Ctenopharyngodon Idella STAT3 Alleviates Auttophagy By Up-regulating Bcl-2 Expression

In mammals,signal transducer and activator transcription 3(STAT3;Signal transducer and activator transcription 3)can be activated by cytokines,and transcriptional regulation of the downstream genes,is a key transcription factor.Recent studies have shown that STAT3 is not only present in the nucleus,but also in the cytoplasm.In the nucleus,STAT3 is activated and targets downstream genes to regulate autophagy;in the cytoplasm,STAT3 interacted with PKR to regulate autophagy by regulating EIF2?.Grass Carp is the fish with the highest sales volume of freshwater fish in the world,especially in Asian countries.Therefore,the research on Grass Carp is of great importance and far-reaching significance.In this study,cells were stimulated by the autophagy inducers rapamycin and tunicamycin,respectively,and the expression of CiIL-6r was quantitatively detected by RT-PCR.The results showed that the expression of CiIL-6r was decreased,but the expression of CiIL-6r was increased after the treatment with human recombinant interleukin-6(rIL-6)for a short time,and it was higher than that in the blank control.We suggested that rIL-6 may inhibit autophagy.Western blot to detect autophagy indicating protein LC3,autophagy inducer(rapamycin and tunicamycin)stimulating cells in experimental LC3?/LC3? ratio of 0.84 and 4.7,respectively,compared with the control,rIL-6 function after a short period of time,LC3?/LC3? ratio is reduced to 0.45 and 0.69,named autophagy is reducing.In addition,we transfected the recombinant plasmid GFP-LC3 into CIK cells,and detected fluorescence by confocal microscopy.It was confirmed that autophagy inducers could induce autophagy,but the autophagy was alleviated after as short period of IL-6.We suggested that rIL-6 inhibits autophagy.To verify whether STAT3 involved in rIL-6 inhibit autophagy process,we respectively use autophagy inducers rapamycin and tunicamycin to gradient time stimulate cell,RT-PCR detection CiSTAT3 expression,according to the results STAT3 has been suppressed,and within 24 hours meet minimum,when cells in autophagy inducer tunicamycin,use the rIL-6 role can increase CiSTAT3 expression.We believe that STAT3 is involved in autophagy.To verify whether or not STAT3 is activated,the recombinant plasmid FLAGtagged CiSTAT3 and GFP-tagged CiSTAT3 were co-transferred into HEK-293 T.The immunoprecipition assay(CO-IP)showed that CiSTAT3 could not form its own dimer without IL-6,while CiSTAT3 formed dimer with IL-6,indicating that STAT3 was activated by IL-6.In mammals,Bcl-2 is a downstream target gene of STAT3,and it is not only involved in the regulation of apoptosis,but also involved in the regulation of autophagy.Fluorescence quantitative results also showed that the expression of CiBcl-2 was decreased under the action of autophagy inducer,and the expression of Bcl-2 reached a peak under the action of specific IL-6 concentration,which was consistent with CiIL-6R and CiSTAT3.Then,to verify whether CiSTAT3 can activate the expression of CiBcl-2 gene,we will co-transfer pCDNA3.1-ORF-CiSTAT3(or pCDNA3.1-Basic)and pGL3-CiBcl-2 into CIK cells to detect the transcriptional activity of the Bcl-2 promoter.The results of Dual-luciferase activity analysis showed that CiSTAT3 could up-regulation the transcriptional activity of CiBcl-2 and enable it to be expressed in large quantities.Bcl-2 is an anti-apoptosis gene,and its expression ratio to Bax can reflect the apoptosis of cells.We used One Step TUNEL Apoptosis Assay Kit to detect the apoptosis of cells.The results showed that the autophagy inducer TM could induce apoptosis,but the apoptosis was reduced after the addition of IL-6 for a short time incubation.In addition,we also used CCK8 to test the cell viability,and the results showed that the autophagy inducer TM reduced the cell viability,but the cell viability increased after a short period of IL-6.