OsKASI-2 is required for the regulation of unsaturation levels of membrane lipids and chilling tolerance in rice.

Chilling stress has seriously limited the global production and geographical distribution of rice. However, the molecular mechanisms associated with plant responses to chilling stress are less known. In this study, we revealed a member of ß-ketoacyl-ACP synthase I family (KASI), OsKASI-2 which confers chilling tolerance in rice. OsKASI-2 encodes a chloroplast-localized KASI enzyme mainly expressed in the leaves and anthers of rice and strongly induced by chilling stress. Disruption of OsKASI-2 led to decreased KAS enzymatic activity and the levels of unsaturated fatty acids, which impairs degree of unsaturation of membrane lipids, thus increased sensitivity to chilling stress in rice. However, the overexpression of OsKASI-2 significantly improved the chilling tolerance ability in rice. In addition, OsKASI-2 may regulate ROS metabolism in response to chilling stress. Natural variation of OsKASI-2 might result in difference in chilling tolerance between indica and japonica accessions, and Hap1 of OsKASI-2 confers chilling tolerance in rice. Taken together, we suggest OsKASI-2 is critical for regulating degree of unsaturation of membrane lipids and ROS accumulation for maintenance of membrane structural homeostasis under chilling stress, and provide a potential target gene for improving chilling tolerance of rice.

Network module function enrichment analysis of lung squamous cell carcinoma and lung adenocarcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the two major subtypes of non-small cell lung cancer that pose a serious threat to human health. However, both subtypes currently lack effective indicators for early diagnosis. METHODS: To identify tumor-specific indicators and predict cancer-related signaling pathways, LUSC and LUAD gene weighted co-expression networks were constructed. Combined with clinical data, core genes in LUSC and LUAD modules were then screened using protein-protein interaction networks and their functions and pathways were analyzed. Finally, the effect of core genes on survival of LUSC and LUAD patients was evaluated. RESULTS: We identified 12 network modules in LUSC and LUAD, respectively. LUSC modules "purple" and "green" and LUAD modules "brown" and "pink" are significantly associated with overall survival and clinical traits of tumor node metastasis, respectively. Eleven genes from LUSC and eight genes from LUAD were identified as candidate core genes, respectively. Survival analysis showed that high expression of SLIT3, ABI3BP, MYOCD, PGM5, TNXB, and DNAH9 are associated with decreased survival in LUSC patients. Furthermore, high expression of BUB1, BUB1B, TTK, and UBE2C are associated with lower patient survival. CONCLUSIONS: We found biomarker genes and biological pathways for LUSC and LUAD. These network hub genes are associated with clinical characteristics and patient outcomes and they may play important roles in LUSC and LUAD.

Shared network pattern of lung squamous carcinoma and adenocarcinoma illuminates therapeutic targets for non-small cell lung cancer.

Background: Non-small cell lung cancer (NSCLC) is a malignant tumor with high mortality. Lung squamous carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the common subtypes of NSCLC. However, how LUSC and LUAD are compatible remains to be elucidated. Methods: We used a network approach to find highly interconnected genes shared with LUSC and LUAD, and we then built modules to assess the degree of preservation between them. To quantify this result, Z-scores were used to summarize the interrelationships between LUSC and LUAD. Furthermore, we correlated network hub genes with patient survival time to identify risk factors. Results: Our findings provided a look at the regulatory pattern for LUSC and LUAD. For LUSC, several genes, such as AKR1C1, AKR1C2, and AKR1C3, play key roles in regulating network modules of cell growth pathways. In addition, CCL19, CCR7, CCL21, and LY9 are enriched in LUAD network modules of T lymphocyte-related pathways. LUSC and LUAD have similar expressed gene expression patterns. Their networks share 46 hub genes with connectivity greater than 0.9. These genes are correlated with patient survival time. Among them, the expression level of COL5A2 in LUSC and LUAD is higher than that in normal tissues, which is closely related to the poor prognosis of LUSC and LUAD patients. Conclusion: LUSC and LUAD share a network pattern. COL5A2 may be a risk factor in poor prognosis in LUSC and LUAD. The common landscape of LUSC and LUAD will help better define the regulation of NSCLC candidate genes and achieve the goals of precision medicine.

Hepatitis C virus core protein-induced miR-93-5p up-regulation inhibits interferon signaling pathway by targeting IFNAR1.

