| Objective:the main characteristic of hepatic ischemia-reperfusion injury(I/RI)is inflammatory reaction.It is very important to understand and explore the mechanism of inflammatory reaction in order to formulate therapeutic strategies for ischemia-reperfusion injury.In this study,we aimed to elucidate the role of the transcription factor YY1 in liver ischemia-reperfusion injury and the micro RNA(MIR)-181a-5p/estrogen receptor mediated by the transcription factor YY1α(ESR1)/epidermal growth factor receptor 2(ERBB2)signaling axis in liver IRI.Method:Part 1: In order to clarify the effects of YY1 and mi R-181a-5p on hepatic ischemia-reperfusion injury in vivo,UCSC database was used to retrieve the promoter sequence of mi R-181 a,and then h TFtarget and ALGGEN databases were used to predict the upstream regulatory transcription factors of mi R-181a-5p.Then we established the liver I/RI mouse model.HE staining,ALT and AST levels and ELISA assay were used to detect hepatocyte injury,apoptosis and inflammatory cytokines(TNF-α,IL-6 and IL-1β)in liver I/RI mice.Immunohistochemistry and q RT-PCR were used to verify the expression of YY1 and mi R-181a-5p in liver tissues of I/RI mice.Furthermore,YY1-overexpressing lentivirus(YY1-oe)was constructed,and the hypoxic reperfusion(H/R)AML12 cell model was established.ELISA was used to detect the levels of TNF-α,IL-6 and IL-1β in the culture supernatants of control and H/R AML12 cells.The expression of YY1 and mi R-181a-5p and the targeting relationship between YY1 and mi R-181a-5p were verified by q RT-PCR,western blot and dual luciferase reporter gene detection.H/R-induced hepatocyte apoptosis and inflammation were detected by flow cytometry and western blot.Part II: The downstream mechanism of mi R-181a-5p in H/R-induced damage was explored,the binding site of YY1 gene in the promoter region of mi R-181 a was predicted by JASPAR database,and the candidate target genes of mi R-181a-5p were analyzed by bioinformatics.Then,the expression of mi R-181a-5p target gene in liver tissues of liver I/RI mice was detected by immunohistochemistry and western blot.The expression of mi R-181a-5p target genes in liver I/RI mice and H/R AML12 cells and the effect of mi R-181a-5p on target genes were verified by dual luciferase reporter gene assay and q RT-PCR.The apoptosis,liver function and inflammatory response of H/R AML12 cells treated with mi R-181a-5p inhibitor or combined with sh-ESR1 were detected by flow cytometry,western blot and ELISA.Part III: In order to verify the hypothesis that ESR1 is involved in liver I/RI mice by regulating ERBB2,we observed the localization and expression of ERBB2 in liver tissues by immunohistochemistry and western blot.To explore whether ESR1 in hepatocytes could regulate ERBB2,we detected the expression of ESR1 and ERBB2 in AML12 cells after lentiviral transduction.The silencing efficiency of sh-ESR1 and the protein expression of ESR1 and ERBB2 in AML12 cells were detected by q RT-PCR and western blot.Flow cytometry,western blot and ELISA were used to detect the apoptosis,liver function indexes and inflammatory response of H/R AML12 cells treated with oe-ESR1 or combined with sh-ERBB2.Part IV: We hypothesized that YY1 regulates apoptosis and inflammation of mouse H/R cells AML12 through mi R-181a-5P/ESR1/ERBB2 axis.To test this hypothesis,q RT-PCR and western blot were used to detect the expression of YY1,mi R-181a-5p,ESR1 and ERBB2 in AML12 cells after different treatments.Flow cytometry and TUNEL staining assay were used to detect the changes of apoptosis level in mouse hepatocytes and liver tissues..The level of apoptosis related protein(Bax,Bcl-2)was detected by western blot.In addition,we measured the levels of liver function indicators and inflammatory factors in the culture supernatants of H/R AML12 cells.Result:Part I: Firstly,the upstream regulatory transcription factor YY1 of mi R-181a-5p was analyzed by bioinformatics.In I/RI mice and H/R AML12 cells,compared to sham-operated mice and control group,the area of liver necrosis in liver I/RI mice was significantly enlarged;while