Expression And Relationship Of APEX1, Jagged1 In Hepatocellular Carcinoma

Objective:To study the expression of APEX1、jagged1 in hepatocellular carcinoma(HCC). To assess the correlation of APEX1,jagged1 expression with clinical pathological characteristics. Then detect the expression and the correlation of Notch1、Notch2、Notch3、Notch4 m RNA with jagged1.Lentivirus-mediated recombinants including LV-APEX1-RNAi(induce APEX1 downregulation) and NC(negative control) were constructed. Lentivirus-mediated recombinants including LV-APEX1-RNAi(induce APEX1 downregulation) and NC(negative control) were constructed. Liver cancer cells SMMC-7721 were infected with LV-APEX1-RNAi and their corresponding NC respectively. The expression of APEX1 m RNA and APEX1 protein were detected with Real-time PCR and western Blot after they were infected with the corresponding recombinants, investigate the role of APEX1 in the invasiveness and metastasis of human liver cancer cells and the potential molecular mechanisms.Methods:1、 To investigate the level of APEX1、jagged1 m RNA in 46 samples of HCC 、corresponding adjacent liver tissues and far-carcinoma tissues,13 samples of normal liver tissues by using Real-time polymerase chain reaction(Real-time PCR),and analysis the relationship between APEX1 and jagged1;detected the Notch1、Notch2、Notch3、Notch4 m RNA expression level in 36 samples of HCC tissues, and evaluate the correlation with jagged1.2、 Using Western Blot, to detect the protein expression of APEX1 in 25 samples of HCCs and corresponding adjacent liver tissue; and to detect the protein expression of jagged1 in 18 samples of HCCs and corresponding adjacent liver tissue.3、 Immunohistochemistry(IHC) and high-throughput tissue microarray technique were applied to detect the protein expression of APEX1、jagged1 in 119 samples of HCC and corresponding adjacent liver tissue. Then we evaluate the correlation in these proteins. The relationships between protein expression and HCC clinicopathological parameters were also analyzed.4、 Lentivirus-mediated recombinants including LV-APEX1-RNAi(induce APEX1 downregulation) and NC(negative control) were constructed. Liver cancer cells SMMC-7721 were infected with LV-APEX1-RNAi and their corresponding NC respectively. The expression of APEX1 m RNA and APEX1 protein were detected with Real-time PCR and western Blot after they were infected with the corresponding recombinants.5、 To study the effect of APEX1 on the proliferation 、migration and invasion of liver cancer cells, SMMC-7721 cells with APEX1 downregulation(SMMC-7721-LV-APEX1-RNAi) and their corresponding controls were detected with the assays of MTT and transwell.6、 The expression of jagged1 were detected in the SMMC-7721-LV-APEX1-RNAi and the corresponding controls with western Blot.Results:1、 The expression of APEX1 m RNA were 2.154±1.644、1.152±0.689、1.352±0.818、0.960±0.039 in HCCs、para-carcinoma tissues、far-carcinoma tissues and normal liver tissues respectively. APEX1 was significantly higher in HCCs than normol tissues. There was statistical significance among each group(P<0.01). The expression of APEX1 in HCCs was associated with tumor differentiation level, capsuler invasion, and postoperative recurrence(P<0.05),not associated with age、 gender、preoperative AFP level、 cirrhosis、HBV infection、 tumor size、tumor number.2、 The result of Western Blot and Immunohistochemistry staining showed that the average level of APEX1 and jagged1 protein in HCCs was higher than corresponding adjacent liver tissues, which with statistically significant difference.3、 The relationships between jagged1 protein expression and HCCclinicopathological parameters shows that the expression of jagged1 protein was associated with tumor differentiation level, preoperative AFP level, tumor invasion and metastasis, Postoperative recurrence, not associated with gender、 age、 tumor size、 cirrhosis、vascular invasion(P<0.05).4、 The spearman rank correlation test showed that the APEX1 expression was positively correlated with jagged1(P?0.05);the expression of jagged1 m RNA was positively correlated with Notch3(r=0.501,P=0.002)and was not correlated with Notch1、Notch2、Notch4.5、 Compared with the control, the expression of APEX1 m RNA and APEX1 protein in SMMC-7721 cells were significantly decreased after being infected with LV-APEX1-RNAi.6、 Compared with the control, the MTT assays showed that APEX1 downregulatin inhibited the proliferation capacity of SMMC-7721 cells; the transwell assays showed that APEX1 downregulatin inhibited the invasion and migration capacity of SMMC-7721 cells(P?0.01).7、 Western Blot results revealed that compared with the control, in SMMC-7721-LV-APEX1-RNAi APEX1 protein expression was decreased, jagged1 protein expression was also decreased.Conclusion:1、 APEX1 promotes the invasiveness and metastasis of human liver cancer cells.2、 The positive expression of jagged1 protein probably promote the progression of HCC.3、 APEX1 might enhance hepatocarcinogesis through the upregulation of jagged1/Notch signaling pathway.