ABSTRACT
Objective To determine the effect of COMMD7 inhibition on invasion and migration in liver cancer stem cells (LCSCs),and investigate the possible mechanism.Methods After LCSCs were infected by shRNA lentiviral vectors of COMMD7,adhesion assay and Transwell assay were used to detect the invasion and migration,and phalloidin staining was employed to observe the morphological changes.Western blotting was adopted to measure the expression of E-cadherin,N-cadherin and Vimentin.Results COMMD7 knockdown significantly inhibited the invasion and migration of LCSCs.The relative cell quantity of adhesion was 1.00 ± 0.12 and 2.35 ± 0.20 respectively in control cells and infected cells,suggesting there were significantly more adhesive cells in the infected group (P ＜ 0.05).The relative cell quantity per visual field of migration was 1.00 ±0.04 and 0.24±0.03,and that of invasion was 1.00 ±0.05 and 0.24 ±0.04 respectively in the control cells and infected cells,and there were significantly less invasive and migrated cells in the infected group (P ＜0.05).What's more,COMMD7 knockdown also induced some morphological changes of cells corresponding to the weakened abilities of migration and invasion.All the changes above were associated with up-regulation of E-cadherin (P ＜ 0.05) and down-regulation of N-cadherin and Vimentin (P ＜0.05),the molecules related to mesenchymal-epithelial transition (MET).Conclusion COMMD7 knockdown inhibits the invasion and migration in LCSCs,which may be through its regulation on the MET course.
ABSTRACT
Objective To investigate the mechanism of COMMD7,a hepatocellular carcinoma gene,promoting human hepatocellular carcinoma cell line (HePG2 cells)proliferation and migration.Methods The specific siRNA (small interference RNA)was designed for COMMD7 gene,siRNA transfected HepG2 cells was set as Si-HePG2 group,and the PTEN inhibitor treating group (Si +BpV-HePG2),the control group (HePG2),the empty vector group (HePG2 was infected empty vector,N-HePG2 group)were also set up.qRT-PCR was per-formed to evaluate the mRNA expression changes of COMMD7 and PTEN,and Western blot was performed to test the expression changes of COMMD7,PTEN,p-AKT,and AKT.MSP method was performed to detect methylation level.CCK8 was performed to test cell proliferation a-bility.Transwell was performed to detect the invasion of cells.Results The results of qRT-PCR showed that the expression of COMMD7 gene in Si-HePG2 group was lower (0.101 times)than the control group and the N-HePG2 group,and the expression of PTEN gene was higher (2.841 times)than the control group and the N-HePG2 group,and all the results above were of statistically singificant difference (P <0.05). The results of Western blot showed that in Si-HePG2 group,the expression of COMMD7 reduced obviously,the expression of PTEN increased and the expression of p-AKT was significantly inhibited.In Si +Bpv-HePG2 group,the expression of PTEN was significantly inhibited and the expression of p-AKT was increased.The result of MSP showed that compared with the control group and the N-HePG2 group,Si-HePG2 group was more obviously demethylated,which indecated that the expression of COMMD7 gene could induce PTEN demethylation.The results of CCK8 showed that the proliferation in Si-HePG2 group was decreased compared with the control group,N-HePG2 group and Si +BpV-HePG2 group,and the difference was singificant (P <0.05).The results of Transwell showed that the numbers of cell permeating septum in Si-HePG2 group,N-HePG2 group,control group and PTEN group were (17.4 ±2.7),(36.2 ±3.2),(41.6 ±4.5)and (47.6 ±1.8)respec-tively,which were obviously decreased with a significant difference (P <0.05).Conclusion siRNA interference decreased the expression of COMMD7.It can induced the demethylation of PTEN gene in HePG2 and increase the expression of PTEN gene to inhibit PI3K/AKT signaling pathway,thus decreasing the proliferation,migration and invasion ability of HePG2 cells.
ABSTRACT
Objective To observe the changes of the cells of human hepatocellular carcinoma (HepG2)using RNA for silencing the expression of COMMD7 gene,and investigate related mechanism of COMMD7 gene promoting HepG2 proliferation.Methods COMMD7 gene shRNA was designed and constructed into COMMD7-shRNA plasmid.HepG2 cells were divided into the HepG2 group,control-shRNA group (empty vectors were infected) and COMMD7-shRNA group (positive vectors were infected).Cells shapes were observed by fluorescence microscope after infecting.The expression of COMMD7 and expression and phosphosylation of extracellular regulated protein kinase1/2 (ERK1/2) and MEK1/2 protein were measured by Western blot.The cell vitality was measured by cholecystokinin octapeptide (CCK-8),and the apoptosis of cell was detected by flow cytometry.The measurement data with normal distribution were presented as (x) ± s.The comparisons among groups were evaluated with the one-way ANOVA,and pairwise comparison was analyzed by the LSD-t test.Results The cells were oval or spindle shapes and displayed green fluorescent after infected successfully.The results of Western blot showed that the relative quantitative expression of COMMD7 protein in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.90 ±0.18,1.03 ±0.05 and 0.23 ±0.03,respectively,with a significant difference among the 3 groups (F =152.08,P ＜ 0.05),and the expression of COMMD7 protein in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =20.74,21.16,P ＜ 0.05).The results of CCK-8 showed that the scores of the HepG2 vitality in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 1.193 ±0.024,1.225 ±0.034 and 1.147 ±0.021,respectively,with a significant difference among the 3 groups (F =6.90,P ＜ 0.05),and the HepG2 vitality in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =3.53,3.69,P ＜ 0.05).The results of flow cytometry showed that the apoptosis rate of HepG2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 6.1％ ± 0.3％,7.8％ ± 0.5％ and 20.9％ ± 1.4％,showing a significant difference among the 3 groups (F =270.80,P ＜0.05),and the apoptosis rate of HepG2 in the COMMD7-shRNA group was significant higher than those in the other 2 groups (t =21.77,19.36,P ＜0.05).The results of Western blot showed that the relative quantitative expression of phosphorylation (p)-ERK1/2 and p-MEK1/2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.932 ±0.046,0.945 ±0.017,0.553 ±0.052 and 0.452 ±0.031,0.468±0.027,0.263 ± 0.022,respectively,showing significant differences among the 3 groups (F =93.61,49.16,P ＜ 0.05),and the relative quantitative expression of p-ERK1/2 and p-MEK1/2 in the COMMD7-shRNA group were significantly lower than those in the other 2 groups (t =11.94,12.17,9.33,8.65,P ＜ 0.05).Conclusions COMMD7 gene can promote HepG2 proliferation via activating ERK/mitogen-activated protein kinase (MAPK) signaling pathway,and its mechanism may be promoting the phosphorylation of expression of ERK1/2 and MEK1/2.