The Mechanism Of Exosomal MiR-335-3p In Cancer-associated Fibroblasts Affecting The Cisplatin Sensitive In Ovarian Cancer

ObjectiveOvarian cancer(OC)is a gynecological malignant tumor with the highest mortality rate.Drug resistance is the bottleneck of ovarian cancer treatment.Recent studies have shown that in addition to the genetic changes of tumor cells,the local microenvironment also plays an important role in the sensitivity of tumor cells to chemotherapy drugs.Cancer associated fibroblasts(CAFs),most of it came from the activation of normal fibroblasts(NFs),as one of the most important mesenchymal cells in the tumor microenvironment,can regulate the occurrence of tumor drug resistance through the interaction of exosomes and tumor cells.Micro RNAs(miRNAs)are non-coding small RNA molecules that play an important role in the occurrence,development and chemotherapy resistance of various tumors.Studies have shown that low expression of miR-335-3p plays a tumor suppressing role in various tumors.But the role of miR-335-3p in ovarian cancer is unclear.The purpose of this study was to investigate the role of exosomal miR-335-3p by CAFs on ovarian cancer cells in cisplatin resistance.Method(1)CAFs and NFs were isolated and cultured from OC tissues and normal ovarian tissues,and the exosomes of their culture supernatants were extracted and co-cultured with ovarian cancer cells SKOV3,respectively.CCK8 was used to detect the changes in SKOV3 cell activity under cisplatin.At the same time,flow cytometry was used to detect the effect of exosomes on apoptosis of SKOV3 cells under cisplatin,and to detect the mRNA expression levels of apoptosis-related proteins and drug resistance-related proteins.(2)Detecting and comparing the expression level of miR-335-3p in the above two exosomes by qRT-PCR;(3)Using CCK8 to detect the cisplatin sensitivity of SKOV3 cells after co-culture with the CAFs overexpressed of miR-335-3p;at the same time,using qRT-PCR to detect ?-SMA and FAP mRNA expression levels in CAFs overexpressed of miR-335-3p;(4)Using fluorescent labeling method to detect whether SKOV3 cells take up miR-335-3p through the exosomal pathway.(5)Using CCK8 to detect the effect of cisplatin sensitivity and flow cytometry to detect the apoptosis of SKOV3 cells after transfecting miR-335-3p mimic.(6)Using database to predict the downstream target gene of miR-335-3p may be ERH,the qRT-PCR and Western blot(WB)to analyze whether miR-335-3p affects the expression level of ERH in SKOV3 cells;and verify with dual luciferase experiment.(7)Selecting the surgically resected primary epithelial ovarian cancer(EOC)tissues and using immunohistochemical staining to detect the expression level of ERH protein,and verifing with WB and qRT-PCR.Besides,analyze the relationship between the expression level of ERH and the clinicopathological characteristics of EOC patients.Using CCK8 detects whether miR-335-3p regulates the cisplatin sensitivity of SKOV3 cells to by inhibiting ERH expression.Results1.Fibroblasts isolated and cultured from OC tissues highly express ?-SMA and FAP.The cisplatin sensitivity of SKOV3 cells co-cultured with CAFs/CAFs-exo was significantly lower than that with NFs/NFs-exo.The CAFs-exo significantly inhibited the apoptosis and increased the expression level of resistance-related protein mRNA and decreased the apoptosis-related protein mRNA of SKOV3 cells.2.The expression level of miR-335-3p in exosomes secreted by CAFs was significantly lower than NFs.Cell function test results show that CAFs transfected with miR-335-3p mimic can increase the cisplatin sensitivity of SKOV3 cells,and the expression levels of ?-SMA and FAP are also significantly reduced in CAFs.3.Fluorescent labeling results showed that miR-335-3p was transferred from CAFs to SKOV3 cells through exosomes.4.Transfection of miR-335-3p mimic can reduce the expression level of ERH in SKOV3 cells.In addition,overexpression of ERH can partially reverse the increased cisplatin sensitivity caused by miR-335-3p mimic of SKOV3 cells.Dual luciferase results show that miR-335-3p can specifically regulate the expression of ERH.5.The expression level of ERH is related to the poor prognostic factors of EOC patients.Interfering the expression of ERH can significantly increase the cisplatin sensitivity and promote apoptosis in SKOV3 cells.ConclusionIn this study,we found that exosomes secreted by CAFs pass miR-335-3p to ovarian cancer cells,and miR-335-3p can enhance the sensitivity to cisplatin by targeting ERH in SKOV3 cells.It reveals the new mechanism of CAFs regulating cisplatin resistance of ovarian cancer cells in the tumor microenvironment,and provides a theoretical basis for targeting CAFs to increase the cisplatin sensitivity of ovarian cancer cells.