Long Non-coding RNA PTENP1 Functions As A Competing Endogenous RNA To Regulate PTEN Level In Gastric Cancer

ObjectiveRecent studies have shown that long non-coding RNAs(lncRNAs)play important roles in tumorigenesis and tumor progression via functioning as competing endogenous RNAs(ceRNAs).Pseudogene transcripts also belong to the category of lncRNAs.PTENP1,the pseudogene of PTEN tumor suppressor,has been found to exert its tumor suppressive impacts through regulation of PTEN expression in several malignancies.However,the expression and biological function of PTENP1 in gastric cancer remain exclusive.Our preliminary work demonstrated that PTENP1 and PTEN were concurrently downregulated in gastric carcinoma and showed a positive correlation,indicating a meaningful regulatory role between them.The aim of this study was to investigate and validate PTENP1 modulates PTEN expression via competitively binding to miR-106 b and miR-93 so as to suppress gastric carcinogenesis by carrying out the cellular and molecular biological assays,in other words,to confirm the existence and significance of PTENP1: miR-106b/miR-93: PTEN ceRNA in gastric cancer cells.Methods1.qRT-PCR was adopted to compare the PTENP1 level in different human gastric cancer cell lines(including AGS,SGC7901,MGC803 and BGC823)with in human gastric epithelial mucosa cell(GES-1).In addition,qRT-PCR and western blot were used to detect the expression of PTEN mRNA and protein in PTENP1 stable-overexpression cells constructed by lentivirus infection,repectively.2.For the PTENP1 over-expression cells,MTT assay was performed to evaluate cell proliferation,and annexin V/PC labeling via flow cytometry was used to examine apoptosis.Furthermore,uncoated and matrigel-coated transwell membranes were carried out to study the impacts of PTENP1 overexpression on cell migration as well as invasion.3.Microcosm Targets,TargetScan and miRanda bioinformatics software were used to identify the seed sequences that match for PTEN-targeting miR-106b~25 cluster(miR-106b?miR-25 and miR-93).This sequence was further validated to be available for targeting PTENP1 transcript by employing Clustal Omega.Through dual-luciferase reporter assay,we analyzed whether miR-106 b and mi R-93 directly regulates PTENP1 expression.4.In order to make it clear that whether PTENP1 regulates PTEN expression through sponging miR-106 b and miR-93(PTENP1:miR-106b/miR-93:PTEN ceRNA network)in gastric cancer cells,we employed a novel concentration gradient assay,in which a series of increasing amounts of the wild-type or mutant PTENP1 expression plasmid(pCDH-PTENP1 or pCDH-mutPTENP1)and a constant amount of miR-106 b or miR-93 mimics were co-transfected into gastric cancer cells.And then,PTEN mRNA levels were analyzed by qRT-PCR.Results1.The PTENP1 mRNA level in gastric cancer cells(MGC803 and BGC823)was significantly lower than that in human gastric epithelial mucosa cell(GES-1).Hence,we selected these two cell lines as the subjects for further study.We ectopically overexpressed PTENP1 in MGC 803 and BGC 823 and subsequently detected that the expression of PTEN mRNA and protein was remarkablely increased compared to the cells infected with empty plasmid(Lenti-NC).2.When PTENP1 upregulation occurred in vitro,MTT assay showed that cells growed relatively slower.Besides,Annexin V-FITC/PI experiment confirmed that overexpression of PTENP1 promoted both early and late apoptotic events in gastric cancer cells.Of course,the total apoptosis rate increased.And,the results of transwell experiment showed PTENP1 overexpression inhibited cell migration and invasion,consistent with the previous work that low PTENP1 level was related with more frequent lymph node metastasis.3.Bioinformatics and Clustal Omega analysis indicated that PTENP1 might be a potential target for miR-106 b and mi R-93,but not for miR-25.As was demonstrated by dual-luciferase reporter assay,miR-106 b and miR-93 mimics visibly reduced the luciferase activity of the reporter containing the wild-type 3'UTR of PTENP1,but had no effect on the mutant.4.The concentration gradient assay showed that the expression of PTEN mRNA increased with the ectopic PTENP1(pCDH-PTENP1)increased,presenting a linear dose-dependent pattern,while PTEN mRNA level was not affected by the increasing amounts of the mutant vector(pCDH-mutPTENP1).Conclusions1.After stable over-expression of PTENP1 in vitro,the PTEN expression upregulated at both the mRNA and the protein level in gastric cancer cells.At the same time,PTENP1 overexpression inhibited cell growth,migration and invasion,but induced apoptosis.Accordingly,PTENP1 played a tumor suppressive role in gastric cancer cells by modulating the expression of PTEN.2.The dual luciferase reporter gene assay and the concentration gradient experiment proved that PTENP1 sponged miR-106 b and mi R-93 thus resulting in the upregulation of PTEN expression.Consequently,the PTENP1:miR-106b/miR-93:PTEN ceRNA network does exist in gastric cancer cells.3.The cellular and molecular biological assays demonstrated that PTENP1 functioned as a competing endogenous RNA to exert its tumor suppressive impacts in gastric cancer cells via the PTENP1:miR-106b/miR-93:PTEN ceRNA network.