Expression Regulation And Anti-cancer Mechanisms Of LncRNA WDR11-AS1 In Hepatocellular Carcinoma

BackgroundLiver cancer is a kind of malignant liver tumors.Primary liver cancer originates from the liver epithelium or mesenchymal tissue,of which 85.5%primary livercancer is hepatocellular carcinoma(HCC).The liver cancer mostly refers to HCC.HCC tumors grow fast and the early symptoms are not obvious.Symptoms often appear in the middle disease stage or the late disease stage.Therefore,the treatment of HCC is difficult and the efficacy is poor.The etiology of HCC has not yet been fully elucidated,and some of its relatively clear molecular mechanisms have not been fully elucidated.According to current research,the occurrence of HCC is a complex process involving multiple causes,such as hepatitis B virus(HBV),hepatitis C virus(HCV),aflatoxin,and alcohol abuse.In human genome,only a small number of genes can be transcribed and translated into proteins and most DNA sequences are transcribed as non-coding RNAs(ncRNAs).NcRNAs account for about 90%of human genes and are previously considered to be "gene garbage".Accumulated evidences demonstrated that ncRNAs play an important regulatory role in human.Among ncRNA,those longer than 200 nt are called long ncRNAs(lncRNAs).lncRNAs have important regulatory functions and are involved in various biological processes and various diseases.In human genome,the number of lncRNAs is much greater than the number of protein coding genes.Up to now,although a small number of lncRNAs’ functions are relatively clear,the functions of most lncRNAs are still unknownWDR11 is located in chromosome 10q25-26,and has been identified as a tumor suppressor gene in human medulloblastoma.WDR11-AS1 is a reverse transcript located around the promoter region of the WDR11 gene.There are no studieson the role of lncRNA WDR11-AS1 and WDR11 in HCC.We found that WDR11 and its reverse transcript are down-regulated in HCC tissues,which suggesting that lncRNA WDR11-AS1 and WDR11 may play tumor suppressive role in HCC.In supports of this,we found that lncRNA WDR11-AS1 and WDR11 can inhibit proliferation,invasion and metastasis of HCC cells.WDR11 can exert its anti-cancer effects by regulating transcription of lncRNAWDR11-AS1 Interestingly,we found that lncRNA WDR11-AS1 can directly interact with PARP1 protein to affect development of HCC.This study might identify new targets for the early diagnosis and treatment of HCC in the future.Methods1.Detecting expression of lncRNA WDR11-AS1 and WDR11 in HCC tissuesTwenty-five HCC patients’tissues were collected from Shandong Cancer Hospital.Total RNAs were isolated from these tissue specimens.RT-qPCR assays were performed detect the expression levels and correlation of IncRNA WDR11-AS1 and WDR11 in these tissues2.The anti-cancer effects of lncRNA WDR11-AS1 and WDR11 in HCC cells(1)The effects of lncRNA WDR11-AS1 and WDR11 on the proliferation ability of HCC cells were measured using cell countingassays(2)The effects of lncRNA WDR11-AS1 and WDR11 on the invasion and metastasis capability of HCC cells were detected by wound healing assays and transwell invasion assays.3.The mechanisms of lncRNA WDR11-AS1 and WDR11 suppressing HCC development(1)RT-qPCR detection in HCC cells confirms the regulatory relationship between lncRNA WDR11-AS1 and WDR11.(2)The subcellular localization of lncRNA WDR11-AS1 was detected by nuclear separation assays.(3)The binding protein(s)of IncRNA WDR11-AS1 was detected by RNA pulldown assays,Mass Spectrometry and RNA-protein co-immunoprecipitation assays assay.(4)The impacts of lncRNA WDR11-AS 1 on PARP1 protein degradation was detected.4.Xenograft assaysThe HCC SK-Hep-1 cell line was used in the xenograft assays of nude mice.Nude mice were randomly divided into three groups(the control group,the WDR11 group,and the lncRNAWDR11-ASigroup).The impacts of lncRNA WDR11-AS1 and WDR11 on xenograft growth were observed.Results1.lncRNA WDR11-AS1 and WDR11 was down-regulated in HCC tissues compared to normal tissues.lncRNA WDR11-AS1 and WDR11 RNA expression levels were positively correlated in HCC tissues and normal tissues.2.The anti-cancer effects of lncRNA WDR11-AS1 and WDR11 in HCC cells(1)Cell proliferation assays indicated that lncRNA WDR11-AS1 and WDR11 can significantly inhibit proliferation of HCC cells.(2)Wound healing assays and transwell assays indicated that lncRNA WDR11-AS1 and WDR11 can evidently inhibit invasion and metastasis of HCC cells.3.The mechanisms of lncRNA WDR11-AS1 and WDR11 suppressing development of HCC(1)In HCC cells,WDR11 plays a regulatory role on transcription of lncRNA WDR11-AS1.(2)lncRNA WDR11-AS1 existed in both nucleus and cytoplasm of HCC cells.(3)RNA pulldown and Mass Spectrometry assays,or RNA-protein co-immunoprecipitation assay indicated that lncRNA WDR11-AS1 directly binds to PARP1 protein in HCC cells(4)lncRNA WDR11-AS1 promotes degradation of PARP1 protein through the ubiquitin-proteasome pathway.4.The xenograft growth rate of nude mice with high expression of lncRNA WDR11-AS1 and WDR11 was slower than controls.Conclusions1.lncRNA WDR11-AS1 and WDR11 are down-regulated in HCC tissues2.The expression of IncRNA WDR11-AS1 and WDR11 are positively correlated in tissues.3.lncRNA WDR11-AS1 and WDR11 can inhibit proliferation,invasion and metastasis of HCC cells.4.lncRNA WDR11-AS1 binds to PARP1 protein and promotes PARP1 degradation through the ubiquitin-proteasome pathway.