Mechanism Research On The Function Of Host Molecule TXNIP In HCV Infection

Hepatitis C virus infection may cause chronic Hepatitis C,which has great impact on host cellular physiology,inducing various cellular signaling pathways and regulating gene expression.The host responses may have positive or negative influence on virus clearance and cell survival.ThroughTranscriptom Microarray,werevealed a series of transcriptome change before and after HCV infection.Thioredoxin-interactingprotein TXNIP is a host gene up-regulated during HCV infection discovered by the Microarray.We verified its differential expression and investigated its effect on HIV infection.It has been found that J6/JFH1 strain HCVcc infection could increase the expression of TXNIP in Huh7 but not in Huh7.5.1 cells.It is unclear that: 1.How does HCV induce upregulation of TXNIP;2.What is the role of TXNP in HCV life cycle;3.Whether HCV infection induced upregulation of TXNIP is involved in the pathogenesis of HCV.We intend to discuss these issues and find the interaction mechanism of TXNIP and HCV.So thatwe can raise awareness for the molecular mechanism of chronic HCV infection and pathogenesis;as well as provide new targets and new ideas for the development of effective anti-HCV drugs and vaccine research.The main results and conclusions are as follows:(1)HCV infection induced gene-expression change in Huh7 cells,the transcription levels of TXNIP was increased.Huh7 cells was infected by HCVcc(MOI=1).After 72 h of infection,the infected cells and uninfected cells were collected with Trizol,Glue grant human Transcriptome array gene chip analysis was preceded after sending the sample to the Ming Information Technology Co.,Ltd.,to compare the difference of transcriptome in Huh7 cell before and after HCVcc infection.871 significantly up-regulated genes(Greater than or equal to 1.5 times),765 down-regulated genes(Greater than or equal to 0.66 times);2339 significantly up-regulated non coding RNA,2479 significantly down-regulated non coding RNA;27 significant changes miRNA,and 210 alternative splicing pattern changes gene were discovered.We can see that,host molecule TXNIP mRNA level increased about 3.7 times in Huh7 cell line after HCVcc infection.Using RT-PCR to verify the mRNA transcription of TXNIP of HCVcc infected Huh7 cells and uninfected Huh7 cells;once again we confirm that transcription level of TXNIP is significantly up-regulated in Huh7 cells after HCVcc infection.For further study on the changes ingene expression pattern after HCVcc infection profile,we performed deep sequencing with gene transcriptome of HCVcc infected Huh7 cell line at 12 h,36 h,60 h,and compared it with the control samples at each time point.The results showed that TXNIP mRNA expression in Huh7 cell was significantly up-regulated after HCV infected at 36 h,60 h and 36 h was the optimal.(2)The expression of TXNIP interference will affect HCV replicationSynthesis of TXNIP specific siRNA,Huh7 or Huh7.5.1 cell lines were transfected with siRNA to knock down TXNIP expression.And then the cells were infected by HCVcc(MOI=0.2),immune fluorescence detection was conducted to detect the infection at 48 h.The results showed that HCVcc infection in TXNIP knockdown cells was significantly decreased compare with cells transfected with non-specific binding siRNA(negative control group)and the non transfected siRNA(blank control group).The constructed TXNIP eukaryotic expression vector were transfected into Huh7 and Huh7.5.1 cells,for cells transfected with GFP eukaryotic express vector were negative control group,cells without any treatment were used as blank control.Then it was infected with HCVcc(MOI=0.2),immunofluorescence detection of HCVcc infection was conducted after 48 h.The results showed that HCVcc infection of TXNIP over expression cells was increased compared with cells transfected with GFP expression plasmid(negative control)and blank control group.To detect the effect of host molecules TXNIP on HCV cell entry,TXNIP targeting siRNA was transfected into huh7.5.1 cells to reduce TXNIP expression,cells was transfected with non-specific binding siRNA as negative control;cells without any treatment were used as blank control.Then infect the cells with the HCV entry model,con1 type HCVpp.After 72 h of culture,green fluorescent labeled HCVpp infected cells were visualizedby the fluorescent microscope directly.The results showed that knocking down TXNIP protein does not affect the cell entry of HCVpp.For studying the impact of host molecule TXNIP onHCV replication,transfect TXNIP targeting siRNA to interfere TXNIP expression in BB7 cell line,the control group is transfected with non specific binding siRNA.Extraction of BB7 cell total RNA was preceded at 72 h after transfection to detect expression level of J6/JFH1 type HCV non structural gene by RT-PCR.The results showed that reduced expression of the host molecule TXNIP affected HCV replication.In summary,TXNIP in the host cell promoted HCV infection.This effect is likely to play a role in HCV replication stage.(3)HCV infection does not induced the upregulation of TXNIP in Huh7.5.1 cell lineIn order to verify HCVcc infection induced TXNIP upregulation is established in other HCV infected cell lines,more detailed studies on Huh7 and Huh7.5.1 were conducted.Compared with Huh7,RIG-1 gene of Huh7.5.1 mutated,so it has defects in IFN synthesis induced by RNA.Huh7 and Huh7.5.1 cells were infected with HCVcc(MOI=0.5),1-4 days infection time samples and the corresponding control samples were collected,respectively.TXNIP mRNA levels changes was detected by RT-PCR and TXNIP protein expression levels was detected by Western blot.The results showed that,mRNA and protein level increase of TXNIP can be detected after HCVcc infection at 24 h in Huh7 cells;however,this does not happen in Huh7.5.1 cells.(4)Interferon induced the up-regulation of TXNIP in Huh7 and Huh7.5.1 cell linesIFN-alpha,IFN-beta,IFN-lambda eukaryotic expression plasmid were constructed,respectively.Then transfect 293 T cell line with this plasmid,the supernatant containing interferon and non-transfected cell culture supernatant were collected.In order to determine the optimal concentration of interferon to treat Huh7 and Huh7.5.1 cells with,cell culture supernatant with different concentrations of interferon was added to HCVcc infected Huh7.5.1 cells,infection levels were detected by immunofluorescence after 2 days.Find out the corresponding concentrations that have 50% antiviral level.Huh7 and Huh7.5.1 cells were treated with the interferons at this concentration.The processing time was 12 h,1-3 days,respectively.The results showed that a TXNIP mRNA level increase was observed after 12 h treatment with interferon.