Jia-Jia Zhou

Epigenetic regulation of miR-124 by hepatitis C virus core protein promotes migration and invasion of intrahepatic cholangiocarcinoma cells by targeting SMYD3

Hepatitis C Virus core protein (HCVc) plays important roles in the development of intrahepatic cholangiocarcinoma (ICC). MicroRNAs (miRNAs) contribute to tumor progression by interacting with downstream target genes. However, the regulation and role of miRNAs in HCV‐related intrahepatic cholangiocarcinoma (HCV‐ICC) is poorly understood. In this study, we found that miR‐124 was down‐regulated in HCV‐ICC and the induction of DNMT1 by HCVc mediated the suppression of miR‐124. Over‐expression of miR‐124 suppressed cell migration and invasion in vitro, and reduced the protein levels of SMYD3 and downstream target genes (c‐Myc and MMP9). Knockdown of SMYD3 inhibited cell migration and invasion resembling that of miR‐124 over‐expression. In conclusion, our studies indicate that low miR‐124 levels mediated by HCVc via DNMT1 promote ICC cell migration and invasion by targeting SMYD3.

Overexpression of miR-122 promotes the hepatic differentiation and maturation of mouse ESCs through a miR-122/FoxA1/HNF4a-positive feedback loop

microRNA‐122 is the only identified liver‐specific miRNA and plays a crucial role in liver development, maintenance of hepatic homeostasis as well as tumourigenesis. In our previous differentiation of ESCs into hepatocytes, microRNA‐122 (miR‐122) was expressed at a relatively low level. Here, we aim to elucidate the effect and underlying mechanisms of miR‐122 during differentiation of ESCs into hepatocytes.

Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells

Increasing evidence has been accumulated indicating the important role of epigenetic regulation in tumor genesis. Previously, we observed that the transfection of hepatitis C virus core (HCVc) protein led to malignant transformation in normal biliary cells, and that tumor suppressor gene RASSF1A was downregulated in many hilar cholangiocarcinoma patients by hypermethylation in the promoter region. In the present study, we found SET and MYND domain-containing protein 3 (SMYD3), a novel histone methyltransferase, was overexpressed in cholangiocarcinoma patients especially in those with HCV infection. Transfection of HCVc into hilar cholangiocarcinoma cell lines QBC939 and FRH0201 could upregulate the expression of SMYD3 and promote cell growth, which was consistent with the results of our clinical research. This phenomenon indicated that SMYD3 was related to the epigenetic regulation of cholangiocarcinoma genesis with HCV infection. Overexpression of SMYD3 could inhibit RASSF1A expression, whereas inhibition of SMYD3 by siRNA improved its expression. Methylation-specific polymerase chain reaction (MS-PCR) results showed the methylation status of RASSF1A promoter was regulated by SMYD3. In conclusion, HCVc could upregulate the methylation status of the RASSF1A promoter through regulation of SMYD3, and histone methylation may affect the DNA methylation of downstream gene by an unknown mechanism.

Hepatitis C virus core protein regulates NANOG expression via the stat3 pathway

HCV Core plays a role in the development of hepatocellular carcinoma. Aberrant expression of NANOG has been observed in many types of human malignancies. However, relationship between Core and NANOG has not been clarified. In this study, we found that Core is capable of up‐regulating NANOG expression. Core‐induced NANOG expression was accompanied by enforced expression of phosphorylated stat3 protein and was attenuated by inhibition of stat3 phosphorylation. ChIP showed that phosphorylated stat3 directly binds to the NANOG promoter. Core‐induced NANOG expression resulted in enhanced cell growth and cell cycle progression. Knockdown of NANOG blocked the cell cycle at the G0/G1 phases and inhibited the cyclin D1 expression. Our findings provide a new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Metabolic phenotypes in pancreatic cancer.

