Yiqi Wu

Hepatitis B virus induces G1 phase arrest by regulating cell cycle genes in HepG2.2.15 cells

BackgroundTo investigate the effect of HBV on the proliferative ability of host cells and explore the potential mechanism.MethodsMTT, colony formation assay and tumourigenicity in nude mice were performed to investigate the effect of HBV on the proliferative capability of host cells. In order to explore the potential mechanism, cell cycle and apoptosis were analysed. The cell cycle genes controlling the G1/S phase transition were detected by immunohistochemistry, westernblot and RT-PCR.ResultsHepG2.2.15 cells showed decreased proliferation ability compared to HepG2 cells. G1 phase arrest was the main cause but was not associated with apoptosis. p53, p21 and total retinoblastoma (Rb) were determined to be up-regulated, whereas cyclinE was down-regulated at both the protein and mRNA levels in HepG2.2.15 cells. The phosphorylated Rb in HepG2.2.15 cells was decreased.ConclusionsOur results suggested that HBV inhibited the capability of proliferation of HepG2.2.15 cells by regulating cell cycle genes expression and inducing G1 arrest.

Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

AIM To investigate the biological features of hepatitis B virus (HBV)-transfected HepG2.2.15 cells. METHODS The cell ultrastructure, cell cycle and apoptosis, and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay. Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation in nude mice, and the pathological analysis of tumor formation was performed. Two cytoskeletal proteins were detected by Western blotting. RESULTS Compared with HepG2 cells, HepG2.2.15 cells showed organelle degeneration and filopodia disappearance under electron microscope. HepG2.2.15 cells proliferated and migrated slowly in vitro, and hardly formed tumor and lung metastasis in nude mice. Flow cytometry showed that the majority of HepG2.2.15 cells were arrested in G1 phase, and apoptosis was minor in both cell lines. Furthermore, the levels of cytoskeletal proteins F-actin and Ezrin were decreased in HepG2.2.15 cells. CONCLUSION HepG2.2.15 cells demonstrated a lower proliferation and invasion ability than the HepG2 cells due to HBV transfection.

The response to interferon is influenced by hepatitis B virus genotype in vitro and in vivo.

PURPOSE To investigate the effectiveness of an interferon administration on different genotypes of hepatitis B virus (HBV) in vitro and in vivo. METHODS In vitro, we transfected plasmids carrying different HBV genotypes including recently identified new genotype I into HepG2 and HuH7 cells, then treated with standard interferon alpha (IFN-α); in vivo, we treated mice with pegylated interferon alpha (Peg-IFN-α) after injection with HBV DNA of different genotypes. The culture supernatants from cell culture and sera from mice were collected and used in hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) assays by ELISA and HBV DNA measurement by PCR. RESULTS Both in cell culture and in mouse model, it was observed that HBV genotypes A and B exhibited significantly better response to IFN-α2a or Peg-IFN-α2a in terms of reduced expression of HBsAg, HBeAg and the HBV DNA level as compared to HBV genotypes C and D. Moreover, the inhibitory effect of IFN-α2a or Peg-IFN-α2a on HBV genotype I was greater than on genotype C or D, but less than genotype A. However, there was no significant response difference between genotypes A and B, C and D, B and I, respectively. CONCLUSION The effectiveness of IFN/Peg-IFN to suppress HBV replication is dependent on different HBV genotypes. IFN/Peg-IFN is more effective on HBV genotype A or B than on genotype C, D or I. Treatment regimens are suggested to be adapted to HBV genotype.