assessment of liver function revealed the presence of up-regulated serum levels of ALT and AST in I/RI mice and in serum and cell culture supernatant as compared to those in control group.Meanwhile,ELISA results demonstrated that the serum levels of inflammatory factors(TNF-α,IL-6 and IL-1β)were all elevated in I/RI mice.I/RI mice presented with significantly lowered expression of YY1,as well as elevated expression of mi R-181a-5p.Transcription factor YY1 target regulates the expression of mi R-181a-5p,over-expression of YY1 repressed mi R-181a-5p expression and reduced H/R-induced hepatocyte apoptosis and inflammation.Part II: Through bioinformatics analyses,we obtained a total of 79 candidate target genes of mi R-181a-5p.Subsequently,these genes were subjected to interaction analysis to construct an interaction network,and the degree of each gene was calculated,following which the ESR1 gene was highlighted as the gene with the highest degree.The Target Scan database was further utilized to predict the binding sites of mi R-181a-5p and ESR1.ESR1 was primarily expressed in the nucleus and cytoplasm,while being reduced in the liver tissues of liver I/RI mice.Meanwhile,the results of Western blot assay demonstrated that relative to AML12 cells of the Con group,those of the H/R group presented with up-regulated levels of ESR1,which is consistent with the results of the liver I/RI mouse models.Furthermore,the results of q RT-PCR demonstrated that mi R-181a-5p mimic led to up-regulated expression of mi R-181a-5p,as well as down-regulated expression of ESR1 in AML12 cells,whereas treatment with mi R-181a-5p inhibitor led to opposite results.Part III: Immunohistochemistry illustrated that ERBB2 was primarily located in the cytoplasm and cell membrane of mouse hepatocytes.Meanwhile,ERBB2 expression was repressed in liver tissues of liver I/RI mice.In addition,Western blot assay results demonstrated ERBB2 expression was down-regulated in H/R-exposed AML12 cells as compared with the Con group,consistent with the results of the liver I/RI mouse model.Over-expression of ESR1 alone down-regulated the levels of liver function indicators as well as inflammatory factors in the supernatant of H/R-exposed AML12 cells,whereas its combination with ERBB2 knockdown resulted in opposite results.Part IV:H/R-exposed AML12 cells treated with oe-YY1 presented with increased expression of YY1,ESR1,and ERBB2,as well as diminished expression of mi R-181a-5p;relative to oe-YY1 alone,its combination with sh-ERBB2 led to down-regulated ERBB2 expression.AML12 cells had repressed apoptosis,down-regulated levels of Bax,and up-regulated levels of Bcl-2 in the presence of YY1 over-expression;relative to YY1over-expression alone,its combination with ERBB2 knockdown resulted in opposite results;According to the results,YY1 over-expression led to down-regulated levels of the aforementioned indicators and factors(ALT,AST,TNF-α,IL-6,and IL-1β)in H/R-exposed AML12 cells;compared to YY1 over-expression alone,its combination with ERBB2 knockdown resulted in up-regulated levels of the indicators and factors.YY1over-expression could repress mi R-181a-5p expression and stimulate ESR1-mediated activation of ERBB2,thereby ameliorating the H/R-induced hepatocyte apoptosis and inflammation.The liver tissues of I/RI mice presented with elevated expressions of mi R-181a-5p and diminished expression of YY1,ESR1,and ERBB2,whereas treatment with oe-YY1 led to the opposite results.Meanwhile,the results of HE staining illustrated augmented necrosis in the liver tissues of I/RI mice,while such liver damage was ameliorated following YY1 over-expression.Furthermore,TUNEL staining assay results demonstrated that cell apoptosis in liver tissues of I/RI mice was accelerated,whereas YY1 over-expression led to reduced cell apoptosis.Conclusions:This study demonstrates that YY1 repressed mi R-181a-5p expression and stimulated ESR1-mediated activation of ERBB2,thereby ameliorating liver I/RI,providing an insight for the development of new targets for liver I/RI. |
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