Study On The Effect And Mechanism Of Signal Transducer And Activ E-ator Of Transcription Factor 3 In Varicella Zoster Virus Replication

Background and ObjectiveVaricella zoster virus (varicella-zoster virus, VZV) is a herpesvirus alpha subfamily, double stranded DNA virus. Humans are generally susceptible to VZV, VZV is mainly transmitted through respiratory tract and close contact, its infection can cause varicella and herpes zoster. Chicken pox is caused by VZV primary infection, mainly seen in children, characterized by systemic herpes and often accompanied by fever. Chickenpox is a highly contagious epidemic disease. The infection rate of susceptible population in the same household is often 100% in which 96% are dominant infection with obvious clinical symptoms, only 4% are recessive infection.VZV primary infection viruses which can cause zoster through reactivation when airframe immunity power is low cannot be cleared out the body completely, but lurk in the sensory nerve sells of spinal ganglion and trigeminal ganglion, with clinical features as unilateral distribution along the limb erythema blister with obvious pain. Varicella and herpes zoster is a clinically common disease, however, as an important part of the pathogenesis of VZV, the study of the infection mechanism of VZV is still relatively scarce.The gene expression of the level control of Small RNA (micro RNA-21) in the post gene transcription, is a kind of small non coding RNA with length 21-24nt, is involved in a variety of biological signal pathway regulation..mi RNA can control many biological processes:such as cell proliferation, differentiation and apoptosis. More and more evidence show the abnormal expression of mi RNA in tumor. In the aspect of mi RNA affecting viral replication, mi RNA were shown to have different effects. MiR-181 inhibited the respiratory syndrome virus; miR-17-29 interference of HBV and miR-501 enhanced HBV production, but not inhibit the replication, the relationship between the two is the same as in the interaction between miR-126 and Coxsackie virus. However, it has not been found that any kind of MI RNA can act on the replication of VZV.Signal transducers and activators of transcription family (signal transducer and activate of transcription, Stats) is a kind of transcription factor activated by polypeptide ligand such as growth factors and cell factors. The family has 7 members, of which Stat3 is one of the more active members. Stat3 is a transcription factor which has carcinogenic potential. Stat3 has 7 main parts ①the conserved amino terminal sequence, is the effect between Stat3 and other transcription factors, and is necessary for Stat3 dimer formation and for functions like Combined with cytokine receptors beta ②Serine phosphorylation sites in 727 place can activate Stat3 causing connection between STAT pathway and Ras pathway. ③Located in the carboxyl terminal tyrosine phosphorylation sites of 705th.The sites after phosphorylation can activate Stat3 shifting into the nucleus to activate transcription of target genes; ④it is located between the highly conserved 400th and 500th amino acids of DNA binding region, ⑤the region in which the two different splicing patterns formed Stat3a and Stat3βwhich contains a transcriptional activation domain;⑥the function of SH3 region between 500th and 600th amino acid is poor in conservation has not been clear yet;⑦ SH2 District which is located between the 600th and 700 amino acid is to make Stat3 molecules and activated receptor form compound, mediate interaction between Ja-nus kinase and STAT, making Stat3 a two mer into the nucleus, regulating gene expression Under normal physiological conditions, the activation of Stat3 is rapid and transient, plays a critical role in physiological function. Under pathological conditions, Stat3 belongs to a cancer causing gene, inhibit apoptosis strongly, promote cell proliferation. In addition, Stat3 plays an important role in the pathogenesis of the virus, such as gamma herpes, card Posey sarcoma herpesvirus (KSHV), and Epstein-Barr virus (EBV) . Recently it is reported that VZV triggers the Stat3 signal transduction pathway, and according to the anti apoptosis protein surviving dependent mechanism activate the Stat3, increase VZV replicationIFNs is a cytokine with important antiviral immune response, in accordance with its origin and function, it can be divided into three categories. Type Ⅰ interferon include IFN-alpha and IFN-beta, can be produced in most cell types, with antiviral activity. IFN-ω, epsilon, kappa, Delta and tau also belongs to the I IFN, but their expressions in cell types are different according to species and specificity. Type Ⅰ interferon by receptor IFNARl/2 to intracellular signaling is involved in the induction of [7 type Ⅰ IFN signaling pathways, such as Jak Stat pathway, CRKL pathway; PI3K pathway; MAPK pathway. Many viruses are very sensitive to interferon on type Ⅰ, type Ⅰ interferon can play the role of [59] in any phase of viral replication.. type Ⅰ interferon antiviral function is the function of protein factors induced by interferon to achieve, the