Introduction The aim of present study was to profile the glucose-dependent and glutamine- dependent metabolism in pancreatic cancer. Methods We performed Immunohistochemical staining of GLUT1, CAIX, BNIP3, p62, LC3, GLUD1, and GOT1. Based on the expression of metabolism-related proteins, the metabolic phenotypes of tumors were classified into two categories, including glucose- and glutamine-dependent metabolism. There were Warburg type, reverse Warburg type, mixed type, and null type in glucose-dependent metabolism, and canonical type, non-canonical type, mixed type, null type in glutamine-dependent metabolism. Results Longer overall survival was associated with high expression of BNIP3 in tumor (p = 0.010). Shorter overall survival was associated with high expression of GLUT1 in tumor (P = 0.002) and GOT1 in tumor (p = 0.030). Warburg type of glucose-dependent metabolism had a highest percentage of tumors with nerve infiltration (P = 0.0003), UICC stage (P = 0.0004), and activated autophagic status in tumor (P = 0.0167). Mixed type of glucose-dependent metabolism comprised the highest percentage of tumors with positive marginal status (P<0.0001), lymphatic invasion (P<0.0001), and activated autophagic status in stroma (P = 0.0002). Mixed type and Warburg type had a significant association with shorter overall survival (P = 0.018). Non-canonical type and mixed type of glutamine-dependent metabolism comprised the highest percentage of tumors with vascular invasion (p = 0.0073), highest percentage of activated autophagy in tumors (P = 0.0034). Moreover, these two types of glutamine-dependent metabolism were significantly associated with shorter overall survival (P<0.001). Further analysis suggested that most of tumors were dependent on both glucose- and glutamine-dependent metabolism. After dividing the tumors according to the number of metabolism, we found that the increasing numbers of metabolism subtypes inversely associated with survival outcome. Conclusion Warburg type, non-canonical type and mixed types of glucose- and glutamine-dependent metabolism comprised of more metabolically active, biologically aggressive and poor prognostic tumors. Moreover, the increasing subtypes and categories of the metabolism in each tumor significantly associated with poor prognosis.

Inhibition of glutamine metabolism counteracts pancreatic cancer stem cell features and sensitizes cells to radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) cells utilize a novel non-canonical pathway of glutamine metabolism that is essential for tumor growth and redox balance. Inhibition of this metabolic pathway in PDAC can potentially synergize with therapies that increase intracellular reactive oxygen species (ROS) such as radiation. Here, we evaluated the dependence of pancreatic cancer stem cells (PCSCs) on this non-canonical glutamine metabolism pathway and researched whether inhibiting this pathway can enhance radiosensitivity of PCSCs. We showed that glutamine deprivation significantly inhibited self-renewal, decreased expression of stemness-related genes, increased intracellular ROS, and induced apoptosis in PCSCs. These effects were countered by oxaloacetate, but not α-ketoglutarate. Knockdown of glutamic-oxaloacetic transaminase dramatically impaired PCSCs properties, while glutamate dehydrogenase knockdown had a limited effect, suggesting a dependence of PCSCs on non-canonical glutamine metabolism. Additionally, glutamine deprivation significantly increased radiation-induced ROS and sensitized PCSCs to fractionated radiation. Moreover, transaminase inhibitors effectively enhanced ROS generation, promoted radiation sensitivity, and attenuated tumor growth in nude mice following radiation exposure. Our findings reveal that inhibiting the non-canonical pathway of glutamine metabolism enhances the PCSC radiosensitivity and may be an effective adjunct in cancer radiotherapy.

Knockdown of NANOG enhances chemosensitivity of liver cancer cells to doxorubicin by reducing MDR1 expression

Multidrug resistance (MDR) is one of the major reasons for the failure of liver cancer chemotherapy, and its suppression may increase the efficacy of chemotherapy. NANOG plays a key role in the regulation of embryonic stem cell self-renewal and pluripotency. Recent studies reported that NANOG was abnormally expressed in several types of tumors, indicating that NANOG is related to tumor development. However, the correlation between NANOG and liver cancer chemoresistance remains uncertain. In this study, RNA interfere technology was employed to knock down NANOG expression in HepG2 human liver cancer cells. We found that the knockdown of NANOG expression in NANOG siRNA-transfected HepG2 cells resulted in decreased colony formation rate and cell migration compared to control HepG2 cells. In addition, HepG2 cells were treated with doxorubicin to evaluate the chemosensitivity to doxorubicin. We found that the doxorubicin sensitivity of HepG2 cells was increased with downregulation of NANOG expression. The expression of MDR1 at both mRNA and protein levels was decreased in HepG2 cells when NANOG was knocked down. These findings suggest that the knockdown of NANOG in HepG2 human cells resulted in decreased MDR1 expression and increased doxorubicin sensitivity, and NANOG could be used as a novel potential therapeutic target to reverse multidrug resistance of liver cancer.

