| Background Stroke can cause local and systemic pathophysiological changes.Tregs and Th17 cells play an important role in the pathological process after stroke.The change of Th17 / Treg ratio plays an important role in local and systemic immune environment after stroke.About 2% of the genes in human body encode proteins,and more than 90% of the genes have transcriptional activity but no translation function,so they are called non coding RNA(nc RNA).At present,micro RNA(miRNA)and lnc RNA(long nocoding RNA)have been widely studied.Micro RNAs are a class of non coding RNAs with a length of 19-25 NT.Their sequences are highly conserved and their expression is time and tissue specific.Lnc RNA is a class of nc RNA molecules with a length of more than 200 NT.It plays a role in various aspects of gene expression regulation through different mechanisms such as RNA base pairing,RNA protein interaction,RNA-DNA interaction,including chromatin modification,transcription regulation,RNA cutting and stability regulation.More and more studies have shown that lnc RNA and micro RNA not only participate in various cell biological processes,but also play an important role in various diseases.At present,researchers have studied the role of nc RNA in regulating the function and differentiation of T cells after stroke,and obtained some evidence.However,we speculate that there may be other regulatory mechanisms.Part one Objective We aimed to detect the key molecules involved in Th17 and Treg differentiation in MCAO model and sham operation model,and analyze their possible mechanisms in regulating Th17 and Treg differentiation under stroke conditions.Methods Mice were randomly divided into two groups,three in each group.One group underwent MCAO modeling(observation group),the other group underwent sham operation(control group).The eye blood was taken 24 hours after modeling.PCR was used to detect the key molecules in the key pathway of Th17 and Treg differentiation in peripheral blood samples of the two groups.According to the results of PCR,the stable and distinct expression molecules were selected as the research objects.PCR was used to detect the differential expression of micro RNA in the blood samples of two groups of mice.The expression of the molecules with obvious difference and stability was selected as the research objects,and the difference of lnc RNA expression in the two groups of blood samples was detected.Subsequently,we detected the difference in the expression of ROR?t and foxp3,the marker of Th17 and Treg respectively,in the blood samples of the two groups.At the same time,the Th17/Treg ratio was analyzed by flow cytometry.Results Compared with the control group,the relative expression of STAT3 and Fox O1 in peripheral blood of the MCAO group was significantly higher than that of the control group.There was no significant difference in the relative expression of SOCS3,PTEN,Smad7 and Smad3.We selected STAT3 as the research object,and detected the expression of potential micro RNAs that regulate the expression of STAT3 by PCR.We found that compared with the control group,the expression of miR-30 a,miR-495 and miR-1192 in the MCAO group was significantly reduced.However,the expression of miR-203 increased significantly.We selected miR-1192 as the research object,and detected the relative expression of lnc RNA which regulated the expression of miR-1192 by PCR.We found that there was no significant difference in the relative expression of RP23-261O3.1,RP23-402K24.2 and C230088H06 Rik between MCAO group and sham operation group.However,the relative expression of Gm4117 was increased,and the difference was statistically significant.After PCR,we found that compared with the control group,the relative expression of ROR?t in the MCAO group was significantly increased,while the relative expression of Foxp3 was decreased.The differences were statistically significant.Flow cytometry showed that the proportion of Th17 increased and the proportion of Treg decreased in the observation group.Th17/Treg ratio in MCAO group was increased.Conclusion After the operation of MCAO,the proportion of Th17 increased and the proportion of Treg decreased.Th17/Treg ratio increased.Many molecules involved in the regulation have changed,suggesting that these molecules form a complex regulatory network,and ultimately regulate the differentiation of Th17 and Treg.Gm4117-miR-1192-STAT3 is a possible regulatory pathway.Part two Objective To investigate the role of Gm4117 in regulating Th17 and Treg differentiation of na?ve CD4 + T cells in vitro.Methods Single cell suspension was prepared from spleen of mice.Na?ve CD4+ T cells were isolated by magnetic bead sorting.The overexpression and interference plasmids of Gm4117 were constructed.The cells were divided into overexpression group,overexpression control group,interference group,interference control group and blank control group.The cells were treated with overexpression lentivirus,overexpression control lentivirus,interference lentivirus,interference control lentivirus and no lentivirus.Then the five groups were divided into three groups.Th17 polarization culture,Treg polarization culture and blank culture were performed in three groups.First of all,PCR was used to detect the relative expression of Gm4117,miR-1192 and STAT3 in 5 groups of cells under various polarization conditions.WB was used to detect the difference of IL-17 A and TGF-? in 5 groups.The ratio of Th17 cells to Tregs was measured by flow cytometry,and the ratio of Th17 / Treg in 5 groups was compared.We detected the ratio of Th17 to Tregs,calculated Th17/Treg ratio,and detected the expression of STAT3 by PCR.We used WB to detect the level of p-STAT3 in 5 groups of cells,and compared the relative gray value.STAT3 can not only directly regulate the differentiation of na?ve CD4+ T cells,but also indirectly regulate the expression of HIF-1?,a key factor of HIF-1 signaling pathway.So we detected the expression and level of HIF-1? by PCR and WB respectively.The expression and level of apoptosis related proteins Bcl-XL,Bcl-2,SOCS1,MCL1 and PIM1 regulated by STAT3 were detected by PCR and WB,respectively.And the level of Caspase-3 was detected by WB.Results The relative expression of Gm4117 and STAT3 in overexpression group and miR-1192 in interference group were higher than those in other groups;conversely,the relative expression levels of Gm4117,STAT3 in interference group and miR-1192 in overexpression group were lower than those in other groups;The relative expression of Gm4117,miR-1192 and STAT3 in interference control group,overexpression control group