Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

CRISPR/CasRx-mediated resistance to Soybean mosaic virus in soybean

Soybean mosaic virus(SMV),an RNA virus,is the most common and destructive pathogenic virus in soybean fields.The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses;hence,this study aimed to engineer SMV resistance in soybean using this system.Specifically,multiple sgRNAs were designed to target positive-and/or negative-sense strands of the SMV HC-Pro gene.Subsequently,the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans.After inoculation with SMV,39.02%,35.77%,and 18.70%of T_(1)plants were confirmed to be highly resistant(HR),resistant(R),and mildly resistant(MR)to SMV,respectively,whereas only 6.50%were identified as susceptible(S).Additionally,qRT-PCR and DAS-ELISA showed that,both at 15 and 30 d post-inoculation(dpi),SMV accumulation significantly decreased or was even undetectable in HR and R plants,followed by MR and S plants.Additionally,the expression level of the CasRx gene varied in almost all T_(1)plants with different resistance level,both at 15 and 30 dpi.Furthermore,when SMV resistance was evaluated in the T_(2)generation,the results were similar to those recorded for the T_(1)generation.These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.

Emerging perspectives on RNA virus-mediated infections:from pathogenesis to therapeutic interventions

RNA viruses continue to pose significant threats to global public health,necessitating a profound understanding of their pathogenic mechanisms and the development of effective therapeutic interventions.This manuscript provides a comprehensive overview of emerging perspectives on RNA virus-mediated infections,spanning from the intricate intricacies of viral pathogenesis to the forefront of innovative therapeutic strategies.A critical exploration of antiviral drugs sets the stage,highlighting the diverse classes of compounds that target various stages of the viral life cycle,underscoring the ongoing efforts to combat viral infections.Central to this discussion is the exploration of RNA-based therapeutics,with a spotlight on messenger RNA(mRNA)-based approaches that have revolutionized the landscape of antiviral interventions.Furthermore,the manuscript delves into the intricate world of delivery systems,exploring innovative technologies designed to enhance the efficiency and safety of mRNA vaccines.By analyzing the challenges and advancements in delivery mechanisms,this review offers a roadmap for future research and development in this critical area.Beyond conventional infectious diseases,the document explores the expanding applications of mRNA vaccines,including their promising roles in cancer immunotherapy and personalized medicine approaches.This manuscript serves as a valuable resource for researchers,clinicians,and policymakers alike,offering a nuanced perspective on RNA virus pathogenesis and the cutting-edge therapeutic interventions.By synthesizing the latest advancements and challenges,this review contributes significantly to the ongoing discourse in the field,driving the development of novel strategies to combat RNA virus-mediated infections effectively.

Correlation between HBeAg and Hepatitis B Virus DNA and RNA Levels in Diverse Liver Disease Cohorts

Objective:To investigate the disparities and associations between HBV DNA and HBV RNA in various liver disease groups with respect to HBeAg status.Methods:Between September 2020 and September 2023,90 patients diagnosed with chronic hepatitis B(CHB),74 patients diagnosed with liver cirrhosis(LC),and 102 patients diagnosed with hepatocellular carcinoma(HCC)from the Department of Gastroenterology or Infection at the First Affiliated Hospital of Xi’an Jiaotong University were selected.HBV DNA,HBV RNA,and HBeAg quantitative tests were conducted using serum samples from the same patients.Results:In the three groups of cases,the HBV RNA load was higher when HBeAg was positive than when HBeAg was negative,and this difference was statistically significant.Only in the HCC group was the HBV DNA load significantly higher when HBeAg was positive than when HBeAg was negative.Additionally,there was a positive correlation between HBV DNA and HBV RNA regardless of HBeAg status.Conclusion:During HBeAg conversion,HBV RNA demonstrates a more sensitive response than HBV DNA.As CHB progresses to LC or HCC,HBV RNA exhibits better diagnostic value than HBV DNA.

