ABSTRACT
Objective@#To investigate the function of virD4 gene in Helicobacter pylori clinical strain SBK. @*Methods@#The virD4 gene segment was obtained through T-A cloning method. The prokaryotic expression vector pET-28a(＋)-virD4 was constructed and transformed into E. coli Rosetta for the expression by induction of IPTG. The recombinant proteins were obtained and purified by KCl dyeing with gel cutting method, and identified via SDS-PAGE analysis. The purified recombinant virD4 protein was used to immunize mice to produce polyclonal antibodies. The titer of the polyclonal antibodies was tested by ELISA and the antigenic specificity was identified by western blot. The purified recombinant virD4 proteins were co-cultured with GES-1 cells followed by detecting the expression of inflammatory cytokines secretion and the ability of cell proliferation. @*Results@#The full length of virD4 gene was 1 728 bp. The sequence shared quite high homology with the virD4 gene of isolate Shi470. The recombinant prokaryotic expression plasmid pET-28a(＋)-virD4 was successfully constructed. The recombinant virD4 proteins were obtained by IPTG induction and purified via KCl dyeing method. SDS-PAGE showed that the relative molecular weight of recombinant virD4 protein was 63 000. The purified proteins were used to immunize mice to obtain the anti-virD4 polyclonal antibodies with the titer 512 000. The reaction between anti-virD4 polyclonal antibodies and recombinant virD4 proteins was highly specific. The recombinant virD4 protein induced inflammatory cytokines secretion and promoted GES-1 cell proliferation. @*Conclusion@#The virD4 gene was successfully cloned and highly expressed in prokaryotic expression system, and its antibodies were prepared. The recombinant virD4 protein can induce cytokine secretion and cell proliferation.
ABSTRACT
Objective@#Abstract: Objective: cancer cells and its effects on the proliferation and migration of gastric cancer cells. @*Methods@#The expression level of LINC00473 in gastric cancer cells was verified by qRT-PCR system. LINC00473 siRNA segment and overexpression vector were separately transfected into gastric cancer cells by the method of lipofection. The proliferation and migration abilities of gastric cancer cells with LINC00473 knockdown or overexpression in vitro were evaluated by cell counting kit-8 (CCK8) assay, colony formation assay and Transwell migration assay. The expression levels of proteins involved in epithelial-mesenchymal transition (EMT) were examined by western blot analysis. @*Results@#The expression levels of LINC00473 were decreased in gastric cancer cells compared with that in human gastric epithelial cell strain GES-1 (P＜0.05). LINC00473 knockdown cells showed significant increased ability for cell growth (F=163.10, P＜0.01) and colony formation (t=3.29, P＜0.05) compared with the knockdown cells in scramble control. The results of Transwell migration assay showed that LINC00473-knockdown enhanced the migratory abilities of gastric cancer cells (t=4.68, P＜0.05). The knockdown of LINC00473 downregulated E-cadherin expression (t=4.08, P＜0.05) and upregulated N-cadherin (t=5.06, P＜0.01), Snail (t=7.69, P＜0.01) and Vimentin (t=3.82, P＜0.05) expression. Compared with the control group, LINC00473 overexpression cells showed significantly decreased cell growth (F=186.00, P＜0.01) and colony formation ability (t=3.22, P＜0.05). The results of Transwell migration assay showed that LINC00473-overexpression reduced the migratory ability of gastric cancer cells (t=5.52, P＜0.05). The overexpression of LINC00473 enhanced E-cadherin expression (t=2.90, P＜0.05) and reduced the expressions of N-cadherin (t=7.44, P＜0.01), Snail (t=2.78, P＜0.05) and Vimentin (t=4.64, P＜0.01). @*Conclusion@#The knockdown of LINC00473 may promote gastric cancer cell proliferation and migration in vitro by regulating EMT.