MCM3AP,a Novel HBV Integration Site in Hepatocellular Carcinoma and Its Implication in Hepatocarcinogenesis

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

Beyond ribosomal function:RPS6 deficiency suppresses cholangiocarcinoma cell growth by disrupting alternative splicing

Cholangiocarcinoma(CCA)is a bile duct malignancy with a dismal prognosis.This study systematically investigated the role of the ribosomal protein S6(RPS6)gene,which is dependent in CCA.We found that RPS6 upregulation in CCA tissues was correlated with a poor prognosis.Functional investigations have shown that alterations in RPS6 expression,both gain-and loss-of function could affect the proliferation of CCA cells.In xenograft tumor models,RPS6 overexpression enhances tumorigenicity,whereas RPS6 silencing reduces it.Integration analysis using RNA-seq and proteomics elucidated downstream signaling pathways of RPS6 depletion by affecting the cell cycle,especially DNA replication.Immunoprecipitation followed by mass spectrometry has identified numerous spliceosome complex proteins associated with RPS6.Transcriptomic profiling revealed that RPS6 affects numerous alternative splicing(AS)events,and combined with RNA immunoprecipitation sequencing,revealed that minichromosome maintenance complex component 7(MCM7)binds to RPS6,which regulates its AS and increases oncogenic activity in CCA.Targeting RPS6 with vivo phosphorodiamidate morpholino oligomer(V-PMO)significantly inhibited the growth of CCA cells,patient-derived organoids,and subcutaneous xenograft tumor.Taken together,the data demonstrate that RPS6 is an oncogenic regulator in CCA and that RPS6-V-PMO could be repositioned as a promising strategy for treating CCA.