The Role Of High Frequency Copy Number Variations Gene PPP1R16A In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is characterized by insidious onset,rapid development and poor prognosis.Due to the unobvious symptoms of early HCC,as well as the strong capacities of cell proliferation ?invasion and metastasis,the patients are usually in advanced stage when diagnosed.Thus they miss the best opportunity for treatment,which leads to high morbidity and mortality of HCC.The development of HCC is a multi-level and complex biological process,involving the alteration of several signaling pathways and genes.Genetically,the hepatocarcinogenesis is not only related to the genetic background of individuals,the environmental factors also play an important role in hepatocarcinogenesis.In China,the hepatitis B virus(HBV)infection is a major risk factor,which accounts for the high incidence of HCC.The integration of HBV viral DNA into the host genome will result in the change of key gene function within certain signaling pathway through a variety of forms,such as point mutation in single gene? copy number variations(CNVs)in the large fragments of genome or chromosome translocation.CNVs is an important form of structural variation in genome,which contains large fragement(?1kb)amplification?deletion and insertion and complex variations.Compared with mutation in single gene,CNVs show much higher mutation rates.It has been found that there exist hot spots of HBV integration in HCC.Meanwhile,there is a significant correlation between high frequency fragments of CNVs with the hot spots of HBV integration in HCC.These results indicate that CNVs may have a crucial role in the development of HBV-related HCC.Thus,the study of CNVs in HCC will be helpful to deepen the understanding of the pathogenesis of HBV related-HCC,and provide new ideas and methods for the diagnosis and treatment of HCC.Through the integration analysis of expression profile microarray data containing HCC tissues and adjacent tissues and high frequency variation of copy number in HCC from literature search,this study sought to find key gene on the high frequency variation fragments of CNVs in HCC.First,we used molecular biological techniques to verify and compare the level of genomic DNA and mRNA in large clinical samples,and analyses the correlation between them.Next,we observed the variation trendency of two levels in the process in the pathnogensis of HCC by clinical validation and bioinformation analysis.We took further in vitro experiments to observe impacts of targeted gene silencing by siRNA on the biological behaviors of HCC cells,such as cell proliferation? cell cycle and apoptosis.By literature retrieval,we confirmed the existence of multiple high frequency variation fragments of copy number in HCC(both amplification and deletion).In combination with gene set obtained from HBV-related expression profiling microarray performed by our research group previously,we found significant up-regulation of PPP1R16A(Protein phosphatase 1 regulatory subunit 16A)mRNA level,and it was located in 8q24.3,one of the high frequency variation fragments of copy number.By real-time fluorescent quantitative PCR,we confirmed that compared to adjacent tumor tissues,PPP1R16 A showed significant amplification in the copy number and upregulation of its mRNA level in HCC tissues,and there existed a correlation between them.The results indicated that PPP1R16 A was overexpressed with copy number amplification in HCC.We found that the CNV and mRNA level of PPP1R16 A gene were increased with the HCC progression by experiment and bioinformation analysis of every stage of HCC.To unveil the mechanism of CNVs in HCC,we transfected HCCLM3 with PPP1R16 A siRNA and found that the silencing of PPP1R16 A could significantly inhibit the proliferation of HCC cells and restrain the cell cycle,which suggested that PPP1R16 A may play an important role in promoting HCC.By KEGG pathway analysis of genes correlated with PPP1R16 A from expression profiling microarray,we found that these mRNAs were enriched in cell cycle signaling pathway.The expression of CDK2?CDK4?SKP2 were significantly down-regulated in HCCLM3 transfected with PPP1R16 A siRNA,which indicated PPP1R16 A may affect cell proliferation and cell cycle by regulation of expression of cell cycle-associated genes.Our study enriched the mechanisms of CNVs in hepatocarcinogenesis through function of gene on high frequency fragments of CNVs in HCC,and also provided new possibility for HCC targeted therapy.