Study Of Proliferative Effects Of HSD17B4 On Hepatocellular Carcinoma Cells And Its Mechanism

Human hepatocellular carcinoma(HCC), which is one of the most malignant cancers in the world, is closely associated with a history of chronic hepatitis caused by viral infection(such as hepatitis B or C virus), metabolic injury, toxic insults or autoimmune reactions. In the great majority of cases, HCC arises in a setting of chronic inflammation and subsequent liver fibrosis and cirrhosis. Additionally, the enhanced production by inflammatory cytokines causes hepatic inflammation and the activation of oncogenes, which then results in liver damage, in fine, promoting HCC progression. Nuclear factor-kappa B(NF-κB) is one of such inflammatory cytokines. NF-κB links inflammation to cancer development and progression. NF-κB is activated and then is freed to enter the nucleus, where the activated NF-κB can promote the expression of its downstream genes with binding sites for NF-κB. Because many downstream genes are involved in inflammation and proliferation, NF-κB activation is a frequent and early event in human liver cancers of viral or nonviral etiologies and has been associated with the acquisition of a transformed phenotype during hepatocarcinogenesis.A relevant aspect of HCC epidemiology is the gender difference in the incidence of this neoplasm. The incidence of HCC is higher in men than in women. Many results suggest that the pathogenesis and development of HCC might be affected by sex hormones, particularly estradiol(E2) levels. E2 can suppress the pathological progression of HCC.17β-Hydroxysteroid dehydrogenase 4(HSD17B4) is ubiquitously found and participates in estradiol metabolism. HSD17B4 can catalyze the conversion of E2, which is the most potent and active form of estrogen, to estrone(E1), which is the less active form of estrogen. HSD17B4 over-expression was found in prostate cancer tissues. So we hypothesized that,whether the expression of HSD17B4 was also increased in HCC? Did it promote the development process of cancer by reducing the level of E2? Is there a link between the inactivation of E2 by HSD17B4 and the activation of proliferative genes?In the present study, we used HCC model of rats, human liver tumor tissue and adjacent tissue, and Hep G2 cells to prove our hypothesized. Firstly, the expression of HSD17B4 was increased in HCC tumor tissue, and the linear positive correlation between the expression of inflammatory factors or proliferative genes and HSD17B4. Secondly, it was demonstrated that HSD17B4, as a NF-κB target gene, could be up-regulated and then promote Hep G2 cell proliferation through decreasing levels of E2. Finally, it was found that the activation of STAT3 was involved in the proliferation of HCC cell promoted by over-expression of HSD17B4. Taken together, our findings indicate for the first time that the higher expression of HSD17B4 plays an important role in aggravated HCC progression and provides a novel therapeutic intervention for HCC.PartⅠStudy on the correlation among HSD17B4 expression, inflammation and proliferation in liver of HCC model ratsObjective: To detect the expression and activation of HSD17B4, the expression of inflammatory factors and genes related proliferation for the evaluation of their correlation.Methods: 1 HCC model rats were induced by Diethylnitrosamine(DEN). 2 Detection of liver damage by HE. 3 Detection of protein expression of p-Akt, p-MEK, p-ERK and HSD17B4 by Western blotting. 4 Detection of expression of HSD17B4 in rats and human HCC liver by immunohistochemistry. 5 Detection of m RNA expression of HSD17B4, IL-6, TNF-α, cyclin D1 and PCNA by Real-time PCR. 6 Detection of E2 level by radioimmunoassay. 