Lamin B1 regulates the growth of hepatocellular carcinoma cells by influencing telomerase activity / 生物工程学报

Lamin B1 (LMNB1) is highly expressed in liver cancer tissues, and its influence and mechanism on the proliferation of hepatocellular carcinoma cells were explored by knocking down the expression of the protein. In liver cancer cells, siRNAs were used to knock down LMNB1. Knockdown effects were detected by Western blotting. Changes in telomerase activity were detected by telomeric repeat amplification protocol assay (TRAP) experiments. Telomere length changes were detected by quantitative real-time polymerase chain reaction (qPCR). CCK8, cloning formation, transwell and wound healing were performed to detect changes in its growth, invasion and migration capabilities. The lentiviral system was used to construct HepG2 cells that steadily knocked down LMNB1. Then the changes of telomere length and telomerase activity were detected, and the cell aging status was detected by SA-β-gal senescence staining. The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments, subsequent histification staining of tumors, SA-β-gal senescence staining, fluorescence in situ hybridization (FISH) for telomere analysis and other experiments. Finally, the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues, and its relationship with clinical stages and patient survival. Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity, cell proliferation, migration and invasion abilities. Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity, shortened telomere length, senesced cells, reduced cell tumorigenicity and KI-67 expression. Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival. In conclusion, LMNB1 is overexpressed in liver cancer cells, and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Relationship Between Dilated Cardiomyopathy and Nuclear Lamina Protein A Gene Mutation in Kazak Ethnics at Xinjiang Area / 中国循环杂志

Objective: To study the relationship between dilated cardiomyopathy and nuclear lamina protein (LMNA) gene mutation in Kazak ethnics at Xinjiang area. Methods: A Kazak familial dilated cardiomyopathy (FDCM) with 31 members was studied. In addition, 160 patients with idiopathic dilated cardiomyopathy (IDCM) with 160 healthy controls were enrolled in our study, and they were divided into 4 groups: IDCM-Kazak, IDCM-Han and Control-Kazak, Control-Han.n=80 in each group. Peripheral blood DNA were extracted, 12 exons with nearby introns of LMNA gene were detected by PCR and the ampliifed products were sequenced and compared with the standard template of CHROMAS and BLAST software to identify mutation sites. LMNA mutation in both Kazak and Han IDCM patients were investigated. Results: A novel LMNA mutation (insC, CGG→CCG) at exon 7 was identiifed in a FDCM proband, it caused an amino acid substitution as Arg to Pro, and a known LMNA polymorphism loci rs4641 (c.1362C>T His454His) was fund at exon 10. In addition, LMNA polymorphism loci rs4641 genotype distribution (χ2=5.16,P=0.036) and allele frequency (χ2=4.50,P=0.034) were statistically different between IDCM-Kazak group and Control-Kazak group; while such differences were no statistic meaning between IDCM-Han group and Control-Han group. Logistic regression analysis indicated that LMNA polymorphism loci rs4641 was related to IDCM occurrence in Kazak ethnics (P=0.025, OR=0.412, 95% CI 0.189-0.896). Conclusion: LMNA polymorphism loci rs4641 was related to IDCM in Kazak ethnics at Xinjiang area, which might be susceptible loci for IDCM occurrence.

Association of progerin-interactive partner proteins with lamina proteins:Mel18 is associated with emerin in HGPS / 北京大学学报(医学版)

Objective :The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a childhood disorder with features of premature aging and is caused by mutations in the lamin A gene resulting in the production of an abnormal protein, termed progerin. To investigate the underlying pathogenic mecha-nism, we studied the nuclear co-localization and association of progerin interactive partner proteins (PIPPs) with lamina proteins. Methods:Both wild-type (WT) and progeria fibroblasts were studied by various methods including eonfocal microscopy, immunopreeipitation and Western blot. Results:All PIPPs discovered so-far co-localized with lamin A/C. In addition, the PIPPs were selectively associated with lamina proteins. An increased immunofluorescent staining signal was found for Mel18 in HGPS as com-pared to WT cells. An association of Me118 with emerin was observed in HGPS, but not in WT cells.Conclusion: Based on these findings, we propose that PIPPs, along with associated lamina proteins may form a pathogenic progerin-containing protein complex.

Nuclear Egress of Herpesviruses / 中国病毒学·英文版

Herpesviruses assemble and fill their capsids in the infected cell nucleus,and must then move this enormous macromolecular assembly across the nuclear membrane and into the cytoplasm.Doing so is a complex,multi-step process that involves envelopment of the capsid at the inner nuclear membrane and de-envelopment by fusion with the outer nuclear membrane.This process is orchestrated by viral proteins,but requires the modification of cellular structures and mechanisms including the nuclear lamina.In this review I summarize recent research on the mechanism of nuclear envelopment and the viral and cellular systems involved in its execution.

Towards understanding lamin gene regulation.

The lamins are components of the nuclear lamina, which forms a fibrous meshwork lining the inner nuclear membrane. Lamina-membrane interactions play a crucial role during nuclear disassembly and reassembly at mitosis, whereas lamina-chromatin association has been proposed to be essential for chromatin organization. The composition of the lamina changes considerably during embryonic development and cell differentiation. Recent studies have provided insights into the regulation of the lamin genes.