AIM: To investigate the mechanism by which hepatitis C virus (HCV) core protein-induced miR-93-5p up-regulation regulates the interferon (IFN) signaling pathway. METHODS: HCV-1b core protein was exogenously expressed in Huh7 cells using pcDNA3.1 (+) vector. The expression of miR-93-5p and interferon receptor 1 (IFNAR1) was measured using quantitative reverse transcription-polymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of miR-93-5p and IFNAR1 were performed using miR-93-5p agomir and antagomir, and pcDNA3.1-IFNAR1 and IFNAR1 siRNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of miR-93-5p. Cellular experiments were also conducted. RESULTS: Serum miR-93-5p level was increased in patients with HCV-1b infection and decreased to normal level after HCV-1b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum miR-93-5p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1b core protein increased miR-93-5p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of miR-93-5p, and IFNAR1 restore could rescue miR-93-5p-reduced STAT1 phosphorylation, suggesting that the miR-93-5p-IFNAR1 axis regulates the IFN signaling pathway. CONCLUSION: HCV-1b core protein-induced miR-93-5p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the miR-93-5p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1b infection.

miR-375 is downregulated by promoter hypermethylation in MIN6 insulinoma cells.

Epigenetics may affect the susceptibility for type 2 diabetes mellitus (T2DM). Aberrant DNA methylation patterns are nowadays recognized as a key epigenetic hallmark of T2DM. Previously, our studies have shown that the hypomethylation of human miR-375 promoter may contribute to the pathogenesis of T2DM. However, no comprehensive study defines the miR-375 promoter methylation patterns present in the established pancreatic ß cell line. To address this matter, we have analyzed the DNA methylation profile of insulinoma MIN6 cells by MassARRAY spectrometry and employed the DNA demethylating drug 5-aza-2'-deoxycytidine (5-aza-CdR) to treat MIN6 cells to explore the methylation patterns of the mmu-miR-375. The expression of mmu-miR-375 in mRNA level was measured by quantitative RT-PCR (qRT-PCR). Methylation analysis reveals that MIN6 cells display hypermethylation at the mmu-miR-375 promoter. Following the decreased methylation of mmu-miR-375, the relative expression of mmu-miR-375 increased gradually after 5-Aza-CdR treatment. In addition, we find that there was an inverse correlation between DNA methylation levels and transcription level of mmu-miR-375. In summary, this is the first report for analyzing mmu-miR-375 promoter methylation using MALDI-TOF MS technology and our results indicate that promoter hypermethylation of the mmu-miR-375 is a common event in MIN6 cells.

Stability of Chronic Hepatitis-Related Parameters in Serum Samples After Long-Term Storage.

BACKGROUND: Serum samples are widely used in clinical research, but a comprehensive research of the stability of parameters relevant to chronic hepatitis and the effect of a relatively long-term (up to 10 years) storage on the stability have rarely been studied. AIMS: To investigate the stability of chronic hepatitis-related parameters in serum samples after long-term storage. MATERIALS AND METHODS: The storage stability of common clinical parameters such as total bile acid (TBA), total bilirubin (TBIL), potassium, cholesterol, and protein parameters such as alanine aminotransferase (ALT), creatine kinase (CK), γ-glutamyltransferase (GGT), albumin, high-density lipoprotein (HDL) and also hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, hepatitis B surface antigen (HBsAg), and chemokine (C-X-C motif) ligand 10 (CXCL10) were tested in serum samples after storing at -20°C or -70°C for 1, 2, 3, 7, 8, and 10 years. RESULTS: Levels of TBA, TBIL, and protein parameters such as ALT, CK, GGT, HDL, and HBsAg decreased significantly, but levels of potassium and cholesterol increased significantly after long-term storage, whereas blood glucose and triglycerides were stable during storage. HBV DNA remained stable at -70°C but changed at -20°C, whereas HCV RNA was stable after 1-, 2-, and 3-year storage. CXCL10 was still detectable after 8-year storage. CONCLUSIONS: Low temperatures (-70°C/80°C) are necessary for storage of serum samples in chronic hepatitis B research after long-term storage.

Effect of entecavir on CD4+ T-cell subpopulations in patients with chronic hepatitis B.

