Differential expression of genes in HepG2 cells caused by UC001kfo RNAi as shown by RNA-seq.

The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles. The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs. The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line. The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (P<0.001). Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lncRNA UC001kfo. LncRNA UC001kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that mRNAs were differentially expressed in the HepG2 cell line after the down-regulation of lncRNA-UC001kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq / 华中科技大学学报（医学）（英德文版）

The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq.HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles.The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR.RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line.Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs.The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line.The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (P＜0.001).Additionally,we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics,cell adhesion,invasion and migration.The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lncRNA UC001kfo.LncRNA UC001kfo may play a role in regulating cancer cell invasion and metastasis.It was suggested that mRNAs were differentially expressed in the HepG2 cell line after the down-regulation of lncRNA-UC001kfo.Some took part in the extracellular matrix,cell adhesion,motility,growth,and localization.The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

Disease progression in Chinese patients with hepatitis C virus RNA-positive infection via blood transfusion.

The majority of patients with hepatitis C virus (HCV) in China were infected via blood transfusion prior to the year 1996. In this systematic retrospective cohort study, disease progression in 804 consecutive patients with transfusion-acquired HCV is investigated. In addition, the occurrence of compensated cirrhosis, decompensated cirrhosis and hepatocellular carcinoma (HCC) is analyzed among these patients, along with the risk factors for disease progression. Patients with cirrhosis or HCC were classified as the serious development group (SD group) and the remaining patients with chronic hepatitis were classified as the hepatitis group (H group). Significant differences were found between the two groups in age at the time of infection, duration of infection and age at the time of observation. SD group patients were significantly older at the time of transfusion (33.73 vs. 23.56 years; P<0.001), with a significantly longer mean duration of HCV infection (21.88 vs. 21.15 years; P=0.029) compared with that in the H group. Male gender and age at the time of transfusion were significant risk factors for HCC (OR=2.48, P=0.031 and OR=1.07, P=0.002, respectively). Age was a significant risk factor for disease progression in older Chinese patients with transfusion-acquired HCV, and there were significant differences in the prevalence of compensated cirrhosis, decompensated cirrhosis and HCC between the age groups (P<0.001), suggesting that more patients with HCV may develop cirrhosis or HCC in their third and fourth decades of infection. Results of the present study will be helpful for predicting disease progression in Chinese patients with HCV infected via blood transfusion.

PU.1 Is Identified as a Novel Metastasis Suppressor in Hepatocellular Carcinoma Regulating the miR-615-5p/IGF2 Axis.

Invasion and metastasis is the major cause of tumor recurrence, difficulty for cure and low survival rate. Excavating key transcription factors, which can regulate tumor invasion and metastasis, are crucial to the development of therapeutic strategies for cancers. PU.1 is a master hematopoietic transcription factor and a vital regulator in life. Here, we report that, compared to adjacent non-cancerous tissues, expression of PU.1 mRNA in metastatic hepatocellular carcinoma (HCC), but not primary HCC, was significantly down-regulated. In addition, levels of PU.1 mRNA in metastatic hepatoma cell lines MHCC97L and MHCC97H were much lower than in non-metastatic Hep3B cells. Transwell invasion assays after PU.1 siRNA transfection showed that the invasion of hepatoma cell lines was increased markedly by PU.1 knockdown. Oppositely, overexpression of PU.1 suppressed the invasion of these cells. However, knockdown and overexpression of PU.1 did not influence proliferation. Finally, we tried to explore the potential mechanism of PU.1 suppressing hepatoma cell invasion. ChIP-qPCR analysis showed that PU.1 exhibited a high binding capacity with miR-615-5p promoter sequence. Overexpression of PU.1 caused a dramatic increase of pri-, pre- and mature miR-615-5p, as well as a marked decrease of miR-615-5p target gene IGF2. These data indicate that PU.1 inhibits invasion of human HCC through promoting miR-615-5p and suppressing IGF2. These findings improve our understanding of PU.1 regulatory roles and provided a potential target for metastatic HCC diagnosis and therapy.

Expressions of miR-22 and miR-135a in acute pancreatitis.

