Tumor Associated Macrophage-derived MicroRNA-10a Promotes Stemness Of Ovarian Cancer

Background:Ovarian cancer accounts for the highest mortality amongst all gynecologic malignancies due to its aggressive malignant biological behavior,as well as its high chemotherapy resistance and recurrence rate,which severely threatens women’s health globally.The stemness of cancer cells is recently regarded as a crucial factor for chemotherapy resistance and recurrence of ovarian cancer.Tumor associated macrophages(TAMs),the largest and the most important cell components of tumor microenvironment,are reported to be closely related to chemotherapy resistance of ovarian cancer.Therefore,we suggest that TAMs may promote the stemness of ovarian cancer cells.MicroRNAs(miRNAs)regulate target genes’expression by binding to their3’UTR region.Several studies have shown that miRNA can regulate the stemness of cancer cells by mediating the crosstalk between TAMs and cancer cells.By analyzing the TCGA database,we discovered that microRNA-10a(miR-10a)was related to ovarian cancer.In conclusion,we believe that TAMs in epithelial ovarian cancer may promote the cancer cell stemness through miR-10a.In the following study,we will prove these hypotheses,so as to further understand the tumorigenesis and development of ovarian cancer,as well as searching for potential theraputic targets.Method:In this study,we will collect ovarian cancer tissue and noncancerous ovarian tissue;culture ovarian cancer cell lines;analyze the relevant data of ovarian cancer in TCGA database using R language program;sort TAMs and ovarian cancer stem-like cells by flow cytometry sorting system;co-cultured ovarian cancer cells and TAMs obtained from ovarian epithelial cancer tissues;detect mi R-10a and stemness markers in cancer and noncancerous tissues,TAMs,cancer cells and co-cultured cells using qRT-PCR and fluorescence in situ hybridization.Results:We identified TAMs from epithelial ovarian cancer tissues and adjacent noncancerous tissues by flow cytometry sorting system,and found that epithelial ovarian cancer tissue contained much higher ratio of TAMs than that in adjacent noncancerous ovarian tissue.Then we co-cultured ovarian cancer cell line(SKOV-3)and TAMs obtained from cancer tissue,and subsequently examined the expression of cancer stemness markers SOX2 and OCT4.A significantly increased transcriptional level of these markers were detected in co-cultured SKOV-3,which proves that TAMs in ovarian cancer promotes the stemness of ovarian cancer cells.By analyzing the relevant data of ovarian cancer extracted from TCGA database,we found that miR-10a is negatively correlated with the overall survival of ovarian cancer patients.We further examined the miR-10a level using fluorescence in situ hybridization and qRT-PCR,and found that miR-10a was significantly higher in ovarian cancer tissue samples in contrast to noncancerous ovarian tissue samples.In addition,we detected the expression of miR-10a in TAMs of ovarian cancer and adjacent noncancerous tissues.Not to our surprise,a significantly elevated expression of miR-10a was discovered in TAMs obtained from ovarian cancer.miR-10a baseline is relatively low in SKOV-3,after co-culturing with TAMs extracted form ovarian cancer,mi R-10a level raised in SKOV-3.Then,we sorted ovarian cancer cells that were obtained from the co-culture system by stemness membrane marker CD44 using flow cytometry sorting system.Compared to CD44-cells,CD44~+cells display a much higher mi R-10a level,which indicates that miR-10a promotes ovarian cancer cell stemness.To sum up,TAMs in ovarian cancer express high level of miR-10a,and the miR-10a they express can later be transported to ovarian cancer cells.Last but not least,the up-regulation of miR-10a stimulates stemness of ovarian cancer cells.Conclusion:Tumor associated macrophage improves the stemness of ovarian cancer,miR-10a may play a role as the mediator of this pathway.