Long Non-coding RNA PVT1 Promotes Malignancy In Human Endometrial Carcinoma Stem Cells Through Negative Regulation Of MiR-195-5p

Objective: Human endometrial carcinoma is one of the most common malignant tumors in the female reproductive system.Therapies for endometrial carcinoma include surgery,radiotherapy,and pharmaceutical(chemicals and hormones)treatment.At present,the most effective targeted treatment for endometrial carcinoma is still unclear and there has been much research into the molecular mechanism underlying the development of endometrial cancer.Here,we sought to determine whether the long-chain noncoding RNA,PVT1,can regulate the malignant behaviors of endometrial carcinoma stem cells(ECSCs);we,then further analyzed the possible molecular mechanism of PVT1 action.We determined that,PVT1 down-regulates mi R-195-5p to promote the expression of fibroblast growth factor receptor 1(FGFR1)and fibroblast growth factor 2(FGF2)and facilitate the malignant behaviors of ECSCs.Methods: First,the expression of PVT1 and mi R-195-5p in human endometrial carcinoma tissues was detected using real-time PCR(RT-PCR)and fluorescence in situ hybridization(FISH),and their expression in clinical samples of endometrial carcinoma were analyzed.Next,the binding sites and effects of expression of PVT1 on mi R-195-5p were detected using a dual-luciferase reporter gene system.The human endometrial carcinoma cell lines Ishikawa,HEC-1A,and RL95-2 were cultured and then Ishikawa-ECSCs were extracted via serum-free suspension culture.Then,the expression of stem cell-related factors was detected by flow cytometry,immunofluorescence,and RT-PCR.The proliferation capability as well as the migration and invasion capabilities of ECSCs were detected separately using Cell Counting Kit 8 & Ed U assays and a Transwell assay;the effects of ECSCs on the growth of endometrial tumors transplanted into nude mice was also evaluated.The expression of PVT1 and mi R-195-5p in ECSCs was detected by RT-PCR.Cell lines with either overexpressed and silenced PVT1 and mi R-195-5p were constructed by cell transfection;then,changes in the proliferation,migration,and invasion capabilities of cells,cell cycle progression,and apoptosis capability were detected by CCK-8 assay,Transwell assay,and flow cytometry,respectively.Finally,the binding sites and effects of mi R-195-5p/FGFR1 and mi R-195-5p/FGF2 expression were detected using dual-luciferase reporter gene system.After PVT1 overexpression and silencing,changes of mi R-195-5p expression were detected by RT-PCR,and the changes in the expression of FGFR1,FGF2,PI3 K,p-AKT1,AKT1,p-Erk ?,Erk ?,p-p38 MAPK and p38 MAPK by Western blot.The effects of PVT1 and mi R-195-5p on the proliferation tumors implanted into nude mice were also measured.Results: PVT1 expression was significantly up-regulated in clinical human endometrial carcinoma tissues and ECSCs.ECSCs were isolated from the endometrial carcinoma cell line Ishikawa by serum-free suspension culture;these ECSCs had stronger proliferation,migration,and invasion capabilities than Ishikawa cells.In addition,ECSCs demonstrated a stronger proliferation capability and higher expression of stem cell-related factors(CD44,CD133,Oct4,Sox2 and Nanog)in the nude-mouse tumor transplantation experiment.Silencing of PVT1 markedly inhibited the proliferation,migration,and invasion capabilities of ECSCs,restrained cell cycle S phase arrest,and promoted apoptosis.mi R-195-5p expression was down-regulated in human endometrial carcinoma tissues and ECSCs.Overexpression of mi R-195-5p had the same effects as silencing PVT1.PVT1 and m IR-195-5p were shown to bind to each other specifically.Silencing of PVT1 decreased expression of FGFR1 and FGF2 by negatively regulating mi R-195-5p,thus controlling the expression of FGFR1,FGF2,PI3 K,p-AKT1,AKT1,p-Erk ?,Erk ?,p-p38 MAPK and p38 MAPK and inhibiting the malignant behaviors of ECSCs.The combination of silencing PVT1 and overexpressing mi R-195-5p suppressed the growth of endometrial tumors transplanted in nude mice.Conclusion: 1.PVT1 and mi R-195-5p are endogenously expressed in human endometrial carcinoma tissues and ECSCs;PVT1 expression is high,while mi R-195-5p expression is low.2.ECSCs can be isolated from the endometrial carcinoma cell line,Ishikawa,by serum-free suspension culture.They can express the pluripotent stem cell markers CD44,CD133,Oct4,Sox2,and Nanog,and have stronger proliferation,migration and invasion capabilities.They also have the ability to generate tumors in nude mice.By negatively regulating mi R-195-5p,PVT1 can promote the proliferation,migration and invasion of ECSCs,promote cell cycle S phase arrest,and inhibit apoptosis.3.mi R-195-5p can bind to the 3'UTR of both the FGFR1 and FGF2 genes.And by negatively regulating mi R-195-5p,PVT1 can thus regulate the expression of PI3 K,p-AKT1,AKT1,p-Erk ?,Erk ?,p-p38 MAPK and p38 MAPK.