Résumé
Objective:To investigate the effects and molecular mechanism of neuropilin and tolloid-like 2 (NETO2) on proliferation, migration, cell cycle, and apoptosis in gallbladder cancer (GBC).Methods:The NETO2 mRNA and protein expression in GBC-SD, ZJU-0430, NOZ GBC cells were detected by quantitative real-time polymerase chain reaction and Western blot. NETO2 overexpression and knockdown stable cell lines were constructed by plasmid transfection. Cell counting kit-8 assay, colony formation assay, transwell assay, flow cytometry and WB assay were performed to evaluate proliferation, migration, cell cycle, apoptosis, epithelial-mesenchymal transition (EMT) and changes of phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.Results:GBC-SD and ZJU-0430 cells with NETO2 gene overexpression and NOZ cells with NETO2 gene knockdown were effectively constructed. NETO2 overexpression in gallbladder cancer cell lines significantly improved cell proliferation and migration, advanced cell cycle progression from G0/G1 to S phase, and inhibited cell apoptosis. In the ZJU-0430 and GBC-SD cells, the clone number increased from (78.5±9.2), (217.0±6.4) to (213.5±10.3), (296.3±9.3)( t=10.98, 6.51; P=0.008, 0.023); The number of migrating cells increased from (198.6±8.4), (233.3±11.0) to (382.7±12.4), (379.0±7.3) ( t=16.98, 16.85, both P<0.001); The total apoptosis rate reduced from (29.7±0.9)%, (35.6±1.1)% to (19.2±0.5)%, (29.1±0.4)% ( t=9.74, 9.05; both P<0.001); The expression of EMT related proteins such as N-cadherin, Vimentin, Snail, and Slug were upregulated, while E-cadherin expression was downregulated. Phosphorylated PI3K (p-PI3K) and Akt (p-Akt) protein expression were significantly increased (all P<0.05). In contrast, NETO2 knockdown had the opposite effect on all these parameters. Conclusion:NETO2 influences the EMT process by regulating the PI3K/Akt signaling pathway, thus promotes GBC cell proliferation, migration and cell cycle progression, and inhibits cancer cell apoptosis.
Résumé
Background and purpose:In recent years, the studies have indicated that long non-coding RNAs (lncRNAs) may play important roles in the initial stage and development of tumors. H19 is one of those lncRNAs, which is already proved to overexpress in a variety of tumors such as bladder cancer, stomach cancer and hepatocellular carcinoma (HCC). And H19 also could promote tumor proliferation and increase tumor cell migration and invasion ability, but neither the expression nor the function of H19 in non-small cell lung cancer(NSCLC) are clariifed. This study aimed to observe the effects of H19 on the proliferation, epithelial-mesenchymal transition (EMT) and invasion ability of NSCLC cell line A549.Methods:H19 was overexpressed by plasmids transfection, then the effect of H19 on the proliferation of A549 was measured by cell counting kit-8(CCK-8), the invasion of A549 cells was detected by Transwell assay, and the changes of cell morphology were observed with an optical microscope, and the expression of EMT-related proteins was detected by Western blot, and the promoter activity of CDH1 was measured by luciferase assay.Results:The proliferation of A549 cells was increased under the overexpression of H19(D value in blank group was 1.64±0.02, in negative control group was 1.59±0.04, in overexpression of H19 group 1.89±0.02,P<0.05), the invasion ability of A549 cells was dramatically enhanced [negative control group (30±6)/vision, overexpression of H19 group (110±7)/vision,P<0.05], and the A549 cells developed longer pseudopodia and had wider intercellular spaces. All these morphology changes indicated that the cells were undergoing the process of EMT, and meantime, the expres-sion of CDH1 was decreased, along with the expression of VIM and SNAI2 elevated, which were also related to the progression of EMT, and H19 also could depress the promoter activity of CDH1 by 60% (P<0.05).Conclusion:The overexpression of H19 induces the EMT, and enhances the proliferation and invasion ability of A549 cells.