Clinical Value of Hepatitis B Virus RNA Detection in Patients with Chronic Hepatitis B Infection

Objective:To study the clinical value of hepatitis B virus pregenomic RNA(HBV-pgRNA)detection in the treatment of hepatitis B.Methods:60 patients with hepatitis B were included in the study.Serum HBV-pgRNA and HBV DNA levels in different phases of infection and during treatment were detected,and serum hepatitis B surface antigen(HbsAg)titer was detected by chemiluminescent immunoassay.DNA was extracted from liver biopsy tissue,and covalently closed circular DNA was detected to predict the therapeutic value in patients.Results:At the initial stage of treatment,the level of HBV-pgRNA in phase I,II,III,and IV showed a gradual decrease.Comparing the levels of HBV-pgRNA before and after treatment,we found that the level of HBV-pgRNA was significantly lower after treatment(P<0.05).Among the indicators for predicting HBsAg seroconversion,the accuracy of HBV-pgRNA level was 85.0%(51/60).Conclusion:The clinical value of HBV-pgRNA detection in the treatment of hepatitis B is high.

RNA结合蛋白ELAVL1的功能及其调控病毒复制的研究进展

胚胎致死异常视觉蛋白1(embryonic lethal abnormal vision like 1, ELAVL1),又被称为人类抗原R(human antigen R, HuR),是一种典型的RNA结合蛋白(RNA binding protein, RBP),通过与3′非翻译区(3′untranslated element, 3′UTR)的AU富含元件(AU-rich element, ARE)结合,在mRNA转录和翻译过程中发挥重要作用。ELAVL1的作用十分广泛,可调控肿瘤的发生发展、凋亡、迁移与侵袭,是许多癌症治疗的关键靶点,还能参与调节编码器官发育和组织稳态的蛋白质和mRNA的水平,也有许多研究表明,ELAVL1通过转录后调控和翻译后修饰来调节病毒复制。本文主要综述了ELAVL1的生物学特点、功能、调控机制及其在病毒复制过程中的调控作用,旨在为进一步研究ELAVL1与畜禽病毒复制之间互作的机制提供理论参考。

RNF20影响RNA病毒感染后的巨噬细胞极化

目的 探究环指蛋白20(RNF20)在抗RNA病毒先天免疫中的作用。方法 在293T人胚肾上皮细胞中过表达RNF20,运用双荧光素酶报告基因实验,检测仙台病毒(SeV)感染诱导的干扰素a4(Ifna4)基因启动子活化。构建Rnf20基因髓系条件性敲除小鼠模型(Rnf20F/F;Lyz2-Cre),运用流式细胞术检测Rnf20F/F;Lyz2-Cre和对照Rnf20F/F小鼠骨髓、脾脏和外周血髓系细胞亚群的比例;利用建立的小鼠模型培养骨髓源巨噬细胞(BMDMs),在SeV和水疱性口炎病毒(VSV)感染后,运用免疫印迹检测转录因子干扰素调节因子3(IRF3)和核因子-κB (NF-κB)p65亚基的磷酸化,通过ELISA检测细胞因子IFN-β、TNF-α、IL-6的表达,通过高通量转录组测序分析转录组的改变,并通过LPS和IL-4分别诱导巨噬细胞M1和M2极化,证实RNF20对于转录组的影响。结果 293T细胞中RNF20过表达不影响SeV感染诱导的Ifna4基因启动子活化。Rnf20F/F;Lyz2-Cre和Rnf20F/F小鼠髓系细胞发育无明显差异,在RNA病毒感染2组小鼠的BMDMs后,IRF3、NF-κB p65的磷酸化和IFN-β、TNF-α、IL-6的表达相似,但是一些与巨噬细胞极化相关的基因表达有显著差异。RNF20的缺失有抑制巨噬细胞M1型极化、促进巨噬细胞M2型极化的趋势。结论RNF20不影响RNA病毒感染诱导的IRF3和NF-κB通路活化,但可通过影响巨噬细胞极化而参与感染后炎症的消退。

