RESUMO
Aim To study the expression of microRNA(miR-34c-5p)in gastric cancer tissues and the mechanism of regulating the invasion and migration of gastric cancer cells. Methods The expression of miR-34c-5p mRNA in human gastric cancer tissues and cells was detected by quantitative real-time polymerase chain reaction(RT-qPCR). An overexpression model of miR-34c-5p was constructed in human gastric cancer cells. The transfection of lentivirus was observed under inverted fluorescence microscope, and the transfection efficiency was verified by RT-qPCR. Scratch test and Transwell assay were used to detect the effects of overexpression of miR-34c-5p on invasion and migration of gastric cancer cells. Western blot was used to detect the expression of MAP2K1, E-Cadherin and Vimentin in gastric cancer cells of each group. Dual-luciferase reporter assay was employed to detect whether MAP2K1 was a target gene of miR-34c-5p. Results The expression of miR-34c-5p mRNA in 10 human gastric cancer tissues was significantly lower than that in adjacent tissues(P<0.05). The expression of miR-34c-5p mRNA in gastric cancer HGC-27 cell was significantly lower than that in normal gastric mucosa GES-1 cell(P<0.01). Compared with blank control group and miR-34c-5p-NC group, miR-34c-5pmimics group significantly increased its miR-34c-5p mRNA expression level(P<0.05). The results of scratch test and Transwell experiment showed that overexpression of miR-34c-5p significantly inhibited the migration and invasion ability of gastric cancer cells(P<0.05, P<0.01). Western blot results showed that the protein expression levels of MAP2K1 and Vimentin significantly decreased and E-Cadherin markedly increased in gastric cancer HGC-27 cells after overexpression of miR-34c-5p(P<0.05). In addition, compared with mimic-NC+miR-34c-5p-mimics group, the lucifase activity of pmiR-MAP2K1-WT plasmid decreased(P<0.01), while the lucifase activity of pmiR-MAP2K1-MUT plasmid was not changed. Conclusions The low expression of miR-34c-5p in gastric cancer tissues and cells may inhibit the invasion and migration of gastric cancer cells through targeted regulation of MAP2K1 expression.
RESUMO
This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL) on carbon tetrachloride(CCl_4)-induced liver fibrosis in rats, analyze and predict its mechanism of action and potential quality markers(Q-marker). Firstly, male SD rats were taken and injected subcutaneously with a 40% CCl_4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis. The rats with liver fibrosis were randomly divided into model group, silybin group(43.19 mg·kg~(-1)), Fuzheng Huayu Capsules group(462.75 mg·kg~(-1)), and TFL groups(100 mg·kg~(-1) and 25 mg·kg~(-1)), with normal rats as a blank group, 10 rats in each group. Except for the blank group, the rats in the other groups were subcutaneously injected with 40% CCl_4-vegetable oil solution of a maintenance dose, once a week. The rats in various treatment groups received corresponding doses of drugs, while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks. At the end of the experiment, blood was collected from the abdominal aorta and the liver tissues were collected. The levels of total bilirubin(TBiL), direct bilirubin(DBiL), indirect bilirubin(IBiL), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum were detected by using an automatic biochemical detector. Masson staining was used to observe the histopathological changes of rat liver. Then, the chemical compositions of TFL were collected, and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI). After screening of disease targets of liver fibrosis by Gene Cards database, the protein-protein interaction was analyzed with use of STRING database, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) enrich analysis were also carried out. Moreover, an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL, model and blank groups to verify the targets. Furthermore, Cytoscape software was used to establish and visualize the network of chemical components, targets and pathways, and predict the potential Q-marker of TFL. The results showed that the levels of TBiL, DBiL, IBiL, ALT, and AST in the model group were significantly higher than those in the blank normal group(P<0.05), and the above levels in the treatment groups were lower than those in the model group, but with no significant differences. Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group, and were relieved to different degrees in the treatment groups. Then, 74 chemical components were screened, which could act on 865 targets such as EGFR and SRC, participating in the regulation of cancer pathways, PI3 K-Akt signaling pathway, HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways. Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified, among which 6 proteins were significantly expressed, including 5 up-regulated proteins and 1 down-regulated protein. The protein expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups. In conclusion, TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1, which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.