This study examined the expressions of miR-22 and miR-135a in rats with acute edematous pancreatitis (AEP) and their target genes in order to shed light on the involvement of miR-22 and miR-135a in the pathogenesis of acute pancreatitis (AP). The in vivo model of AEP was established by introperitoneal injection of L-arginine (150 mg/kg) in rats. The miRNA microarray analysis was used to detect the differential expression of miRNAs in pancreatic tissue in AEP and normal rats. The in vitro AEP model was established by inducing the rat pancreatic acinar cell line (AR42J) with 50 ng/mL recombinant rat TNF-α. Real-time quantitative RT-PCR was employed to detect the expression of miR-22 and miR-135a in AR42J cells. Lentiviruses carrying the miRNA mimic and anti-miRNA oligonucleotide (AMO) of miR-22 and miR-135a were transfected into the AR42J cells. The AR42J cells transfected with vehicle served as control. Western blotting was used to measure the expression of activated caspase3 and flow cytometry analysis to detect the apoptosis of AR42J cells. Targets of miR-22 and miR-135a were predicted by using TargetScan, miRanda, and TarBase. Luciferase reporter assay and quantitative real-time RT-PCR were performed to confirm whether ErbB3 and Ptk2 were the target gene of miR-22 and miR-135a, respectively. The results showed that the expression levels of miR-22 and miR-135a were obviously increased in AEP group compared with the control group in in-vivo and in-vitro models. The expression levels of miR-22 and miR-135a were elevated conspicuously and the expression levels of their target genes were reduced significantly in AR42J cells transfected with lentiviruses carrying the miRNA mimic. The apoptosis rate was much higher in the TNF-α-induced cells than in non-treated cells. The AR42J cells transfected with miRNA AMOs expressed lower level of miR-22 and miR-135a and had lower apoptosis rate, but the expression levels of ErbB3 and Ptk2 were increased obviously. It was concluded that the expression levels of miR-22 and miR-135a were elevated in AEP. Up-regulating the expression of miR-22 and miR-135a may promote the apoptosis of pancreatic acinar cells by repressing ErbB3 and Ptk2 expression in AEP.

Expression profile of altered long non-coding RNAs in patients with HBV-associated hepatocellular carcinoma.

In China, hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC), which is called HBV-related HCC (HBV-HCC), but the pathogenesis has not been clearly elucidated. Long non-coding RNAs (lncRNAs) have been paid increasing attention to, as an important regulatory molecule involved in many biological processes, as well as a variety of diseases. This study examined lncRNA that might play an important role in HBV-HCC pathogenesis by conducting lncRNA and mRNA profile comparison between HBV-HCC and normal liver tissues. The differentially expressed lncRNA and mRNA profiles between HBV-HCC and normal liver tissues were analyzed by microarrays. The potential protein-encoding gene regulated by lncRNA, and the biological function (gene ontology, pathway analysis) of the target gene were investigated. The results showed that the expression levels of lncRNA and mRNA in HBV-HCC tissues were different from those in normal liver tissues. As compared with normal liver tissue, 837 (4.30%) lncRNAs exhibited more than two-fold change (P<0.05); 325 were up-regulated, and 512 were down-regulated; 991 (5.70%) mRNAs exhibited more than 2-fold change (P<0.05); 733 were up-regulated and 258 were down-regulated in HBV-HCC tissue. Besides, there were 7 lncRNAs with above 10-fold elevation, 6 lncRNAs with above 10-fold decrease, 18 mRNAs with above 10-fold elevation and 11 mRNAs with above 10-fold decrease. 444 (53.05%) lncRNAs had their corresponding mRNAs, some of which were adjacent to lncRNAs. The biological analysis showed that the target gene of differentially expressed lncRNAs took part in the important biological regulatory function. Target gene-related pathway analysis revealed the pathways in carcinoma and mitogen-activated protein kinase (MAPK) signaling pathways significantly changed in the HBV-HCC tissues as compared with normal liver tissues (P<0.05). It was suggested that as compared with normal liver tissues, the expression of lncRNAs in HBV-HCC tissues changed significantly, and lncRNAs played a key role in the pathogenesis of HBV-HCC probably by mainly regulating the carcinoma-related signaling pathway and MAPK signaling pathways.

Role of long non-coding RNAs in gene regulation and oncogenesis.