UNLABELLED:  Background and aims. CD4+ T cells play an important role in response to hepatitis B virus (HBV) infection. We investigated the change in CD4+ T-cell subpopulations and viral load in patients with chronic HBV infection who were treated with entecavir. MATERIAL AND METHODS: Thirty patients with chronic HBV infection were enrolled according to the criteria recommended by the Chinese Society of Infectious Diseases and the Chinese Society of Hepatology. The expressions of signature transcription factors and cytokines of CD4+ T-cell subpopulations were measured in chronic hepatitis B (CHB) patients treated with entecavir treatment. RESULTS: Entecavir treatment significantly attenuated hepatitis B virus DNA load and affected the CD4+ T-cell subsets in CHB patients. A dramatic decrease in the Th17 and Treg cell frequencies and expressions of their related cytokines were found in CHB patients with entecavir treatment. In contrast, entecavir treatment caused a remarkable increase in the Th2 cell frequencies and expressions of their related cytokines. CONCLUSION: Our results suggested that Th17 and Treg cells were the more sensitive subtypes to entecavir- induced inhibition of HBV replication compared to Th1 and Th2 cells in chronic HBV patients.

STAT3­regulated long non­coding RNAs lnc­7SK and lnc­IGF2­AS promote hepatitis C virus replication.

Long non­coding RNAs (lncRNAs) are a class of RNAs that do not code protein but are important in diverse biological processes. In previous years, with the application of high­throughput sequencing, a large number of lncRNAs associated with virus infections have been identified and intensively investigated, however, there are few studies examining the association between lncRNAs and HCV replication. Previous studies have demonstrated that signal transducer and activator of transcription 3 (STAT3) is activated by the hepatitis C virus (HCV) and in turn increases the replication of HCV. However, the detailed molecular mechanism is only partially understood. In the present study, using human lncRNA polymerase chain reaction (PCR) arrays, it was identified that lnc­IGF2­AS, lnc­7SK, lnc­SChLAP1 and lnc­SRA1 are upregulated by STAT3. In addition, among these four lncRNAs, only lnc­IGF2­AS and lnc­7SK were involved in HCV replication. Transfection of siRNA lnc­7SK and siRNA lnc­IGF2­AS partially inhibited the replication of HCV in Huh7 cells. Data also indicated that when transfected with siRNA lnc­7SK and siRNA lnc­IGF2­AS, the expression of phosphatidylinositol 4­phosphate (PI4P), which was identified to be associated with HCV replication, was reduced. Thus, the present study identified two new types of lncRNAs, lnc­IGF2­AS and lnc­7SK, which can be upregulated by STAT3 and are involved in HCV replication by regulating PI4P.

The STAT3-regulated long non-coding RNA Lethe promote the HCV replication.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a class of RNAs that do not code protein but play important roles in diverse biological processes. In recent years, with the application of high-throughput sequencing, a great deal of lncRNAs associated with virus infections have been discovered and intensively studied, but there are few studies about the relationship between lncRNAs and HCV replication. METHODS: We identify that several lncRNAs can be upregulated and downregulated by phosphorylated STAT3 by using human PCR array method. And among these lncRNAs, lnc-Lethe was involved in the HCV replication. Transfection of siRNA Lethe partially blocked the replication of HCV in Huh7 cells. RESULTS: In the present study, we have established that phosphorylated STAT3 can promote the HCV replication. Data also indicated that when transfected with siRNA Lethe, the expression levels of PKR, OAS and IRF1, which were all ISGs, were all up regulated. CONCLUSIONS: Based on our findings from Lethe knockdown, we have identified that Lethe, which was upregulated by activated STAT3, may promoting the replication of HCV through a negative regulatory mechanism of type I IFN response.

Hepatitis C virus represses the cellular antiviral response by upregulating the expression of signal transducer and activator of transcription 3 through sponging microRNA­122.

MicroRNAs (miRNAs) are small, non­coding RNAs that inhibit the expression of target protein coding genes at the post­transcriptional level. miR­122 is a liver specific miRNA. Notably, miR­122 is used by the hepatitis C virus (HCV) for triggering viral replication by interacting with the 5' untranslated region of the HCV RNA. The present study demonstrated that miR­122 inhibited the expression of signal transducer and activator of transcription 3 (STAT3), an antivirus response repressor. The HCV RNA acted as an 'miRNA sponge', which upregulated the expression of STAT3 by sealing miR­122. Subsequently, it was confirmed that this miR­122 sponge function of HCV RNA repressed the expression of polyinosinic­polycytidylic acid­stimulated type I interferons. The present study provided a deeper understanding of the complex role of miR­122 in the progression of HCV infection and supported the miR­122 inhibition strategy in anti­HCV infection treatment.

