The Different Expression of Gene Profiles on Hepatocellular Carcinoma Cells with Different Intracellular Hepatitis C Viral Load

( Chia-yen Dai ),( Shu-chi Wang ),( Meng-hsuan Hsieh ),( Cheng-fu Yang ),( Ching-i Huang ),( Chung-feng Huang ),( Ming-lun Yeh ),( Jee-fu Huang ),( Wang-long Chung ),( Ming-lung Yu ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: The different hepatitis C virus (HCV) replication has been reported among individual hepatocytes in chronic HCV infection by identifying hepatocytes with different HCV RNA levels. We have previously established a fluorescence-activated cell sorting (FACS) protocol to study the effects of different intracellular viral loads in HCV-infected cells. The present study aimed to further study the gene expression on different hepatocellular carcinoma (HCC) cells with different HCV viral load. Methods: The JFH1-EYFP viral florescence intensity was used to sort the high and low viral load cells after 5 days infection in vitro which has been shown in our previous study that infected cells efficiently and accurately discriminated between high- and low-viral load cell populations. The next generation sequence-RNA sequence was used to clarify the mRNA and miRNA gene network between HCV-high and HCV-low infected cells of the HCC cell line. Venn diagram summarizing the probe sets that were differentially expressingbetween the Huh7.5.1 versus each differential viral load cell population and miRDB and miRTar databases were used to predict HVL and LVL/S2 unique miRNA target genes. Results: By analyzing the NGS dataset and miRNA microarray dataset, of the significant transcripts, three miRNA were unique for the LVL/S2 cells and nine miRNA unique for the HVL. Twenty-three miRNA were common for all 3 viral load groups. We verified them by q-PCR and data confirmed the array data expression level. We found that high viral loads were associated with cell inflammation- and cell death-associated pathway; and the low viral loads were associated many stress response- and cell adhesion molecular (CAMs)-related genes. Conclusions: With the established cell sorting protocol, we have demonstrated that different gene network between HCV-high and HCV-low infected cells in JFH1-EYFP infectious cells exists. Our results may provide a boarder gene regulation map between high and low viral load cell populations.

Serial Serum MHC Class I Chain-Related a Levels in Predicting Hepatocellular Carcinoma in Chronic Hepatitis C Patients with Curative Antiviral Treatment

( Ming Lung Yu ),( Chung-feng Huang ),( Chia-yen Dai ),( Jee-fu Huang ),( Wan-long Chuang ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: MHC class I chain-related A (MICA) genetic variants and its serum level (sMICA) were associated with hepatitis C virus (HCV) related hepatocellular carcinoma (HCC) in untreated cohorts. The dynamic changes of serial sMICA levels regarding anti-HCV treatment and consequent HCC development is elusive. Methods: Single nucleotide polymorphism rs2596542 of MICA and serial sMICA levels were tested in chronic hepatitis C (CHC) patients with sustained virological response after antiviral treatment. Forty-two patients who developed HCC and another 84 age-, sex- and cirrhosis-propensity score matched non-HCC controls were compared. Serial sMICA levels were measured at three-time points: within 6 months of pretreatment (pre-sMICA), 6 months after the end of treatment (post-sMICA) and last visit before HCC occurrence or not (last-sMICA). Results: Compared to patients without HCC occurrence, those with HCC had lower platelet counts, higher levels of post-sMICA (197.4+398.0 pg/mL vs. 57.6+89.6 pg/mL, P=0.03) and last-sMICA (320.4+508.4 pg/mL vs. 37.7+140.2 pg/mL, P<0.001). Cox regression analysis revealed that last-sMICA is the only factor predictive of HCC development (hazard ratio [HR]/ 95 % confidence intervals [CI.]: 2.27 (per 1 log pg/mL increase)/1.672-3.082, P<0.001). Patients without HCC had a significantly decreased trend of sMICA levels during follow-up (trend P=0.001). In contrast, HCC patients had an increased trend of sMICA levels (trend P=0.024). MICA rs2596542 GG genotype carriers without HCC had a significantly decreased trend of sMICA levels during follow-up (trend P<0.001). However, HCC patients who carried GG genotype had a substantially increased trend of sMICA levels (trend P=0.06). Nevertheless, both trends were not observed in A allele carriers with or without HCC development. t three-time points: within 6 months of pretreatment (pre-sMICA), 6 months after the end of treatment (post-sMICA) and last visit before HCC occurrence or not (last-sMICA). Conclusions: Serial sMICA levels could serve as a surrogate marker for HCC development in CHC patients with SVR. The clinical utility is restricted to MICA rs2596542 GG genotype carriers. CA (320.4+508.4 pg/mL vs. 37.7+140.2 pg/mL, P<0.001). Cox regression analysis revealed that last-sMICA is the only factor predictive of HCC development (hazard ratio [HR]/ 95 % confidence intervals [CI.]: 2.27 (per 1 log pg/mL increase)/1.672-3.082, P<0.001). Patients without HCC had a significantly decreased trend of sMICA levels during follow-up (trend P=0.001). In contrast, HCC patients had an increased trend of sMICA levels (trend P=0.024). MICA rs2596542 GG genotype carriers without HCC had a significantly decreased trend of sMICA levels during follow-up (trend P<0.001). However, HCC patients who carried GG genotype had a substantially increased trend of sMICA levels (trend P=0.06). Nevertheless, both trends were not observed in A allele carriers with or without HCC development. t three-time points: within 6 months of pretreatment (pre-sMICA), 6 months after the end of treatment (post-sMICA) and last visit before HCC occurrence or not (last-sMICA).

