Long Non-coding Rna Pvt1 Promotes Non-small Cell Lung Cancer Cell Proliferation Through Epigenetically Regulating Lats2 Expression

Background: Long non-coding RNAs(lncRNAs)are a novel class of transcripts with no protein coding capacity,but with diverse functions in cancer cell proliferation,apoptosis,and metastasis.The lncRNA PVT1 is 1716 nt in length and located in the chr8q24.21 region,which also contains the myelocytomatosis(MYC)oncogene.Previous studies demonstrated that MYC promotes PVT1 expression in primary human cancers.However,the expression pattern and potential biological function of PVT1 in non-small cell lung cancer(NSCLC)is still unclear.Objective: The aim of this study is to detect PVT1 expression in NSCLC and to evaluate the role and potential mechanisms of PVT1 in the development and progression of NSCLC.Methods:(1)The lncRNAs expression profiles of NSCLC patient from Gene Expression Omnibus(GEO)were analyzed.(2)Real-time quantitative PCR(qRT-PCR)assays was performed to detect the expression of PVT1 in NSCLC tissues and cells.(3)MTT and clone formation assays were used to evaluate the viability of NSCLC cells transfected with si-PVT1.(4)Nude mouse experiment was performed to observe the effect of A549 cell transfected with si-PVT1 on tumor ability in vivo.(5)QRT-PCR and western blotting assays were used to explore the downstream target genes of PVT1 in NSCLC cells.(6)RIP and CHIP assays were performed to evaluate the potential mechanism that PVT1 how to regulate the downstream target gene.(7)The biological effect of LATS2 on NSCLC cells were explored by MTT,clone formation and flow cytometry analysis.(8)Western blotting experiment was conducted to study the downstream molecular pathway of LATS2.Results:(1)We analyzed the profiles of NSCLC patient from Gene Expression Omnibus(GEO)and found that PVT1 was upregulated in NSCLC tissues.(2)The results revealed that PVT1 expression was upregulated in NSCLC tissues and cells by qRT-PCR assay.Statistical analyses revealed that PVT1 expression levels in NSCLC were significantly correlated with tumor size,advanced pathological stage,and lymph node metastasis,also were correlated with poor prognosis of NSCLC.(3)MTT and colony formation assays demonstrated that growth of NSCLC cells transfected with si-PVT1 was attenuoated compared with control cells.Flow cytometry analysis revealed that knockdown of PVT1 expression induced apoptosis and induced cell cycle arrest at G1–G0 phase in NSCLC cells.(4)The results of nude mouse experiment indicated that knockdown of PVT1 expression could suppress tumor growth in vivo.(5)The results of western blotting and qRT-PCR assays revealed that LATS2 expression at protein and m RNA levels were increased in si-PVT1-transfected cells.(6)RIP and CHIP assays demonstrated that PVT1 could recruit PRC2 complex to the promoter of LATS2 and inhibited transcription.(7)MTT and colony formation assays of ectopically expressed LATS2 in NSCLC cells demonstrated that the NSCLC cell viability was inhibited dramatically.Flow cytometry analysis revealed a G1–G0 cell cycle arrest and increased apoptosis rate in A549 cells transfected with pCDN-LATS2.(8)The results of western blotting showed that Mdm2 expression was decreased and p53 expression was increased in A549 cells transfected with pCDNA-LATS2.Conclusion:Our results firstly demonstrated that PVT1 contributed to NSCLC cell proliferation,partly though EZH2-medicated suppression of the LATS2/MDM2/P53 pathway.Our study may provide a novel strategy for targeting the PVT1/EZH2/LATS2 axis as a new therapeutic application for NSCLC patients.