7 Analysis of expression correlation between HSD17B4 and inflammatory factors, or proliferatve gene by spearman correlation test.Results: 1 The stablishment of HCC model of ratsHE staining and blood biochemical indexes showed grave damage and a trend of deterioration in HCC rat liver tissue. The Akt and MEK/ERK phosphorylation levels were increased significantly in liver of HCC model rats compared to the NC rats. The results confirmed that HCC model of rats had been established.2 The expression of HSD17B4 was increased in liver of HCC model of rat and human liver tumor tissueThe protein expression of HSD17B4 was higher 2 times in HCC group than NC group by Western blotting and immunohistochemical, and the m RNA expression of HSD17B4 was higher 3.5 times. While the immunohistochemisty images also shown that HSD17B4 expressed in tumor tissues more than that in tumor adjacent tissue of livers from HCC patients. These demonstrated HSD17B4 expression was increased in HCC rat liver.3 E2 levels of liver and serum were decreased in HCC model of ratsThe level of E2 was significantly reduced in liver tissue and serum of rats with HCC by radio-immunity.4 The linear positive correlation between the expression of inflammatory factors and HSD17B4The m RNA expression of IL-6 and TNF-α in liver was significantly higher in HCC group by real-time PCR. The results of Pearson correlation test showed the linear positive correlation between the expression of inflammatory factors and HSD17B4(IL-6: r2 = 0.6233, P < 0.05 and TNF-α: r2 = 0.2281, P < 0.05).5 The linear positive correlation between the expression of HSD17B4 and genes related to proliferationThe m RNA expression of cyclin D1 and PCNA was significantly higher in HCC group by real-time PCR. The results of Pearson correlation test showed the linear positive correlation between the expression of HSD17B4 and proliferation-related genes(cyclin D1: r2 = 0.4053, P < 0.01 and PCNA: r2 = 0.4925, P < 0.01).Summary: Expression and activity of HSD17B4 in HCC liver was significantly increased. There was a positive correlation between the expression of HSD17B4 and proliferation-related genes in livers of HCC model of rats.Part II NF-κB-increased expression of HSD17B4 promotes Hep G2 proliferation via inactivating estradiolObjective: To investigate the role of HSD17B4 in hepatoma cell proliferation progression induced by TNF-α and the possible link among NF-κB, HSD17B4 and E2.Methods: 1 Detection of HSD17B4-promoting proliferation by CCK-8 and Brd U incorporation. 2 Detection of the expression of cyclin D1, PCNA, HSD17B4, IL-6 and IL-8 by Western blotting. 3 Detection of the m RNA expression of HSD17B4, IL-6 and IL-8 by real-time PCR. 4 Detection of NF-κB binding to site A of HSD17B4 upstream, and the HSD17B4 transcriptional activation by reporter gene assay and mutation assay. 5 Demonstration of specificity of NF-κB binding to site A by Ch IP and Oligo pull down experiments. 6 Detection of E2 by radioimmunoassay. 7 Detection of HSD17B4 expression in human HCC liver tissues by immunofluorescence. 8 Demonstration of specificity of NF-κB binding to site A in HCC liver cancer and adjacent by situ hybridization combined immunofluorescence.Results: 1 Increased HSD17B4 is involved in the Hep G2 proliferationHSD17B4 over-expression increased the alive cell number by Brd U incorporation and the protein levels of cyclin D1 and PCNA. In contrast, Brd U incorporation and the protein contents were significantly alleviated by the knockdown of endogenous HSD17B4 using si RNAs. These results indicated that HSD17B4 affects Hep G2 proliferation.2 Inecreased HSD17B4 expression in HepG2 is associated with NF-κB activationHep G2 cells were treated with TNF-α to activate NF-κB, with PDTC to inhibit TNF-α activation, or with si RNA to knockdown NF-κB expression TNF-α caused an increase and PDTC caused a decrease in the expression of HSD17B4, and the NF-κB targets IL-6 and IL-8 were accompanied by NF-κB activation and inhibition. Furthermore, decreases in activated NF-κB, HSD17B4 and the NF-κB targets IL-6 and IL-8 were observed following NF-κB knockdown by si RNA. These results suggest that the activation of NF-κB promoted HSD17B4 expression.3 The distal NF-κB element in the human HSD17B4 promoter responds to activated NF-κBThe luciferase activity of the region site AB-wt of the HSD17B4 promoter was increased by TNF-α treatment and decreased by PDTC pretreatment before TNF-α treatment. However, no significant changes in the luciferase activity of the proximal region site B-wt of the HSD17B4 promoter were observed by identical treatments, suggesting that site A mediates the TNF-α-induced transactivation of HSD17B4. While the mutant fragments also implies site A is