Clinical implications of hepatitis B surface antigen quantitation in the natural history of chronic hepatitis B virus infection.

BACKGROUND: HBsAg quantitation may be useful for managing patients with hepatitis B virus (HBV) infection. OBJECTIVES: We explored the clinical implications of HBsAg quantitation for patients with HBsAg levels >250IU/ml (Abbott Diagnostics). STUDY DESIGN: Two hundred and thirty-three HBV-infected patients comprising 29 immune tolerance cases, 49 treatment-naïve HBeAg-positive chronic hepatitis B (CHB) cases, 91 inactive HBV carrier cases, and 64 treatment-naïve HBeAg-negative CHB cases were analyzed. HBsAg was quantified by the Architect HBsAg assay (Abbott Diagnostics) after a 1:500 automated dilution. RESULTS AND CONCLUSIONS: HBsAg (log10IU/ml) was established for immune tolerance (4.50±0.43), HBeAg-positive CHB (4.17±0.66), inactive HBV carrier (3.32±0.44), and HBeAg-negative CHB (3.23±0.40); (p=4.92×10(-35)). No significant difference was observed between inactive HBV carrier and HBeAg-negative CHB (p=0.247). The proportions of HBsAg <2000IU/ml for inactive HBV carrier and HBeAg-negative CHB were 51.6% and 59.3%, respectively (p=0.341). Positive correlations between HBsAg and HBV DNA were observed for immune tolerance (p=1.23×10(-4)) and HBeAg-positive CHB (p=0.003), but not for HBeAg-negative CHB (p=0.432). A negative correlation between HBsAg and age was observed for immune tolerance (p=0.030), HBeAg-positive CHB (p=0.016), and inactive HBV carrier (p=0.001), but not in HBeAg-negative CHB (p=0.249). No significant differences between HBsAg and ALT for HBeAg-positive (p=0.338) or HBeAg-negative CHB (p=0.564) were observed. For patients with HBsAg quantitation >250IU/ml, HBsAg may reflect HBV DNA replication for HBeAg-positive cases. HBsAg is not a suitable marker for evaluating hepatitis activity and distinguishing between cases of HBeAg-negative CHB and inactive HBV carrier state.

Factors influencing inadequate or suboptimal response to adefovir with or without genotypic resistance.

The aim of the study was to explore the factors in inadequate-responders to treatment with adefovir (ADV) with or without genotypic resistance. The reverse-transcriptase (RT) gene of hepatitis B virus (HBV) was sequenced in 161 patients with inadequate-response to ADV and analyzed for HBV genotypes using a phylogenetic approach. Seventy-six patients (47.2%) were found to carry the rtA181V/T/S or rtN236T residue substitution, and most of them had viral rebound. In the patients with viral rebound and ADV genotypic resistance, 19 (25.7%) showed rtA181V/T/S + rtN236T substitutions. In the other patients, it was found that HBV genotypes and cirrhosis influenced the selection of ADV-resistant positions by univariate analysis and multiple logistic regression analysis. The rtN236T was more frequent in patients with genotype B, and the rtA181V/T/S was more common in patients with genotype C (χ(2) = 11.543, P = 0.001). Multiple logistic regression analysis showed that the rtN236T and time resistant strains occurred during ADV-treatment were statistically significant for influencing rtA181 variation types (P = 0.007 and P = 0.024, respectively), and the occurrence of rtA181T was found to be significantly earlier than rtA181V. In conclusion, genotypic resistance was not detected in the majority of primary nonresponders to ADV when compared to the patients with viral rebound. The different HBV genotypes influence the selection of ADV-resistant mutation positions. The rtA181T occurs more frequently in patients with the rtN236T and it occurs earlier when compared to the rtA181V. These findings suggest that early judgment of adequate response and making a decision for interference in patients treated with ADV are of importance in clinical practice.

Association of TBX21 promoter polymorphisms with type 1 autoimmune hepatitis in a Chinese population.