Time-degenerative Factors and the Risk of Hepatocellular Carcinoma after Antiviral Therapy among HCV Patients: A Model for Prioritization of Treatment

( Ming-lung Yu ),( Chung-feng Huang ),( Ming-lun Yeh ),( Jee-fu Huang ),( Chia-yen Dai ),( Wan-long Chuang ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Age and hepatic fibrosis are the factors that increase the risk of hepatocellular carcinoma (HCC) over time. We aimed to explore their impac at the initiation of antiviral therapy on HCC among chronic hepatitis C (CHC) patients. Methods: A total of 1281 biopsy-proven CHC patients receiving interferon- based therapy were followed for a mean period of 5.5 years. Results: The 5-year cumulative incidence of HCC did not differ between non-SVR and SVR patients who were <40 years old (7.7 % vs. 0.5%, P=0.1), but was significantly higher in non-SVR patients between 40 and 55 years old (18.0% vs. 1.3%, P<0.001) and >55 years old (15.1% vs. 7.9%, P=0.03). Compared with SVR, non-SVR was independently predictive of HCC in patients 40-55 years old (hazard ratio [HR]/95% confidence intervals [CI]: 10.92/3.78-31.56, P<0.001) and >55 years old (HR/CI: 1.96/1.06-3.63, P=0.03) but not in patients <40 years old (HR/CI: 2.76/0.41-18.84, P=0.3). The 5-year cumulative incidence of HCC did not differ between non-SVR and SVR patients whose fibrosis stage was F0-1 (4.6% vs. 1.9%, P=0.25) but was higher in non-SVR patients with F2-3 (21.4% vs. 4.3%, P<0.001) or F4 (33.5% vs. 8.4%, P=0.002). Compared with SVR, non-SVR was independently predictive of HCC in patients with F2-3 (HR/CI: 4.36 /2.10-9.03, P<0.001) and F4 (HR/CI: 3.84/1.59-9.30, P=0.03) but not in those with F0-1 (HR/CI: 1.53/ 0.49-4.74, P=0.47). Conclusions: Delayed HCV clearance for patients with CHC > 40 years old or with a fibrosis stage > 2 increases the risk of HCC over time.

Efficacy and Safety of 12 Weeks of Daclatasvir, Asunaprevir Plus Ribavirin for the Treatment of HCV Genotype 1b Infection without Baseline NS5A Resistance-Associated Variants (DARING)-Interim Report