环状RNA hsa_circ_0085576调控微小RNA-498/B细胞特异性莫洛尼鼠白血病病毒整合位点1轴对口腔鳞状细胞癌细胞迁移和侵袭的影响

目的探究环状RNA hsa_circ_0085576对口腔鳞状细胞癌(OSCC)细胞迁移和侵袭的影响及其分子机制。方法使用实时荧光定量聚合酶链反应(qRT-PCR)和蛋白印迹法检测OSCC细胞中hsa_circ_0085576、微小RNA-498(miR-498)以及B细胞特异性莫洛尼鼠白血病病毒整合位点1(BMI-1)的表达水平。使用CCK-8、划痕实验、Transwell实验以及qRT-PCR、蛋白印迹法分别检测SCC-15细胞增殖活力、迁移及侵袭能力以及相关基因和蛋白的相对表达量。结果OSCC细胞中hsa_circ_0085576与BMI-1表达上调,miR-498表达下调(P<0.05)。下调hsa_circ_0085576表达或过表达miR-498后SCC-15细胞的增殖活性、划痕愈合率、侵袭细胞数目以及细胞周期蛋白D1、波形蛋白表达水平下调,miR-498和E-钙黏蛋白表达水平上调(P<0.05);抑制miR-498表达可减弱下调hsa_circ_0085576表达对OSCC细胞增殖、迁移及侵袭的抑制作用;上调BMI-1表达可减弱过表达miR-498对OSCC细胞增殖、迁移及侵袭的抑制作用。结论下调hsa_circ_0085576表达可通过激活miR-498/BMI-1轴抑制OSCC细胞增殖、迁移及侵袭。

Digoxigenin(DIG)标记RNA探针检测侵染花生的Potyviruses

以花生条纹病毒(PStV)和花生斑驳病毒(PMV)克隆cDNA为模板,体外转录合成Digo-xgenin(DIG)标记PStV—I_9、PStV-I_(131)、PStV—I_(57-7)和PMV—A_(15)四种RNA探针。在点杂交反应中,PStV—I_9RNA探针能检测出0.25ng提纯PStV和花生病汁液62500倍稀释液中PStV。PMV—A_(16)RNA探针能检测到0.67ng提纯PMV和菜豆病汁液5120倍稀释液中PMV。两种病毒RNA探针均有高度特异性。PStV—I_(57-7)和PStV—I_(131)两种RNA探针具有与PStV—I_9RNA探针相同敏感性,但特异性稍差。

Hepatitis D virus dual-infection among Chinese hepatitis B patient related to hepatitis B surface antigen,hepatitis B virus DNA and age

The screening practices for hepatitis D virus(HDV)are diverse and nonstandardized worldwide,and the exact prevalence of HDV is uncertain.AIM To estimate HDV prevalence and investigate viral marker quantity trends in patients with hepatitis D.METHODS We collected 5594 serum samples from patients with hepatitis B in Jilin Province,China(3293 males and 2301 females,age range of 2 to 89 years).We then conducted tests for hepatitis B surface antigen(HBsAg),hepatitis B Virus(HBV)DNA,anti-hepatitis D antigen(HDAg),and HDV RNA.RESULTS We found that the prevalence of anti-HDAg and HDV RNA among hepatitis B patient were 3.6%(3.2-4.2%)and 1.2%(0.9-1.5%),respectively,87.69%of hepatitis D patients were 51-70 years old.HDV infection screening positive rate of patients with HBV DNA levels below 2000 IU/mL(2.0%)was higher than those above 2000 IU/mL(0.2%).Among anti-HDAg positive patients,the HDV RNA positive rate was positively correlated with the HBsAg level and anti-HDAg level.There was a weak correlation between HBsAg and anti-HDAg levels among hepatitis D patients.CONCLUSION Our study highlights the importance of considering multiple factors when assessing the severity of HDV infection,comprehensive evaluation of patients’clinical and laboratory parameters is necessary for proper diagnosis and treatment.

Serum hepatitis B virus RNA is a predictor of HBeAg seroconversion and virological response with entecavir treatment in chronic hepatitis B patients

