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.

Effect of the interaction between M-CSF and MCM7 on DNA replication in HeLa cells

Objective To explore the effect of the interaction between microphage colony-stimulating factor(M-CSF)and minichromosome maintenance protein-7(Mcm7)on DNA replication in HeLa cells.Methods pCMV/nuc/myc,pCMV/nuc/GFP and pCMV/nuc/M-CSF vector were stably transfected into HeLa cells by Lipofectamine,respectively.After screening with G418,the expression and localization of M-CSF in HeLa cells were verified by RT-PCR,Western blot and immunofluorescence staining.The statue and interaction between intracellular M-CSF and Mcm7 in HeLa cells was analyzed by co-immunoprecipitation.The effect of the interaction between M-CSF and Mcm7 on DNA replication was analyzed by a mammalian cell or cell-free DNA replication system in vitro.Results The results indicated that the M-CSF-transfected HeLa cells stably express both M-CSF mRNA and protein,and that M-CSF protein is located to the nuclei of HeLa cells mentioned above.To further analyze the status and interaction between intracellular M-CSF and Mcm7,the Mcm7 from HeLa cells was precipitated with anti-Mcm7 antibody and followed by Protein A/G PLUS agarose.The precipitation was blotted with anti-M-CSF monoclonal antibody.The results show that M-CSF was coprecipitated with Mcm7,so intracellular M-CSF existed in Mcm7-bound state.The DNA replication experiments reveal that a higher percentage of the replicating nuclei is present either in unsynchronized or in both synchronized G1 and S phase M-CSF-transfected HeLa cells,compared with both pCMV/nuc-transfected and un-transfected HeLa cells,which suggests that interaction between M-CSF and Mcm7 promote both the initiation and elongation of DNA replication.Conclusions M-CSF directly interacts with Mcm7.The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.

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.

Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-Rye Hybrids

Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres, Furthermore, a mitotically unstable dicentric chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese Spring-lmperial rye 3R addition line. An unstable multicentric chromosome was found in the progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including movement and disappearance of terminal heterochromatin occurred in the progeny of wheat- rye addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were observed in the progeny ofa monosomic 4R addition line, a ditelosomic 5RS addition line and a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric DNA （TTTAGGG）n, derived from a monosomic 4R addition line are stable and transmissible to the next generation. The results indicated that centromeres and terminal heterochromatin can be profoundly altered in wheat-rye hybrid derivatives.