Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter‐κB/p65 in hepatoma cells

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real‐time quantitative RT‐PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor‐κB (NF‐κB) mediated by siRNA targeting NF‐κB/p65 mRNA or PDTC (an inhibitor of NF‐κB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF‐κB binding site of the Capn4 promoter was mutant, suggesting that NF‐κB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF‐κB/p65 to promote migration of hepatoma cells. J. Med. Virol. 82:920–928, 2010. © 2010 Wiley‐Liss, Inc.     

miR-335靶向Sp1对人骨肉瘤MG-63细胞增殖的调控

 目的：探讨 miR-335对人骨肉瘤MG-63细胞增殖的调控是否通过靶向抑制Sp1基因的表达实现。方法：利用萤光素酶报告基因实验验证 miR-335能否靶向作用于Sp1基因的3’-非翻译区(untranslated region, UTR);利用real-time PCR和Western blotting分析Sp1 mRNA和蛋白表达的变化;通过 MTT法分析MG-63细胞增殖水平。结果：萤光素酶报告基因实验结果显示,miR-335可特异性结合于Sp1基因的3’-UTR。Real-time PCR和Western blotting结果显示,转染miR-335可减少Sp1蛋白的表达,但对mRNA表达无显著影响;转染Sp1表达质粒可上调Sp1 mRNA和蛋白表达。MTT结果显示,miR-335可抑制MG-63细胞增殖,转染Sp1表达质粒可部分逆转miR-335对MG-63细胞增殖的抑制作用。结论: miR-335可能通过靶向Sp1基因抑制人骨肉瘤MG-63细胞增殖。     

鼠胎肝干细胞生物学特性与人端粒酶反转录酶基因介导的影响

背景：研究表明,感染人端粒酶反转录酶基因后的干细胞具有稳定表达高水平端粒酶活性,且能使细胞增殖旺盛,这为建立基因工程的永生化干细胞系奠定了基础。 目的：探讨人端粒酶反转录酶基因感染对体外培养鼠胎肝干细胞增殖及细胞周期的影响。 方法：体外培养鼠胎肝干细胞,经重组腺相关病毒作为载体介导人端粒酶反转录酶基因感染,用RT-PCR、Western blot检测鼠胎肝干细胞人端粒酶反转录酶基因和蛋白的表达,CCK-8法、细胞生长曲线检测细胞生长增殖情况,流式细胞仪测定细胞周期分布的变化。 结果与结论：与对照组、空载病毒组相比,基因感染组人端粒酶反转录酶基因和蛋白水平均有表达,细胞的生长速度明显增快,G0/G1期细胞减少,S期细胞数增多。结果表明以重组腺相关病毒作为载体介导人端粒酶反转录酶基因感染能促进体外培养的鼠胎肝干细胞增殖,对其培养有优化作用。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues

The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.     

Effects of telomerase modulation in human hematopoietic progenitor cells

Loss of telomeric repeats has been causally linked to replicative senescence and aging in human cells. In contrast to normal somatic cells, which are telomerase-negative, hematopoietic stem cells have low levels of telomerase, which can be transiently upregulated upon cytokine stimulation. To examine whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we overexpressed telomerase in CD34+ and AC133+ cord blood (CB) cells using retroviral vectors containing hTERT, the catalytic component of telomerase. Although the hTERT-transduced CB cells exhibited significantly elevated telomerase activity (approximately 10-fold), the mean telomere length was only increased up to 600 bp, which was in contrast to hTERT-transduced fibroblast cells gaining more than 2-kb telomeric repeats. Moreover, ectopic telomerase activity did not prevent overall telomere shortening, which was in the range of 1.3 kb in serum-free expansion culture. We also blocked endogenous telomerase activity by ectopic expression of dominant-negative hTERT. Whereas CB cells with absent telomerase activity showed reduced absolute numbers of colony-forming cells, we observed increased rates only for burst-forming units erythroid when the enzyme was overexpressed. These results suggest that telomere shortening in human hematopoietic progenitor cells cannot be compensated by increased levels of telomerase alone and is likely to be dependent on other factors, such as telomere binding proteins. Furthermore, telomerase function seems to be directly associated with the proliferative capacity of stem cells and may exert an additional role in lineage differentiation.     

Could HBx Protein Expression Affect Signal Pathway Inhibition by Gefitinib or Selumetinib,a MEK Inhibitor,in Hepatocellular Carcinoma Cell Lines?

Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as β-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and β-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and β-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.