Preliminary Study Of Abnormal Expression Of RCBTB2 In Hepatocellular Carcinoma And Its Biological Effect

Hepatocellular carcinoma(HCC)is one of the most common maliganant cancers worldwide,its molecular pathological mechanism is very complex.Studies found that there were large amounts of gene mutations and copy number variantions(CNVs)in HCC,activation of oncogenes and the inactivation of tumor suppressor genes(TSGs)were one of the key factors for the development of HCC.So there is a need to search for and identify novel and potent oncogenes or TSGs to elucidate the molecular mechanism of HCC and develop new target therapies.To identify novel targets,we used bioinformatic methods to analyze the CNV and gene expression data.We downloaded a CNV dataset which contained 442 samples from TCGA,and screened CNV regions by GISTIC.The results showed 13q14.2 and 1p36.31 deleted primarily in genome,and these two regions were responsible for coding of 48 genes.We also downloaded 6 mircoarry datasets from NCBI GEO database,and conducted a large scale meta-analysis to find out the genes specifically differerntly expressed in HCC.The results showed RCBTB2 gene on 13q14.2 was the most specifically down-regulated expressed.However,the function of RCBTB2 has yet to be elucidated clearly,it needs to be further discussed.In this study,we examined the expression of RCBTB2 in 123 pairs HCC samples and their matched adjacent normal liver tissues by IHC,and analyzed the correlation of RCBTB2 and the clinicopathological features.We also confirmed the expression level of RCBTB2 in 8 pairs of samples chosen at random by western blot.These results demonstrated RCBTB2 protein level was significantly decreased in HCC tissues and was correlated with histologic grade,vascular invasion and TNM staging.To determine the biological function of RCBTB2 in HCC,we conducted cell colony experiment and the results revealed that when overexpressed RCBTB2 decreased colony forming ability of HepG2 cells.Cell cycle experiment demonstrated that when overexpressing RCBTB2,cells were arrested in G0/G1 phase.To test whether RCBTB2 influences the formation of primary cilia,we transfected shRNA-RCBTB2 into NIH3T3 cells and observed the formation of primary cilia by immunofluorescence.The results proved that knockdown of RCBTB2 gene expression disrupted ciliogenesis,the percentage of cells with primary cilia was reduced in shRNA-RCBTB2 transfected cells.In conclusion,bioinformatics analysis showed that 13q14.2 was significantly deleted in HCC,accompanying with a decrease of RCBTB2 mRNA expression.IHC and western blot proved that RCBTB2 was significantly low expressed in HCC tissues which was correlated with the poor clinical features.Cell experiment demonstrated RCBTB2 inhibited cell proliferation and affected the formation of primary cilia.Our findings laid a foundation for further research of RCBTB2 biological function and provided a new insight into HCC pathological mechanism and targeted therapy.