BACKGROUND Characteristics of alterations of serum hepatitis B virus(HBV) RNA in different chronic hepatitis B(CHB) patients still cannot be fully explained. Whether HBV RNA can predict HBeAg seroconversion is still controversial.AIM To investigate whether HBV RNA can predict virological response or HBeAg seroconversion during entecavir(ETV) treatment when HBV DNA is undetectable.METHODS The present study evaluated 61 individuals who were diagnosed and treated with long-term ETV monotherapy at the Department of Infectious Diseases of Peking University First Hospital(China) from September 2006 to December 2007.Finally, 30 treatment-naive individuals were included. Serum HBV RNA were extracted from 140 μL serum samples at two time points. Then they were reverse transcribed to cDNA with the HBV-specific primer. The product was quantified by real-time quantitative PCR(RT-PCR) using TAMARA probes. Statistical analyses were performed with IBM SPSS 20.0.RESULTS Level of serum HBV RNA at baseline was 4.15 ± 0.90 log10 copies/mL. HBV RNA levels showed no significant difference between the virological response(VR)and partial VR(PVR) groups at baseline(P = 0.940). Serum HBV RNA significantly decreased among patients who achieved a VR during ETV therapy(P < 0.001). The levels of HBV RNA in both HBeAg-positive patients with seroconversion group and those with no seroconversion increased after 24 wk of treatment. Overall, HBV RNA significantly but mildly correlated to HBsAg(r =0.265, P = 0.041), and HBV RNA was not correlated to HBV DNA(r = 0.242, P =0.062). Furthermore, serum HBV RNA was an independent indicator for predicting HBeAg seroconversion and virological response. HBeAg seroconversion was more likely in CHB patients with HBV RNA levels below4.12 log10 copies/mL before treatment.CONLUSION The level of serum HBV RNA could predict HBeAg seroconversion and PVR during treatment. In the PVR group, the level of serum HBV RNA tends to be increasing.

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA

Hepatitis B virus(HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host mi RNAs and may deregulate mi RNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viralderived mi RNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBVderived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBVinduced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

RNAi:antiviral therapy against dengue virus

Dengue virus infection has become a global threat affecting around 100 countries in the world.Currently,there is no licensed antiviral agent available against dengue.Thus,there is a strong need to develop therapeutic strategies that can tackle this life threatening disease.RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process.Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication.This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.

2012年-2022年TBTV、TVDV、TBTVsatRNA和TVDVaRNA在云南主要烟区的发生与分布

烟草丛顶病(tobacco bushy top disease,TBTD)是一种由烟草丛顶病毒(tobacco bushy top virus,TBTV)及其卫星RNA(tobacco bushy top virus satellite RNA,TBTVsatRNA)和烟草扭脉病毒(tobacco vein distorting virus,TVDV)及其伴随RNA(tobacco vein distorting virus associated RNA,TVDVaRNA)共同侵染引起的危险性病害,曾在云南很多烟区造成严重损失。本研究对烟草丛顶病及其相关病原物在云南烟草上的发生情况进行了多年的调查研究,力图对烟草丛顶病及其相关病原物的发生与分布进行追踪和分析。结果表明,2012年-2022年,烟草丛顶病在云南各大烟区仅零星发生,2019年以来已很难发现。烟草丛顶病的相关病原物主要在曾经暴发流行过病害的楚雄、保山和大理发现,且TBTV、TVDV、TBTVsatRNA和TVDVaRNA在烟草上并不总是同时被检出,其中引起烟草丛顶病典型症状的TBTV+TVDV+TBTVsatRNA+TVDVaRNA检出率在5%以下,TVDV的检出率最高,达30.44%,TBTVsatRNA的检出率最低,为7.68%。TBTV、TVDV、TBTVsatRNA和TVDVaRNA不能同时感染同一烟株可能是当前烟草丛顶病很少发生的主要原因。

Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.

High-throughput RNA interference screens integrative analysis: Towards a comprehensive understanding of the virus-host interplay