Knockdown of long non‑coding RNA HOTAIR sensitizes hepatocellular carcinoma cell to cisplatin by suppressing the STAT3/ABCB1 signaling pathway

Long non-coding RNA HOX transcript antisense RNA (HOTAIR) has been demonstrated to exhibit oncogenic activity in several types of cancer, including hepatocellular carcinoma (HCC). However, the association between HOTAIR and HCC multidrug resistance remains uncertain. The present study aimed to investigate the role of HOTAIR in HCC chemoresistance; it was found that knockdown of HOTAIR expression in HCC Huh7 cells resulted in decreased cell proliferation and increased chemosensitivity to cisplatin. Furthermore, expression levels of ATP binding cassette subfamily B member 1 (ABCB1) mRNA and protein were decreased in Huh7 cells upon HOTAIR-knockdown. In addition, HOTAIR-knockdown reduced the levels of phosphorylated signal transducer and activator of transcription 3 (STAT3), and inhibition of STAT3 phosphorylation reduced HOTAIR-mediated ABCB1 expression. Together, these findings indicated that knockdown of HOTAIR in Huh7 cells decreased STAT3 activity and ABCB1 expression, and increased chemosensitivity to cisplatin. Thus HOTAIR could serve as a novel potential therapeutic target to reverse multidrug resistance in HCC.

Hepatitis C virus core protein regulates OCT4 expression and promotes cell cycle progression in hepatocellular carcinoma

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatocellular carcinoma. octamer-binding protein 4 (OCT4) is critically essential for the pluripotency and self-renewal of embryonic stem cells. Abnormal expression of OCT4 has been detected in several human solid tumors. However, the relationship between HCV core and OCT4 remains uncertain. In the present study, we found that HCV core is capable of upregulating OCT4 expression. The effect of HCV core-induced OCT4 overexpression was abolished by RNAi-mediated scilencing of HCV core. In addition, HCV core-induced OCT4 overexpression resulted in enhanced cell proliferation and cell cycle progression. Inhibition of OCT4 reduced the CCND1 expression and induced G0/G1 cell cycle arrest. Furthermore, OCT4 protein directly binds to CCND1 promoter and transactivates CCND1. These findings suggest that HCV core protein regulates OCT4 expression and promotes cell cycle progression in hepatocellular carcinoma providing new insight into the mechanism of hepatocarcinogenesis by HCV infection.

Transfection of HCVc improves hTERT expression through STAT3 pathway by epigenetic regulation in Huh7 cells.

Previous studies showed that transient transfection of HCVc improved hTERT expression in hepatoma cell lines and it was noteworthy that phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and DNA methyltransferases (DNMTs) were up regulated simultaneously. This study was designed to investigate the role of epigenetic regulation in the process of hTERT up regulation after HCVc transfection. Q‐PCR and Western blot were used to analyze the expression of pSTAT3, DNMT1, and hTERT after the transfection of HCVc in hepatoma cell line Huh7. Proliferation and hTERT activity of Huh7 after HCVc transfection were examined by CCK8 and ELISA, respectively. Then, we blocked the JAK/STAT3 pathway or inhibited DNMT1 expression to investigate the regulation of pSTAT3, DNMT1, and hTERT. Methylation status of the promoter of hTERT gene was monitored by MS‐PCR. Cell proliferation, hTERT expression level and activity of hTERT were promoted after HCVc transfection. The expression of pSTAT3 and DNMT1 were up‐regulated simultaneously. DNMT1 and hTERT were down‐regulated after blocking JAK/STAT3 pathway and the expression of hTERT weakened with DNMT1 inhibition. MS‐PCR showed HCVc transfection increased the methylation level of hTERT promoter, and this effect was weakened after blocking the JAK/STAT3 pathway or with the treatment with DNMT1 inhibitor. HCVc transfection improved hTERT expression via epigenetic regulation. JAK/STAT3 pathway could be one of the essential factors in regulating DNMT1 expression during this process. J. Cell. Biochem. 113: 3419–3426, 2012.