The T-box transcription factor T-bet is a key regulator for the lineage commitment in CD4 Th1 cells and CD8 T cells by activating the hallmark production of interferon-γ, and its expression level is linked to autoimmune diseases. T-1993C and T-1514C polymorphisms in the TBX21 gene (encoding T-bet) promoter can affect transcription activity. We investigated the distributions of these functional polymorphisms in 84 adult patients with type 1 autoimmune hepatitis (AIH-1) and 318 healthy controls. Intracellular T-bet staining of polarized CD4 Th1 cells from healthy controls corresponding to T-1993C genotypes were analyzed by flow cytometry. The -1993C allele frequency was 3.0% in AIH-1 and 11.8% in controls (p = 0.000 25). Individuals carrying the -1993C allele had a decreased risk to AIH-1 compared with those without the -1993C allele (p = 0.0016, odds ratio [OR] = 0.22, 95% confidence interval = 0.09-0.56). No association was found between the T-1514C polymorphism and AIH-1. The onset age of AIH-1 was also accelerated among -1993TT homozygotic individuals (p = 0.013). The fractions of T-bet positive Th1 cells in the -1993TT homozygotes were 2.2-fold higher than those in -1993CC homozygotes (p = 0.002). Our results suggest that the T-1993C polymorphism in the TBX21 promoter influences susceptibility to AIH-1 in a Chinese population.

Association of TBX21 T-1993C polymorphism with viral persistence but not disease progression in hepatitis B virus carriers.

AIM: Transcription factor T-bet is responsible for the differentiation of naive T lymphocytes, and its expression level is linked with different responses to some viral infections, including hepatitis B virus (HBV) infection. In this report we examine whether promoter polymorphisms of the TBX21 gene (encoding T-bet) are associated with susceptibility to HBV persistence or disease progression in chronic HBV carriers. METHODS: Three previously reported promoter polymorphisms, T-1993C, T-1514C and G-1499A, were analyzed by polymerase chain reaction restriction fragment length polymorphism analysis. Two common polymorphisms, T-1993C and T-1514C, were selected for genotyping in 1074 chronic HBV carriers, 310 spontaneously recovered controls and 374 HBV naive controls. Of 1074 HBV carriers, 234 were considered to be asymptomatic carriers and 840 were found to have chronic progressive liver disease including cirrhosis and hepatocellular carcinoma. Haplotypes were constructed for each subject and associations with the susceptibility to persistent HBV infection were estimated by logistic regression. RESULTS: The -1993C allele in the TBX21 promoter was significantly more common among chronic HBV carriers compared with recovered controls (chi(2) = 6.65, P = 0.01). In contrast, the frequency of TT haplotype at positions -1993/-1514, was significantly higher in recovered controls than chronic HBV carriers (P = 0.0027, odds ratio = 1.57; 95% confidence interval, 1.16-2.12). In HBV carriers, the TBX21 promoter polymorphisms were not linked to disease progression. CONCLUSION: The TBX21 promoter polymorphisms do not appear to be determinant of disease progression in Chinese HBV carriers. The T-1993C polymorphism in the TBX21 promoter influences susceptibility to persistent HBV infection.

[Distribution and characteristics of hepatitis B virus genotypes in Uighur patients with chronic hepatitis B in Xinjiang province of China].

OBJECTIVE: To study genotype distribution and the characteristics of hepatitis B virus (HBV) in Uighur patients with chronic hepatitis B (CHB) in Xinjiang, China. METHODS: Type specific primers and PCR were used to detect the HBV genotypes of 127 Uighur CHB patients in Xinjiang. Genotyping results were confirmed by PCR product sequencing. RESULTS: Among the 127 patients, the proportions of genotype D, B, C and B/D, C/D, B/C/D were 39.4% (50/127), 22.0% (28/127), 16.5% (21/127) and 9.4% (12/127), 8.7% (11/127) and 3.9% (5/127), respectively. The distribution of the HBV genotypes showed no significant differences between male and female patients (x2 = 8.058, P > 0.05), between HBeAg positive and negative patients (x2 = 6.033, P > 0.05), and between patients of different ages (x2 = 3.137, P > 0.05). CONCLUSION: Genotype D HBV is predominant in Uighur patients with chronic hepatitis B in Xinjiang. The distribution of various HBV genotypes shows no significant differences between these Uighur patients with different HBeAg positivity, sex and age.