Detection of hepatitis B virus DNA in paraffin-embedded intrahepatic and extrahepatic cholangiocarcinoma tissue in the northern Chinese population

This study explored the importance of hepatitis B virus infection in cholangiocarcinoma pathogenesis in northern China. The clinical data of 66 patients with cholangiocarcinoma were analyzed. The hepatitis B virus gene was amplified using nested polymerase chain reaction, and the hepatitis B virus-related antigen was detected using immunohistochemistry in formalin-fixed, paraffin-embedded tissue from patients with intrahepatic cholangiocarcinoma (n = 23) and extrahepatic cholangiocarcinoma (n = 43). Hepatitis B surface antigen seropositivity was found in 52.2% (12/23) of intrahepatic cholangiocarcinoma cases and 13.9% (6/43) of extrahepatic cholangiocarcinoma cases. Hepatitis B virus DNA (X region) was detectable in 34.8% (8/23) of intrahepatic cholangiocarcinoma cases. Hepatitis B surface antigen and/or hepatitis B core antigen was detectable in 30.4% (7/23) of intrahepatic cholangiocarcinoma cases. All cases with detected viral protein were also positive for hepatitis B virus DNA. In contrast, no hepatitis B virus antigens or hepatitis B virus gene was detected in any of the 43 extrahepatic cholangiocarcinoma cases. Our findings strongly suggest that chronic hepatitis B virus infection is a significant risk factor for intrahepatic cholangiocarcinoma, but not for extrahepatic cholangiocarcinoma, in northern China. Hepatitis B virus infection is potentially independently associated with intrahepatic cholangiocarcinoma.

Increased expression of kindlin‐2 is correlated with hematogenous metastasis and poor prognosis in patients with clear cell renal cell carcinoma

Kindlin‐2 is involved in activating the integrin signaling pathway which plays an important role in regulating cancer cell invasion. However, the role of kindlin‐2 may vary among cancer types. The aim of this study was to explore the possible association between kindlin‐2 and clear cell renal cell carcinoma (ccRCC), and its potential role in the prognosis of ccRCC. Immunohistochemistry assays were used to examine kindlin‐2 expression levels in cancer tissues obtained from 336 patients with ccRCC. The correlation between kindlin‐2 expression levels and pathologic variables was then analyzed. In addition, the association between kindlin‐2 expression levels and survival time was analyzed by Kaplan–Meier survival curves and log‐rank tests. Of 336 ccRCC patients, 199 had high levels of kindlin‐2 expression, while 137 had low kindlin‐2 expression levels. Patients at a late stage of ccRCC (stage III or IV) were more likely to have high kindlin‐2 expression levels than those at an early stage (stage I or II) (χ2 = 4.72, P = 0.03). Patients with high levels of kindlin‐2 expression had higher risk of hematogenous metastasis (χ2 = 6.70, P = 0.01) than those with low levels of kindlin‐2 expression. In addition, the survival time was significantly shorter for patients with high levels of kindlin‐2 expression than for those with low levels of kindlin‐2 expression (P = 0.001 for overall survival [OS] and P = 0.002 for disease‐free survival [DFS]). Multivariate survival analysis based on the Cox proportional hazards model showed that high kindlin‐2 expression levels had a hazard risk (HR) of 1.76 for OS (95% CI 1.19–2.62, P = 0.005) and an HR of 1.47 for DFS (95% CI = 1.05–2.06, P = 0.026). By comparison, lymph node metastasis had an HR of 1.48 for OS (95% CI 1.04–2.10, P = 0.029) and an HR of 1.41 for DFS (95% CI 1.04–1.93, P = 0.029). This study provided strong evidence that increased kindlin‐2 expression might be involved in promoting tumor invasiveness and leading to a poor prognosis of ccRCC.

EF24 Suppresses Invasion and Migration of Hepatocellular Carcinoma Cells In Vitro via Inhibiting the Phosphorylation of Src

Diphenyl difluoroketone (EF24), a curcumin analog, is a promising anticancer compound that exerts its effects by inhibiting cell proliferation and inducing apoptosis. However, the efficacy of EF24 against cancer metastasis, particularly in hepatocellular carcinoma (HCC), remains elusive. In this study, the effect of EF24 on HCCLM-3 and HepG2 cell migration and invasion was detected by wound healing and transwell assay, respectively. The results revealed that EF24 suppressed the migration and invasion of both HCCLM-3 and HepG2 cells. Furthermore, EF24 treatment decreased the formation of filopodia on the cell surface and inhibited the phosphorylation of Src in both cell lines, which may help contribute towards understanding the mechanism underlying the suppressive effect of EF24 on HCC migration and invasion. Additionally, the expression of total- and phosphorylated-Src in primary HCC tissues and their paired lymph node metastatic tissues was detected, and phosphorylated-Src was found to be associated with HCC lymph node metastasis. The results of this study suggest that Src is a novel and promising therapeutic target in HCC and provide evidence to support the hypothesis that EF24 may be a useful therapeutic agent for the treatment of HCC.