OBJECTIVE: This article aims to review recent studies on the biological characteristics of long non-coding RNAs (lncRNAs), transcription regulation by lncRNAs, and the results of recent studies on the mechanism of action of lncRNAs in tumor development. DATA SOURCES: The data cited in this review were mainly obtained from the articles listed in PubMed and HighWire that were published from January 2002 to June 2010. The search terms were "long non-coding RNA", "gene regulation", and "tumor". STUDY SELECTION: The mechanism of lncRNAs in gene expression regulation, and tumors concerned with lncRNAs and the role of lncRNAs in oncogenesis. RESULTS: lncRNAs play an important role in transcription regulation by controlling chromatin remodeling, transcriptional control, and post-transcriptional controlling. lncRNAs are involved in many kinds of tumors and play key roles as both suppressing and promoting factors. CONCLUSION: lncRNAs could perfectly regulate the balance of gene expression system and play important roles in oncogenic cellular transformation.

[A cross-sectional survey of occult hepatitis B virus infection in HIV-infected patients in acquired immune deficiency syndrome area].

OBJECTIVE: To assess the prevalence of occult HBV infection in HIV-infected patients inacquired immune deficiency syndrome area. METHODS: Serum samples were obtained from 97 HIV-infected patients who transmitted by paid blood donation. ELISA was used to detect HBV erologic markers (HBsAg, Anti-HBs, HBeAg, anti-HBe and anti-HBc) and HCV antibody. Flow Cytometry were used to detect CD4+ T cell count. Nested PCR was used to amplify surface protein region of HBV DNA. RESULTS: Ninety two patients were HBsAg negative in the 97 HIV-infected patients (94.85%). Twenty seven patients were co-infected with occult hepatitis B virus infection in the 92 HBsAg negative patients (29.35%). Seventy three patients were co-infected with HCV in the 92 HBsAg negative patients(79.35%). CD4 cell count of subjects with occult HBV infection were significantly lower (212.11 +/- 133.1 cells/mm3 versus 318.9 +/- 172.2 cells/mm3, respectively, P < 0.01). A significantly higher prevalence of isolated anti-HBc was observed in HIV-infected subjects co-infectioned with occult HBV infection [62.96% (13 of 27) versus 18.46% (15 of 65), P < 0.01]. No statistical significant association could be established between the age, sex and whether co-infected with HCV. CONCLUSION: It is found that occult HBV infection did occurs in HIV-infected patients. Individuals co-infected with HIV and occult HBV infection are more likely to have isolated anti-HBc than subjects with HIV alone. Co-infection with HIV and occult HBV is more likely to occue in subjects with lower CD4.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro.

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over-length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 mug/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 microg and 2.0 microg plasmid pXF3H-PAP, the levels of HBV nucleocapside-associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 mug plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%. CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.

[Optimal lamivudine treatment duration and the relationship between its early response and long term effect].

OBJECTIVE: To investigate the relationship between the method of administration of lamivudine and the therapeutic effect of the treatment in patients with chronic hepatitis B virus (HBV) infection. METHODS: One hundred and seventy-nine patients were given lamivudine 100 mg daily for 1 to 3 years. The relationships of the therapeutic effect and the early response, YMDD mutants and duration of treatment were analyzed. RESULTS: Alanine aminotransferase normalization rate, the negativity rate of HBV DNA and HBeAg, and HBeAg sero-conversion all were increased gradually with prolonged treatment. At the end of 1 year, HBV DNA negativity rate (57.0%) reached its peak, HBeAg negativity rate (39.7%) and HBeAg sero-conversion rate (16.8%) were higher than those at the end of 3 months (chi2 = 28.489, 33.238, 12.690, P<0.01). The lower the HBV DNA level was at the end of 3 months, the higher the HBV DNA negativity and HBeAg sero-conversion rates were at the end of 52 weeks and at the end of the 6 months follow-up. When the duration of treatment reached 1 year and 1.5 years, HBV DNA rebound rate in the patients (40.0% and 40.0% respectively) with HBeAg sero-conversion was obviously less (chi2 = 12.424, 10.237, P<0.01) than in those without sero-conversion (88.2% and 85.0% respectively). CONCLUSION: Lamivudine therapy for HBV infection is safe and effective. The optimal duration of treatment was 1.5 years. The early responders had better therapeutic effects. HBV DNA positivity persisting at the end of 3 months medication or no HBeAg sero-conversion in 1 year predicts poor therapeutic effects.