play a competitive sponge role of microRNA-93-5p in SCLC.(2)Downstream of mir-93-5p:Smad7 and p21,were differentially expressed in sensitive and drug-resistant SCLC cell lines.(3)cESRP1 can regulate the expression of Smad7 and p21 through combining mir-93-5p.(4)TGF-β can negatively regulate the cESRPl-mir-93-5p-p21/smad7 axis.(5)Blocking TGF-β signaling pathway can increase the sensitivity of SCLC to chemotherapeutic drugs.(6)CESRP1 can affect the chemotherapy effect and prognosis of SCLC patients by regulating the p21/Smad7 TGF-β-EMT signaling pathway.ConclusionCESRP1 is involved in chemotherapeutic resistance of SCLC through regulating TGF-β signaling pathway by competitively binding to miRNA-93-5p.