Viruses are extremely heterogeneous entities; the size and the nature of their genetic information, as well as the strategies employed to amplify and propagate their genomes, are highly variable. However, as obligatory intracellular parasites, replication of all viruses relies on the host cell. Having co-evolved with their host for several million years, viruses have developed very sophisticated strategies to hijack cellular factors that promote virus uptake, replication, and spread. Identification of host cell factors(HCFs) required for these processes is a major challenge for researchers, but it enables the identification of new, highly selective targets for anti viral therapeutics. To this end, the establishment of platforms enabling genome-wide high-throughput RNA interference(HT-RNAi) screens has led to the identification of several key factors involved in the viral lifecycle. A number of genome-wide HT-RNAi screens have been performed for major human pathogens. These studies enable first inter-viral comparisons related to HCF requirements. Although several cellular functions appear to be uniformly required for the life cycle of most viruses tested(such as the proteasome and the Golgi-mediated secretory pathways), some factors, like the lipid kinase Phosphatidylinositol 4-kinase Ⅲα in the case of hepatitis C virus, are selectively required for individual viruses. However, despite the amount of data available, we are still far away from a comprehensive understanding of the interplay between viruses and host factors. Major limitations towards this goal are the low sensitivity and specificity of such screens, resulting in limited overlap between different screens performed with the same virus. This review focuses on how statistical and bioinformatic analysis methods applied to HTRNAi screens can help overcoming these issues thus increasing the reliability and impact of such studies.

Eight key long non-coding RNAs predict hepatitis virus positive hepatocellular carcinoma as prognostic targets

BACKGROUND Hepatitis B virus,together with hepatitis C virus,has been recognized as the leading causes of hepatocellular carcinoma(HCC).Long non-coding RNAs(lncRNAs)have been suggested in increasing studies to be the potential prognostic factors for HCC.However,the role of combined application of lncRNAs in estimating overall survival(OS)for hepatitis virus positive HCC(VHCC)is uncertain.AIM To construct an lncRNA signature related to the OS of VHCC patients to enhance the accuracy of prognosis prediction.METHODS The expression patterns of lncRNAs,as well as related clinical data were collected from 149 VHCC patients from The Cancer Genome Atlas database.The R package was adopted to obtain the differentially expressed lncRNAs(DElncRNAs).LncRNAs significantly associated with OS were screened by means of univariate Cox regression analysis,so as to construct a least absolute shrinkage and selection operator(LASSO)model.Subsequently,the constructed lncRNA signature was developed and validated.Afterwards,the prognostic nomogram was established,which combined the as-established lncRNA signature as well as the clinical features.Meanwhile,subgroup analysis stratified by the virus type was also performed.Finally,the above-mentioned lncRNAs were enriched to corresponding pathways according to the markedly coexpressed genes.RESULTS A total of 1420 DElncRNAs were identified,among which 406 were significant in univariate Cox regression analysis.LASSO regression confirmed 8 out of the 406 lncRNAs,including AC005722.2,AC107959.3,AL353803.1,AL589182.1,AP000844.2,AP002478.1,FLJ36000,and NPSR1-AS1.Then,the prognostic risk score was calculated.Our results displayed a significant association between the risk model and the OS of VHCC[hazard ratio=1.94,95%confidence interval(CI):1.61-2.34,log-rank P=2e-10].The inference tree suggested that the established lncRNA signature was useful in the risk stratification of VHCC.Furthermore,a nomogram was plotted,and the concordance index of internal validation was 0.763(95%CI:0.700-0.826).Moreover,the subgroup analysis regarding etiology confirmed this risk model.In addition,the Wnt signaling pathway,angiogenesis,the p53 pathway,and the PI3 kinase pathway were the remarkably enriched pathways.CONCLUSION An eight-lncRNA signature has been established to predict the prognosis for VHCC,which contributes to providing a novel foundation for the targeted therapy of VHCC.

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

AIM: To investigate the inhibitory effects of hepatitis B virus (HBV) replication and expression by transfecting artificial microRNA (amiRNA) into HepG2.2.15 cells. METHODS: Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays (TRFIA). HBV DNA replication was examined by ? uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS: The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection (P < 0.01 for all). The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% ± 4.7% and 39.9% ± 6.7%, respectively, at 72 h in amiRNA- HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection (P <0.01 for all). In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups (P < 0.01 for all, vs negative control). The copies of HBV DNA were inhibited by 33.4% ± 3.0%, 60.8% ± 2.3% and 70.1% ± 3.3%, respectively. CONCLUSION: In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

AIM： To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA （siRNA） pGenesiI-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS：pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays （TRFIA）. HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS： The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% （P 〈 0.01）, and by 38.67% （P 〈 0.05） and 42.86% （P 〈 0.01） at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR （P 〈 0.05）. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil- HBV X targeting the HBV X coding region.