and blank group were in the middle,and there was no significant difference between them.The Western-Bolt detection results of IL-17 A and TGF-? in the 5 groups showed that IL-17 A in overexpression group was significantly increased,while TGF-? was decreased;IL-17 A was significantly decreased and TGF-? was increased in interference group,the differences were statistically significant.There was no significant difference among the other three groups.The differentiation of Th17 and Treg was similar in 5 groups under different polarization conditions.In the over expression group,the ratio of Th17 cells increased,the ratio of Tregs decreased,and the ratio of Th17/Treg increased;the ratio of Th17 cells decreased,the ratio of Tregs increased,and the ratio of Th17/Treg decreased in the interference group;the ratio of Th17,Tregs and Th17/Treg ratio in the interference control group,over expression control group and blank group were all in the middle level.Under no polarization condition,compared with the blank group,co-transfection with Gm4117 interference lentivirus and miR-1192 inhibitor,there was no significant difference in Th17 and Treg cell ratio and Th17/Treg ratio between the two groups.There was no significant difference in STAT3 expression.WB was used to detect the level of p-STAT3 in the five groups.The results showed that the relative gray value of p-STAT3 in the overexpression group increased,while the relative gray value of pSTAT3 in the interference group decreased,and the difference was statistically significant.There was no significant difference among the other three groups.The expression of HIF-1? was detected by PCR in 5 groups.The results showed that the expression of HIF-1? in the overexpression group was increased,while the expression of HIF-1? in the interference group was decreased,and the difference was statistically significant.There was no significant difference among the other 3 groups.WB method was used to detect the level of HIF-1? in the five groups.The results showed that the relative gray value of HIF-1? in the overexpression group was increased,while the relative gray value of HIF-1? in the interference group was decreased,and the difference was statistically significant.There was no significant difference among the other three groups.The expression and level of apoptosis related proteins Bcl-XL,Bcl-2,SOCS1,MCL1,PIM1 and caspase-3 regulated by STAT3 were detected by PCR and WB,respectively.The results showed that the expression of Bcl-x L,Bcl-2,SOCS1,MCL1 and PIM1 in the overexpression group was increased,while the expression of Bcl-x L,Bcl-2,SOCS1,MCL1 and PIM1 in the interference group was decreased,the difference was statistically significant,and there was no significant difference among the other three groups.WB method was used to detect the levels of Bcl-x L,Bcl-2,SOCS1,MCL1 and PIM1 in the five groups.The results showed that the relative gray values of Bcl-x L,Bcl-2,SOCS1,MCL1 and PIM1 in the overexpression group increased,while the relative gray values of Bcl-x L,Bcl-2,SOCS1,MCL1 and PIM1 in the interference group decreased,and the difference was statistically significant.WB method was used to detect the level of Caspase-3 in the five groups.The results showed that the relative gray value of Caspase-3 in the overexpression group was decreased,while the relative gray value of Caspase-3 in the interference group was increased.Conclusion Overexpression of Gm4117 can negatively regulate miR-1192,eliminate the negative regulation of STAT3,and finally regulate na?ve CD4 + T cells to differentiate into Th17.Interference with Gm4117 positively regulates miR-1192 and negatively regulates the expression of STAT3,and finally regulates the differentiation of na?ve CD4 + T cells into Treg,which can be blocked by miR-1192 inhibitor.Therefore,we suggest that Gm4117 regulates Th17 and Tregs differentiation by negatively regulating miR-1192 expression.Gm4117 regulates the differentiation of Th17 and Treg,not only through the direct regulation of STAT3,but also through the indirect regulation of HIF-1 signaling pathway.In addition,the expression of STAT3 related antiapoptotic protein makes the regulation of Th17 / Treg balance better maintained.Part three Objective To study the effect of gm4117 on Th17 and Treg differentiation of na?ve CD4 + T cells in vivo.Methods Mice were randomly divided into 5 groups: overexpression group,overexpression control group,interference group,interference control group and blank control group,3 mice each group.The corresponding lentivirus was injected into mice via tail vein.After two weeks of injection,MCAO model was established.Then,the expression of Gm4117,miR-1192 and STAT3 in the blood of five groups were detected by PCR;the ratio of Th17 cells and Treg cells in the blood of each group was detected by flow cytometry.Results The relative expression of Gm4117 and STAT3 in overexpression group and miR-1192 in interference group increased,the difference was statistically significant;the relative expression of Gm4117 and STAT3 in the interference group and miR-1192 in the overexpression group were decreased,the difference was statistically significant;the relative expression of Gm4117,miR-1192 and STAT3 in the interference control group,overexpression control group and blank group was in the middle,and there was no significant difference between them.In the over expression group,the ratio of Th17 cells increased,the ratio of Tregs decreased,and the ratio of Th17/Treg increased;in the interference group,the ratio of Th17 cells decreased,the ratio of Tregs increased,and the ratio of Th17/Treg decreased;the ratio of Th17 cells,the ratio of Treg cells and the ratio of Th17/Treg in the interference control group,over expression control group and blank group were in the middle,and there was no significant difference between them.We used western bolt to detect the blood of each group of mice.The results showed that in the overexpression group,IL-17 A was significantly increased,TGF-? was decreased,the difference was statistically significant;IL-17 A was significantly decreased,and TGF-? was increased in the interference group,the difference was statistically significant.The expression of the two proteins in blank group,interference control group and overexpression control group was in the middle.There was no significant difference among the three groups.Conclusion In vivo,Gm4117 can also negatively regulate miR-1192,thus weakening its negative regulatory effect on STAT3,and regulating na?ve CD4 + T cells to differentiate into Th17. |
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