The AT1/Raf/ERK1/2 signaling pathway is involved in Angiotensin II-enhanced proliferation of hepatic carcinoma cells.

Angiotensin II (Ang II) has been strongly associated with biological behavior in human malignant tumors; nevertheless, its function in hepatic carcinoma growth and progression is still not well understood. This study investigates the effect and mechanism of Ang II on the HepG2 and Hep3B hepatic carcinoma cell lines in vitro. The effect of Ang II on HepG2 and Hep3B cell viability was examined by cell counting kit-8 assay (CCK-8). Quantitative real-time PCR and Western blot analysis detected the expression of angiotensin type 1 and type 2 receptors (AT1 and AT2), total extra-cellular signal-regulated kinases 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) and Bcl-2 and c-Myc. Ang II significantly promoted HepG2 cell proliferation by affecting AT1 and AT2 expression and induced ERK1/2 pathway activation. This was reversed by treating HepG2 and Hep3B cells with AT1 blockers; candesartan, Raf inhibitor sorafenib, and ERK1/2 inhibitor PD98059. Ang II also up-regulated the expression of Bcl-2 and c-Myc in HepG2 cells, and our results suggest that Ang II has a positive role in HepG2 and Hep3B cell proliferation through the AT1/Raf/ERK1/2 signaling pathway.

[Mechanism of angiotensin II (Ang II) on the proliferation of human hepatoma cell line HepG2 cells].

Objective: To study the effect and mechanism of angiotensin (Ang II) on the proliferation of human hepatocellular carcinoma HepG2 cells. Methods: The effects of different concentrations of Ang II's (10(-8)-10(-4) mol/L) on proliferated hepatocellular carcinoma HepG2 cells were detected by CCK-8 assay. The expression of angiotensin II type 1 receptor (AT1) protein and activation of ERK1/2 protein in hepatocellular carcinoma HepG2 cells after processing with Ang II were assayed by Western blot. The cells were pretreated with candesartan (AT1 receptor antagonist), sorafenib (Raf kinase inhibitor) and PD98059 (ERK1/2 inhibitor) for 1.5 h and then Ang II (10(-6) mol/L) was added. CCK-8 assay was used to determine whether it could reverse the proliferation of Ang II, and ERK phosphorylation levels were detected by Western blot. The changes in Bcl-2 and c-myc gene expression before and after Ang II processing were detected by Rt-PCR. According to different data, t-test, one-way analysis of variance or SNK method were used for statistical analysis. Results: HepG2 cells treated with different concentrations of Ang II promoted cell proliferation after 24h and 48h. After 24 h, cell vitality was strongest with Ang II concentration 10(-5) mol/L and the absorbance value was 0.990 8±0.097 8; and again after 48 h, the cell viability was strongest with Ang II concentration 10(-6) mol/L and the absorbance value was 1.302 7 ± 0.030 9. Moreover, the pro-proliferation effect of Ang II on HepG2 cells blocked candesartan, sorafenib and ERK1/2 isolated inhibitors. After treatment with 10(-6) mol/L Ang II, Western blot showed that Ang II significantly promoted AT1 receptor expression and phosphorylation of ERK1/2 protein confirmed that Ang II activated the AT1/RAF/ERK1/2 signaling pathway. In addition, Rt-PCR detection showed that the downstream of Bcl-2 and c-myc genes expressions rose significantly when the concentration of Ang II ranged from 10(-8) to 10(-6) mol/L. Conclusion: Ang II can promote the proliferation of HepG2 cells by activating AT1/Raf /ERK1/2 signaling pathway and enhance the downstream of Bcl-2 and c-myc gene expression.

LncRNA H19 promotes the development of hepatitis B related hepatocellular carcinoma through regulating microRNA-22 via EMT pathway.

OBJECTIVE: To explore the relationship between long non-coding RNA (lncRNA) H19 expression and prognosis of hepatitis B-related hepatocellular carcinoma (HBV-related HCC), and its underlying mechanism. PATIENTS AND METHODS: Expression level of lncRNA H19 in 36 HBV-related HCC tissues and para-cancerous tissues was detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The relationship between lncRNA H19 expression and prognosis of HBV-related HCC was analyzed by Kaplan-Meier method. Serum DNA levels of HBV were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR). For in vitro experiments, lncRNA H19 expression in HCC cell line, HBV-related HCC cell line and normal liver cell line was detected by qRT-PCR. After plasmids construction, the effects of lncRNA H19 on cell viability, migration, and invasion were detected by cell counting kit-8 (CCK-8), colony formation and transwell assay, respectively. Finally, protein levels of epithelial-mesenchymal transition (EMT) pathway-related genes were detected by Western blot. RESULTS: LncRNA H19 was highly expressed in HBV-related HCC tissues. The expression of lncRNA H19 was positively correlated with lymph node metastasis and distant metastasis, whereas negatively correlated with the overall survival of HBV-related HCC patients. Results of in vitro experiments showed that lncRNA H19 knockdown significantly downregulated cell proliferation and invasion. However, lncRNA H19 knockdown significantly upregulated apoptosis of HBV-related HCC cells. Western blot results demonstrated that lncRNA H19 remarkably decreased the protein expressions of EMT pathway-related genes, including N-cadherin, Vimentin, ß-catenin and MMP-9. In addition, rescue experiments demonstrated that lncRNA H19 remarkably promoted malignant development of HBV-related HCC via regulating microRNA-22. CONCLUSIONS: LncRNA H19 promotes malignant development of HBV-related HCC through regulating microRNA-22 via EMT pathway.