important for activation of HSD17B4 promoter activity. This implies that site A in the region of HSD17B4 promoter was required for NF-κB binding to active HSD17B4 expression.Ch IP assay and an oligonucleotide pull-down assay were performed, an increase in site A DNA fragments was associated with TNF-α treatment and the increase was prevented by PDTC pretreatment before TNF-α treatment, whereas site B DNA fragments were not significantly affected by identical treatments. The proteins that bind specially to the biotinylated double-stranded oligonucleotides containing putative site A or site B were precipitated with streptavidin-agarose beads and determined by western blot using an anti-NF-κB p65 antibody. These results indicate that the putative site A in the HSD17B4 promoter is an enhancer element that binds to NF-κB to mediate the TNF-α-induced transcription of HSD17B4 in Hep G2 cells.4 NF-κB-upregulated expression of HSD17B4 decreases E2 levelsTNF-α treatment and HSD17B4 over-expression reduced the levels of E2 in the culture medium compared with their respective controls. Hep G2 cells were treated with E1 or E2, we found that the cell proliferation by E2. These results indicate that NF-κB at least partially up-regulate HSD17B4-aggravated hepatoma cell proliferation by inactivating E2.5 HSD17B4 expression is incremental and its promoter site A is co-located with more NF-κB in liver tumor tissues from HCC patientsThe inmmunofluorecence images shown that HSD17B4 expressed in tumor tissues more than that in tumor adjacent tissue of livers from HCC patients. We assessed the co-location of NF-κB and putative NF-κB binding element(Site A) at HSD17B4 promoter in liver tissues from HCC patients by FISH combing IF. The tumor tissues have more activated NF-κB entering nuclei and binding to the site A sequence of HSD17B4 promotor when compared to tumor adjacent tissues, in paralled with the increase in HSD17B4 expression. These results indicate that enhanced HSD17B4 expression by activated NF-κB is associated with the development of HCC.Summary: HSD17B4 is the target gene of NF-κB in HCC cells. The activated NF-κB by TNF-α promoted the expression of HSD17B4. HSD17B4 plays a role in promoting proliferation of Hep G2 by inactivation of E2.Part III HSD17B4 promoted the proliferation of Hep G2 cells by the activation of STAT3Objective: To determine the molecular mechanism that HSD17B4 promoted the proliferation of HCC.Methods: 1 Detection of HSD17B4 location by inmmunofluorecence. 2 Detection of the expression of p-STAT3, p-Akt, p-MEK and p-ERK by Western blotting. 3 Detection of the expression of HSD17B4 and p-STAT3 in human HCC liver tissues by immunohistochemistry. 4 Correlation analysis between IOD of HSD17B4 and IOD of p-STAT3.Results: 1 Over-expression of HSD17B4 was located in the cytoplasm HSD17B4 and PMP70 overlaped to produce yellow fluorescence in control group, and the same conditions, HSD17B4 and GAPDH did not overlap to yellow fluorescence. It indicated that HSD17B4 was located in the peroxisome in control.Over-expressed HSD17B4 and PMP70 did not overlap to produce yellow fluorescence in control group, but HSD17B4 and GAPDH overlaped to produce yellow fluorescence. It indicated that some HSD17B4 were located in the cytoplasm.2 Over-expression of HSD17B4 promoted the activation of STAT3 The phosphorylation level of STAT3 was positive regulated by the expression of HSD17B4. The results of Pearson correlation test showed the linear positive correlation between the IOD of HSD17B4 and p-STAT3(r2 = 0.6359, P < 0.01).3 Over-expression of HSD17B4 promoted the phosphorylation of MEK/ERK and AktThe phosphorylation level of Akt, ERK and ERK was positive regulated by the expression of HSD17B4, and the expression of p-STAT3 was decreased by the inhibitors of Akt and ERK/ERK. The evidence showed HSD17B4 promoted proliferation by activation of STAT3.Summary: Highly expressed HSD17B4 was located in cytoplasm of Hep G2 cells and inactivated E2, the later leaded to the activation of STAT3 via the promotion of Akt and ERK signal pathway, and then results in proliferation of HCC.Conclusions: Highly expressed HSD17B4 in HCC liver promotes the cell proliferation via inactivation of E2.