| Objectives: Hepatocellular carcinoma is one of the most common malignant tumors in patients with chronic liver disease,the main causes of poor prognosis are lymphatic metastasis and recurrence.Proliferation,migration and invasion of HCC cells are the early biological characteristics of metastasis or recurrence.The purpose of this study was to investigate the expression of the JNK/p53/ANXA7 signaling pathway in hepatocellular carcinoma cells and its effect on the proliferation,migration and invasion ability of hepatocellular carcinoma cells.Furthermore,it was further investigated whether JNK or ANXA7 affected the lymphatic adhesion of liver cancer through ATF2 and its related lncRNA.To reveal the pathogenesis of biological behavior in the early stage of hepatocellular carcinoma metastasis and provide a new target for clinical treatment.Methods: 1.The expression levels of JNK,p53 and ANXA7 in Hca-p cells were down-regulated and the down-regulation efficiency was verified.(1)Using Lipofectamine 2000,the synthesized sh RNA-JNK,sh RNA-p53,sh RNA-ANXA7 and unrelated sequences(control-sh RNA-treated)were transfected into mouse ascites hepatoma cell line Hca-P.The GFP expression,qRT-PCR and Western blot were used to verify the transfection efficiency.(2)The transfected cells were screened with G418 for 28 days to establish a stable cell line.2.To investigate the expression of JNK/p53/ANXA7 signaling pathway in Hca-P cells.JNK,p53,ANXA7 were down regulated and JNK inhibitor were used.The expression of genes and proteins in the JNK/p53/ANXA7 signaling pathway was detected by qRT-PCR,Western blot and immunofluorescence compared with the Hca-P group and the control-sh RNA-treated group.3.Effects of the JNK/p53/ANXA7 signaling pathway on proliferation,migration and invasion of Hca-P cells.(1)The proliferation capacity of Hca-P,control-sh RNA-treated,sh RNA-JNK,sh RNA-p53,sh RNA-ANXA7 and JNK inhibitor-treated groups was measured by CCK8 assay.(2)The migration and invasiveness of the above six groups of cells were detected by Transwell assay.4.The effect of down-regulation of JNK or ANXA7 on ATF2 expression.The expression of ATF2 at gene and protein levels in control-sh RNA-treated,sh RNA-JNK and sh RNA-ANXA7 groups was detected by qRT-PCR,Western blot and immunofluorescence.5.The effect of down-regulation of JNK or ANXA7 on lymphatic adhesion.The lymphatic adhesion of control-sh RNA-treated,sh RNA-JNK and sh RNA-ANXA7 groups was detected by the lymphatic adhesion assay.6.The effect of down-regulation of JNK or ANXA7 on the expression of ATF2-related lncRNA NONMMUT14121.1.The expression of lncRNA NONMMUT114121.1 in control-sh RNA-treated,sh RNA-JNK and sh RNA-ANXA7 groups was detected by qRT-PCR.Results: 1.Downregulating the expression of ANXA7,p53 or JNK in Hca-P cells by sh RNA.Through qRT-PCR and Western blot verification at gene and protein levels,the down-regulated ANXA7,p53 or JNK cell lines have been successfully established(P<0.05).2.The JNK/p53/ANXA7 signaling pathway is expressed in Hca-P cells.(1)Compared with Hca-P group and control-sh RNA-treated group,JNK,p53 or ANXA7 decreased in protein level and gene level after JNK was down regulated or JNK inhibitor was used(P<0.05).JNK,p53 or ANXA7 decreased at protein and gene levels after downregulation of p53(P<0.05).When ANXA7 was down-regulated,the expression of ANXA7 at both protein and gene levels was decreased(P<0.05),while JNK or p53 showed no significant changes.(2)Immunofluorescence results showed that the fluorescence brightness of JNK,p53 or ANXA7 was decreased in thesh RNA-JNK group and the JNK-inhibitor-treated group,compared with the Hca-P group and the control-sh RNA-treated group.In the sh RNA-p53 group,the fluorescence brightness of JNK,p53 or ANXA7 decreased.In the sh RNA-ANXA7 group,the fluorescence brightness of ANXA7 decreased,while the fluorescence brightness of JNK or p53 did not change significantly.It was proved that the JNK/p53/ANXA7 signaling pathway was expressed in Hca-P cell.3.Effects of JNK/p53/ANXA7 signaling on proliferation,migration and invasion of Hca-P cells.(1)The results of CCK8 assay showed that: compared with Hca-P group and control-sh RNA-treated group,the ability of cell proliferation decreased significantly after down regulating JNK,p53,ANXA7 or using JNK inhibitor(P<0.05).(2)The results of Transwell assay showed that after 24 h of cell culture with down-regulated JNK,p53,ANXA7 or using JNK inhibitor,the number of cells penetrating the membrane or matrix adhesive was significantly lower than that in the Hca-P group and the control-sh RNA-treated group,with statistically significant differences(P<0.05).4.The effect of JNK or ANXA7 on ATF2 expression.(1)The results of qRT-PCR and Western blot showed that the expression of ATF2 in gene and protein level decreased significantly after JNK or ANXA7 were down regulated compared with the control-sh RNA-treated group(P<0.05).(2)The results of immunofluorescence showed that compared with the control-sh RNA-treated group,the fluorescence intensity of ATF2 in the sh RNA-JNK and sh RNA-ANXA7 group decreased,which was consistent with Western blot.5.The effect of JNK or ANXA7 on lymphatic adhesion.Compared with the control-sh RNA-treated group,the cell number of lymph adhesion decreased after JNK or ANXA7 were down regulated(P<0.05).6.The effect of JNK or ANXA7 on the expression of ATF2-related lncRNA NONMMUT14121.1.Compared with the control-sh RNA-treated group,the expression of lncRNA NONMMUT114121.1 in the sh RNA-JNK group was significantly reduced(P<0.01),while the expression of lncRNA NONMMUT114121.1 in the sh RNA-ANXA7 group was significantly increased(P<0.01).Conclusions: 1.The JNK/p53/ANXA7 signaling pathway is expressed in HCC cells.2.Inhibition of the JNK/p53/ANXA7 signaling pathway can inhibit the proliferation,migration and invasion of HCC cells.3.The down-regulation of JNK or ANXA7 inhibited the expression of ATF2 and the lymphocyte adhesion of HCC cells.4.The JNK/ANXA7 signaling pathway may affect the biological function of HCC cells through ATF2.5.The down-regulation of JNK can inhibit the expression of ATF2-related lncRNA NONMMUT114121.1,and the down-regulation of ANXA7 promoted the expression of ATF2-related lncRNA NONMMUT114121.1,which may be involved in the early biological development of HCC metastasis. |
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