The Role Of Hydroxysterol Sulfotransferase SULT2B1b In The Development Of Hepatocellular Carcinoma And Its Underlying Mechanism

Hydroxysteroid sulfotransferase2B1b (SULT2B1b) is one of the most important steroid-metabolizing enzyme, which is highly selective for the sulfation of3β-hydroxysteroids. Recent studies found that oxysterols play an important role in the pathogenesis of cancer. Although previous reports have suggested that SULT2B1b is correlated with endometrial cancer, breast cancer, prostate cancer and esophageal carcinoma, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. Studies about biological functions of SULT2Bb in the liver demonstrated that SULT2B1b was involved in the oxysterol sulfation, lipid metabolism, liver regeneration after partial hepatectomy and it promoted hepatocytes proliferation, which mechanism was mainly through regulating LXR signal pathway. Accordingly, we proposed the hypothesis that SULT2B1b might correlated with the development of liver cancer, which progression might affected by regulating SULT2B1b levels. In the present study, We investigated the relationship between SULT2B1b and malignant phenotype of hepatocellular carcinoma both in vitro and in vitro, and clarified its possible mechanism from cell proliferation and epithelial-mesenchymal transition perspectives. Therefore, there were two parts involved in our experiment, for one part, to study the effect of SULT2B1b on the cell proliferation and angiogenesis in the hepatocellular carcinoma cells both in vitro and in vivo. For another, to investigate the regulatory function of SULT2B1b with transcription factor protein/DNA array and analyze the relationship between SULT2B1b and hepatocellular carcinoma epithelial-mesenchymal transition.Part Ⅰ. The effect and mechanisms of hydroxysteroid sulfotransferase SULT2B1b on cell proliferation and angiogenesis in hepatocellular carcinoma cells both in vitro and in vivo. In the present study, we found that the SULT2B1b protein level in Hepal-6cells was much higher than normal mouse liver tissue and primary mouse hepatocytes. Besides, adenovirus-mediated SULT2B1b overexpression obviously promoted the growth of the mouse hepatocarcinoma cell line Hepal-6, while lentivirus-mediated SULT2B1b interference apparently inhibited its growth as assessed by the CCK-8assay. Likewise, inhibition of SULT2Blb expression induced cell-cycle G2/M arrest and apoptosis in Hepal-6cells by upregulating the expression of FAS and p-JNK, downregulating the expression of cyclinB1, BCL2and MYC in vitro and in vivo both at the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in Hepal-6nude mouse xenografts. Moreover, SULT2Blb was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721and BEL-7402cells. Knock-down of SULT2Blb inhibited cell growth and cyclinB1levels in human hepatocarcinoma SMMC-7721and BEL-7402cells. Besides, knock-down of SULT2Blb suppressed BEL-7402cells xenograft growth in vivo. In addition, Endothelial cell, bEnd.3. proliferation was inhibited by SULT2B1b interference conditioned medium. Knock-down of SULT2B1b expression significantly decreased microvessel density in Hepal-6nude mouse xenografts. Those results demonstrated that SULT2Blb promoted hepatocellular carcinoma cell proliferation and angiogenesis in vitro and in vivo.Part II. The effect and regulation function of SULT2Blb on epithelial-mesenchymal transition in hepatocellular carcinoma. We detected transcription factor activities of BEL-7402cells with SULT2Blb interference with345spots protein/DNA array. The difference with two-fold increase or decrease was statistically significant. The results showed that168spots were significant, in which32spots were up regulated and136spots were down regulated including ZEB1and Snail transcription factors associated with EMT. Aside from that, We investigated the relationship between SULT2B1b and epithelial-mesenchymal transition (EMT) in human hepatocarcinoma cell line BEL-7402with high expression level of SULT2B1b and mouse primary hepatocytes with low expression level of SULT2B1b respectively, and explored its underlying mechanism further. Western blot results showed that, SULT2Blb interference inhibited EMT by up-regulating zonula occludens-1(ZO-1), down-regulating ZEB1protein level in BEL-7402hepatocarcinoma cells. Aside from that, SULT2Blb overexpression promoted TGF-0induced EMT in mouse primary hepatocytes by regulating ZEB1and Snaill transcriptional factors, which decreased E-cadherin level and increased vimentin level accordingly. Those results suggested that SULT2B1b participate EMT process throuth affecting epithelial-mesenchymal markers, which regulated by EMT associated transcriptional factors in hepatocellular carcinoma.In conclusion, our findings suggested SULT2Blb highly expressed in hepatocellular carcinoma tissues and correlated with malignant phenotype of hepatocellular carcinoma cells. SULT2Blb interference induced G2/M cell-cycle arrest and apoptosis, suppressed tumorigenicity and angiogenesis by up-regulating the expressions of FAS and p-JNK, down-regulating the expressions of cyclinB1, BCL2and MYC both in vitro and in vivo. In addition, SULT2Blb were involved in EMT process by regulating transcription factors and its downstream genes associated with EMT markers. Our findings illustrated the role of SULT2B1b in the development of hepatocellular carcinoma. We proposed that SULT2Blb may be employed as a novel therapeutic target for hepatocellular carcinoma.