High load hepatitis B virus replication inhibits hepatocellular carcinoma cell metastasis through regulation of epithelial-mesenchymal transition.

OBJECTIVES The aims of this study were to investigate the effect of hepatitis B virus (HBV) replication on the metastatic ability of hepatocellular carcinoma (HCC) cells and to explore a potential mechanism from the perspective of epithelial-mesenchymal transition (EMT). METHODS Two short-interfering RNAs (siRNAs) against the HBV S gene were used to inhibit HBV replication in HepG2.2.15 cells. To evaluate the level of HBV replication and interference efficiency, HBV antigen and HBV DNA were detected by ELISA and quantitative PCR (Q-PCR). Invasion and metastatic abilities were compared between different groups by wound healing and trans-well assays. Immunofluorescent staining and Western blotting were utilized to detect EMT markers. RESULTS Both siRNAs effectively inhibited HBV replication in HepG2.2.15 cells. Compared to control HepG2.2.15 cells, cells transfected with the siRNAs showed characteristics of the mesenchymal phenotype and augmented their ability to invade and metastasize. Inhibition of HBV replication suppressed E-cadherin and induced a switch to vimentin expression. Western blots confirmed the decrease in E-cadherin expression. The level of E-cadherin expression was also lower in HepG2 cells than in HepG2.2.15 cells. CONCLUSIONS siRNAs were able to effectively inhibit HBV replication in vitro. A high load of HBV replication may inhibit the invasion and metastatic ability of HCC cells by reversing the EMT process.

Plasma Gelsolin Induced Glomerular Fibrosis via the TGF-β1/Smads Signal Transduction Pathway in IgA Nephropathy

Glomerular fibrosis has been shown to be closely related to the progression and prognosis of IgA nephropathy (IgAN). However, mechanism underlying IgAN glomerular fibrosis remains unclear. Recently, our study showed that plasma gelsolin (pGSN) was decreased in the serum of an IgAN mouse model and that pGSN deposition was found in the glomeruli. Another cytokine, TGF-β1, which is closely related to glomerular fibrosis, was also found to be highly expressed in the glomeruli. In the present study, we report that pGSN induces glomerular fibrosis through the TGF-β1/Smads signal transduction pathway. This is supported by the following findings: human mesangial cells (HMCs) show remarkable morphological changes and proliferation in response to co-stimulation with pGSN and polymeric IgA1 (pIgA1) from IgAN patients compared to other controls. Moreover, ELISA assays showed that more TGF-β1 secretion was found in HMCs supernatants in the co-stimulation group. Further experiments showed increased TGF-β1, Smad3, p-Smad2/3, Smad4, and collagen 1 and decreased Smad7 expression in the co-stimulation group. Our present study implied that the synergistic effect of pGSN and pIgA induced glomerular fibrosis via the TGF-β1/Smads signal transduction pathway. This might be a potential mechanism for the glomerular fibrosis observed in IgAN patients.

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin

Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer.

Corrigendum to High load hepatitis B virus replication inhibits hepatocellular carcinoma cell metastasis through regulation of epithelial-mesenchymal transition: International Journal of Infectious Diseases [20 (2014)] 37-41

Tianzhen Wang , Yinji Jin , Ran Zhao , Yiqi Wu, Yuhua Zhang , Di Wu, Dan Kong , Xiaoming Jin *, Fengmin Zhang ** Department of Pathology, Basic Medical Science College, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081, China Department of Obstetrics and Gynecology, First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Gynecology, Third Affiliated Hospital of Harbin Medical University, Harbin, China Heilongjiang Provincial Key Laboratory for Infection and Immunity, Harbin Medical University, Harbin, China Department of Microbiology, Basic Medical Science College, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081, China