Effect of TGF-beta/smad signaling pathway blocking on expression profiles of miR-335, miR-150, miR-194, miR-27a, and miR-199a of hepatic stellate cells [HSCs]

Aim: The aim of this study was to determine the effect of inhibition of TGF-beta/smad signaling on the expression profiles of miR-335, miR-150, miR-194, miR-27a, miR-199a of hepatic stellate cells [HSCs]

Background: Liver fibrosis is excessive deposition of extracellular matrix proteins due to ongoing inflammation and HSC activation that occurs in most types of chronic liver diseases. Recent studies have shown the importance of microRNAs in the pathogenesis of chronic liver diseases

Methods: In this study, for inhibition of TGF-beta smad-signaling pathway, expressing Smad4 shRNA plasmids were transfected into HSCs. Subsequently, using Real Time-PCR, we measured the expression levels of miR-335, miR-150, miR-194, miR-27a and miR- 199a

Results: Gene expression analysis showed that downregulation of Smad4 by vector Smad4shRNA significantly increased the expression levels of miR-335 [P<0.01] and miR-150 [P<0.001] and decreased the expression level of miR-27a [P<0.05]

Conclusion: The results of this study suggest that blocking TGF-beta smad-signaling can also differentially modulate microRNA expression in support of activation and fibrogenesis of HSCs

Evaluation of hepatitis C virus intrafamilial transmission among families with one index case, a pilot study from Fars province, Iran

Aim: Our aim was to survey the rate and risk factors for Hepatitis C virus interfamilial transmission among families withone index case

Background: The role of intrafamilial transmission in Hepatitis C virus epidemiology is still debated

Patients and methods: A cross-sectional study was conducted on 34 families [236 members] of HCV infected patientsfrom Fars province, spring to summer 2013. All subjects were first evaluated for the risk factors of exposure and then theirserum was checked for the presence of HCV antibody and the genome, using ELISA and PCR. The genotype of all PCRpositive cases was also determined by a commercial assay. Two independent sample t test and Chi-Square test were used tocompare groups together

Results: In 18 out of 34 families, HCV antibody was detected [52.9%] in new members. Among them, HCV transmissionin 11 families [32%] was also confirmed by PCR. Having a history of intravenous drug abuse [P=0.006] and incarceration[P=0.01] showed to be important risk factors for interfamilial transmission. Hence, blade/needle sharing [P=0.016] justfollowing molecular assay and sex [P=036] only in the serologic analysis were also determined as significant risk factors.Furthermore, based on serologic results, medium socioeconomic state was further associated with this manner oftransmission [P=0.019 and P=0.328]. Interestingly, among relatives, 13 cases were brothers while just 5 cases werecouples. The genotypes 3a and 1a were more prevalent among the population

Conclusion: In conclusion, our finding highlighted a noticeable role of interfamilial transmission for HCV spread andsupports the significant role of close relatives, especially brother relationship in this spread. Hence, the socioeconomic statewas associated with the transmission rate of virus in the family

[Effect of HCV core protein on expression of alpha-SMA in human stellate cell, LX-2 cell line]

Objective: Hepatitis C virus [HCV] is considered to be a worldwide health problem. In most cases, HCV infection becomes chronic and may proceed to fibrosis, cirrhosis, and hepatocellular carcinoma. Many pathological effects in cells may occur by viral proteins. The purpose of this study is to evaluate the effect of the HCV core protein on cells to induction of the fibrogenesis process

Methods: We use the LX-2 cell line that originated from hepatic stellate cells. Plasmid which expressed HCV core protein was transfected to the cells. After 72 h, RNA was extracted and treated with DNase, followed by synthesis of cDNA. Positive control cells were treated with the leptin fibrotic hormone. We used real-time PCR to measure and statistically analyze alpha-SMA gene expression

Results: The HCV core protein significantly increased alpha-SMA gene expression [p<0.05]. There was more alpha-SMA gene expression in cells treated with leptin compared to cells treated with the HCV core protein

Conclusion: HCV infection is an impressive factor in the development of chronic hepatitis to hepatic fibrogenesis. The HCV core protein can induce a fibrogenesis process in HCV infection

Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells

In this study, to clarify the SMAD4 blocking impact on fibrosis process, we investigated its down regulation by shRNA on activated human LX-2 cell, in vitro. Liver fibrosis is a critical consequence of chronic damage to the liver that can progress toward advanced diseases, liver cirrhosis and hepatocellular carcinoma [HCC]. Different SMAD proteins play as major mediators in the fibrogenesis activity of hepatic stellate cells through TGF-beta pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear. vector expressing verified shRNA targeting human SMAD4 gene was transfected into LX-2 cells. The GFP expressing plasmid was transfected in the same manner as a control group while leptin treated cells were employed as positive controls. Subsequently, total RNA was extracted and real-time PCR was performed to measure the mRNA levels of SMAD4, COL-1A1, alpha-SMA, TGF-beta and TIMP-1. Furthermore, trypan blue exclusion was performed to test the effect of plasmid transfection and SMAD4 shutting-down on cellular viability. The results indicated that the expression of SMAD4was down-regulated following shRNA transfection into LX-2 cells [P<0.001]. The gene expression analysis of fibrotic genes in LX-2 cells showed that SMAD4 blocking by shRNA significantly reduced the expression level of fibrotic genes when compared to control plasmids [P<0.001]. Vector expressing SMAD4-shRNA induced no significant cytotoxic or proliferative effects on LX-2 cells as determined by viability assay [P<0.05]. The results of this study suggested that knockdown of SMAD4 expression in stellate cell can control the progression of fibrogenesis through TGF-beta pathway blocking

[Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector]

In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient. The partial NS3 [pNS3] gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line. After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively. This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine

Prediction of hepatitis B virus lamivudine resistance based on YMDD sequence data using an artificialneural network model

Hepatitis B virus [HBV] infection is an important health problem worldwide with critical outcomes. The nucleoside analog lamivudine [LMV] is a potent inhibitor of HBV polymerase and impedes HBV replication in patients with chronic hepatitis B. Treatment with LMV for long periods causes the appearance and reproduction of drug-resistant strains, rising to more than 40% after 2 years and to over 50% and 70% after 3 and 4 years, respectively. Artificial neural networks [ANNs] were used to make predictions with regard to resistance phenotypes using biochemical and biophysical features of the YMDD sequence. The study population comprised patients who were intended for surgery in various hospitals in Tehran-Iran. An ACRS-PCR method was performed to distinguish mutations in the YMDD motif of HBV polymerase. In the training and testing stages, these parameters were used to identify the most promising optimal network. The ideal values of RMSE and MAE are zero, and a value near zero indicates better performance. The selection was performed using statistical accuracy measures, such as root mean square error [RMSE], coefficient of determination [R2], and mean absolute error [MAE]. The main purpose of this paper was to develop a new method based on ANNs to simulate HBV drug resistance using the physiochemical properties of the YMDD motif and compare its results with multiple regression models. The results of the MLP in the training stage were 0.8834, 0.07, and 0.09 and 0.8465, 0.160.04 in the testing stage; for the total data, the values were 0.8549, 0.115, and 0.065, respectively. The MLP model predicts lamivudine resistance in HBV better than the MLR model. The ANN model can be used as an alternative method of predicting the outcome of HBV therapy. In a case study, the proposed model showed vigorous clusterization of predicted and observed drug responses. The current study was designed to develop an algorithm for predicting drug resistance using chemiophysical data with artificially created neural networks. To this end, an intelligent and multidisciplinary program should be developed on the basis of the information to be gained on the essentials of different applications by similar investigations. This program will help design expert neural network architectures for each application automatically