ESRP1 Regulates CARM1 To Modulate Chemoresistance Of Small Cell Lung Cancer

BackgroundSmall cell lung cancer(SCLC)accounts for about 15%-20%of all lung cancer patients.It belongs to highly malignant neuroendocrine tumors.It has the characteristics of rapid progress,easy metastasis,and poor prognosis.At present,the clinical treatment method for SCLC is mainly chemotherapy,and the first-line chemotherapy scheme is cisplatin plus etoposide.Most patients with SCLC are very sensitive at the beginning of chemotherapy but will gradually develop resistance to chemotherapy.This is a major problem in clinical treatment of SCLC.Therefore,elucidating the mechanism of chemoresistance in SCLC will help to find new ways to solve chemoresistance.PurposePrevious research by our group has shown that circular RNA circESRP1 can inhibit the development of chemotherapy resistance in SCLC.Therefore,we speculate that the ESRP1 protein formed by translation of circESRP1 precursor mRNA can also participate in the regulation of chemoresistance in SCLC.We firstly discovered that the expression of ESRP1 in chemotherapy-resistant cells of SCLC was significantly lower than that of chemotherapy-sensitive cells by qRT-PCR and Western Blot experiments,and further verified the role of ESRP1 in chemoresistance of SCLC by in vivo and in vitro experiments.Then predicted the downstream targets of ESRP1 splicing regulation through biological information to analyze the molecular mechanism of ESRP1 in the regulation of chemoresistance in SCLC,and to provide new clues for the discovery of new molecular markers and therapeutic targets for SCLC chemoresistance.Materials and methods1.The qRT-PCR and Western Blot experiments were used to analysis and compare the expression of ESRP1 in SCLC chemoresistant cells(H69AR and H446DDP)and chemosensitive cells(H69 and H446).2.SCLC tumor tissue samples were collected to compare the expression differences of ESRP1 in SCLC tumor tissues and adjacent normal tissues by qRT-PCR and Western Blot experiments.3.The effects of ESRP1 on chemoresistance of SCLC in vitro were determined using CCK8 experiment,flow cytometry apoptosis and cell cycle arrest experiments.4.The effects of ESRP1 on chemoresistance of SCLC in vivo were determined by subcutaneous tumor formation experiments in nude mice.5.The biological information was used to analyze the types of alternative splicing events that ESRP1 can regulate.It was further determined through RIP experiments that ESRP1 could bind to CARM1 pre-mRNA.The rescue experiment proves that ESRP1 can participate in the regulation of chemoresistance of SCLC through alternative splicing of CARM1.6.Bioinformatics predicts that CARM1 can arginine methylate smad7 protein.The Co-IP assay was performed to verify that smad7 and CARM1 can be combined with each other.Western Blot experiments explored the effect of ESRP1/CARM1 axis on the TGF-β/smad signaling pathway.Results1.ESRP1 is lowly expressed in SCLC chemo-resistant cells and tumor tissues;The expression level of ESRP1 in SCLC tissues is positively correlated with the overall survival of patients.2.Overexpression of ESRP1 can inhibit chemoresistance of SCLC and induce apoptosis and G0/G1 cell arrest.Conversely,down-regulating ESRP1 can promote chemoresistance of SCLC and inhibit apoptosis and G0/G1 Phase cell arrest.3.Overexpression of ESRP1 can inhibit the growth of SCLC and promote the chemosensitivity of SCLC in vivo.4.ESRP1 regulates the alternative splicing of CARM1 and inhibits chemoresistance of SCLC by reducing CARM1FL transcript expression.5.CARM1 mediates arginine methylation of smad7 protein and affects TGF-β/smad signaling pathway activation.ConclusionESRP1 mediates the arginine methylation of smad7 protein by regulating the selective splicing of CARM1,inhibits the TGF-β/smad signaling pathway,and then promotes the chemosensitivity of SCLC.