more research including PKR, ADAR, OAS (2c,5c-oligoadenylate synthetase), RNase L and Mx. In addition to interferon induced antiviral protein, type I interferon can also play the function by regulating immune cells such as NK cells, T cells and other important.According to the basis of the study above, we speculate that in the case of VZV infection, whether there is connection among VZV, miRNA,IFN and Stat3. This is a topic worthy of study, its significance lies in the research of mechanism of VZV replication.In the early stages of the disease, the research can give active treatment to patients especially for elderly patients with herpes zoster, in order to reduce the incidence of postherpetic neuralgia, and provide new ideas for clinical treatment.Methods1)Compare the uninfected with VZV in human embryonic lung fibroblasts (HELF) cells with infected, using real-time fluorescence quantitative polymerase chain reaction (q RT-PCR), comparing the expressional difference of micro RNA-21, Stat3, survivn.2) According to the consistence of OnM,25nM,50nM, 100nM,200nM increase the analog microRNA-21 in HELF.Detect the virus titer.3)Transfect the analog of microRNA-21 in HELF cell lines, compare partial samples transfected mi R-control, using the qRT-PCR, GAPDH as the internal control, to observe the differences between the expression of m RNA of Stat3 and anti survivn.4) Transfect the analog of microRNA-21 and silent Stat3 gene in HELF cells when infected by VZV. They were divided into 4 groups, namely 21+shStat3 (analog +Stat3 gene silencing cells were transfected with + microRNA-21),21+shcontrol (cell were transfected into +mi RNA -21-simulation and +control gene silencing), control +shStat3 (RNA + Mi cells were transfected with control+Stat3 silencing gene), control + shcontrol (cell transfection+Mi RNA control + control gene silence). The expression of Stat3 protein is detected by Western blotting method (to judge whether the Stat3 is effectively knocked out).Observe the expression of microRNA-21 among groups, and the changes of virus titer.5) VZV infected HELF cells, the expression of Stat3 was detect by WB;6) The VZV titer in VZV+HELF and VZV+HELF +upStat3 two groups was detected by using plaque assay, protein expression of 11 FN (OAS, PKR, IRF3 and STAT1) were detected by qRT-PCR. The same experiment was done in VZV+HELF and VZV+HELF +siStat3.7) in VZV+HELF, VZV+HELF+upSTAT3 group and VZV+HELF+siSTAT3 group, respectively, according to the 0,25,50 100U/ml accession to the IFN, so as to detect the virus titer. In VZV+HELF, VZV+HELF+upSTAT3 group and VZV+HELF+siSTAT3 group three, were added to 25ng/ml IFN Ab, detection of viral titer.RESULT1) The expression level of microRNA-21 in HELF after VZV infection increases. (P= 0.000). Both Stat3 and survivn are all increase in HELF with VZV.2) In HELF cells, the virus titer increased with the analogue of microRNA-21 concentration, there is a positive correlation between them. But when the concentration of the analogue of microRNA-21 is 200nM, there is no significant increase in viral titers.3) Overexpression of microRNA-21 and activation in vitro Associated with the Stat3 signal transduction pathway. In the HELF cells of transfected microRNA-21 analog,the mRNA expression of Stat3 (P= 0.000) and survivn (P=0.000) were significantly increased.4)MicroRNA-21 has higher expression in 21+shStat3 and 21+ shcontrol, significantly better than the other two groups; in 21+shStat3 and control+ shStat3, the expression of Stat3 is extremely low, indicating the effective knockout. The average titer in 4 groups are 3.47 × 102 PFLV ml (21+shStat3),1.48 × 107 PFU/ml (21+shcontrol),1.82 × 102 PFU/ml (control+shStat3),3.23 × 104 PFU/ml (control +shcontrol). Overexpression of miR-21 stimulates VZV replication, until the silence of Stat3 gene.5) After VZV infection, the expression of Stat3 is significantly. The over expressionof Stat3 significantly increased VZV infection (upSTAT3 VS up control, P<0.001),at the same time, interferon response gene (OAS (P= 0.000), PKR (P= 0.000), IRF3 (P= 0.000) and STAT1 (P= 0.000) in upStat3 have a remarkabledecline.VZV 24 hours after the HELF infection, knockdown of STAT3 group virus titer decreased significantly (p=0.000), and the I IFN gene OAS (P= 0.000), PKR (P= 0.000, IRF3 (P=0.000) and STAT1 (P= 0.000) increased significantly.6)In normal HELF and Stat3 knockdown cells, interferon in a dose dependent (25,50, 100 U/ml) method to reduce the VZV replication. With 25ng/ml interferon antibody treated cells, VZV titer reached a high level.Conclusion1) Both the microRNA-21 and Stat3 promot VZV replication. The overexpression of microRNA-21 increasethe activation of Stat3, then promot VZV replication.2) Stat3 expression can significantly inhibit IFN of VZV infection.When the Stat3 was in silence, the response of IFN-I to VZV infection was improved. Stat3 inhibiti the response of IFN-I mediated antiviral in the course of VZV infection.3) Stat3 can promote VZV replication through inhibition the response to IFN-I mediated antiviral. MicroRNA-21 increasethe activation of Stat3,both of them promote the VZV replication.