( Ming-lung Yu ),( Chao-hung Hung ),( Yi-hsiang Huang ),( Cheng-yuan Peng ),( Chun-yen Lin ),( Pin-nan Cheng ),( Rong-nan Chien ),( Shih-jer Hsu ),( Chen-hua Liu ),( Jee-fu Huang ),( Chung-feng Huang 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: The current study aims to elucidate the treatment efficacy (defined as undetectable HCV RNA throughout 12 weeks of post-treatment follow-up, SVR12) and safety DCV/ASV plus ribavirin for 12 weeks in HCV-1b patients without NS5A RAS. Methods: This is a single-arm, open-label phase 2 study. Seventy directly acting antivirals (DAA)-naïve HCV-1b patients without L31/Y93 RAS are planned to receive daclatasvir (60 mg/ day) and asunaprevir (100 mg twice daily) plus weight-based ribavirin (1000-1200 mg/day) for 12 weeks. After treatment they were followed up for 12 weeks. Results: As of 31 Oct 2017, 58 eligible patients are allocated to treatment, with a mean age of 59.3 years and female predominance (67.2%, 39/58). The mean HCV RNA was 5.87+0.77 log10 IU/mL; 23 patients (39.7 %) had significant hepatic fibrosis (>F2). In the modified intention-to-treat analysis, the rate of undetectable HCV at week 1, week 2, week 4, week 8 and endof- treatment was 25 % (14/56), 84.8 % (39/46), 100 % (46/46), 100 % (38/38) and 100 % (27/27), respectively. Undetectable HCV RNA were observed in all of the patients with HCV RNA assessable 4 weeks (SVR4, 18/18) and 12 weeks (SVR12, 12/12) post treatment. None of the 18 patients who completed the 12-week treatment experienced relapse during post-treatment follow-up. The most common adverse event was fatigue (78.3 %), followed by pruritus (65.2 %) and dizziness (52.2 %), of which were considered as ribavirin related. None of the participating subjects withdrew treatment or follow-up throughout the trial peroid. Three serious adverse events were reported which included urosepsis, appendicitis and left ureteral stone. All were unrelated to the investigating drugs. Conclusions: 12 weeks of DCV/ASV plus ribavirin was highly effective and safe in HCV-1b patients without NS5A RAS in the interim analysis. The satisfactory results would be anticipated in the full patient set.

Significant down-regulation of growth hormone receptor expression revealed as a new unfavorable prognostic factor in hepatitis C virus-related hepatocellular carcinoma

( Ching-chih Lin ),( Ta-wei Liu ),( Ming-lun Yeh ),( Yi-shan Tsai ),( Pei-chien Tsai ),( Chung-feng Huang ),( Jee-fu Huang ),( Wan-long Chuang ),( Chia-yen Dai ),( Ming-lung Yu ) 대한간학회 2021 Clinical and Molecular Hepatology(대한간학회지) Vol.27 No.2

Background/Aims: Growth hormone (GH) is the main regulator of somatic growth, metabolism, and gender dimorphism in the liver. GH receptor (GHR) signaling in cancer is derived from a large body of evidence, although the GHR signaling pathway involved in the prognosis of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV)-related HCC, remains unclear. We aimed to explore the expression of GHR and analyze its association with clinicopathologic features and prognosis of patients with chronic hepatitis C and HCC. Methods: The expression of GHR mRNA was investigated by quantitative real-time polymerase chain reaction in paired tumors and adjacent non-tumorous (ANT) liver tissues of 200 patients with chronic hepatitis C and HCC. Western blotting and immunofluorescence assays using the HCV-infected Huh7.5.1 cell model was performed. Results: GHR mRNA was significantly lower in HCV-HCC tissues than in corresponding ANT liver tissues. GHR mRNA and protein levels also decreased in the HCV-infected Huh7.5.1 cell model. Notably, lower GHR expression was associated with age of >60 years (P=0.0111) and worse clinicopathologic characteristics, including alpha-fetoprotein >100 ng/mL (P=0.0403), cirrhosis (P=0.0075), vascular invasion (P=0.0052), pathological stage II-IV (P=0.0002), and albumin ≤4.0 g/dL (P=0.0055), which were linked with poor prognosis of HCC. Most importantly, the high incidence of recurrence and poor survival rates in patients with a low ratio of tumor/ANT GHR (≤0.1) were observed, indicating that low expression levels of GHR had great risk for development of HCC in patients with chronic hepatitis C. Conclusions: Our study demonstrates a significant down-regulation of GHR expression as a new unfavorable independent prognostic factor in patients with chronic hepatitis C and HCC. (Clin Mol Hepatol 2021;27:313-328)