| Objective:To investigate the expression changes of micro RNA-153 and micro RNA-137and their regulatory roles in the process of hepatocyte apoptosis induced by Na As O2.Methods:Human normal liver cells（LO2,WRL-68 cells）were cultured in vitro.According to different treatment methods,it is divided into control group,arsenic treatment group,arsenic+negative transfection group,arsenic+micro RNAs upregulated group and arsenic+micro RNAs downregulated group.Cell apoptosis,proliferation and cell cycle were detected by flow cytometry and Real-time Cell Analyzer（RTCA）.The expression of endoplasmic reticulum stress-related and epigenetic-related proteins was detected by Western blot.Real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）was used to detect the expression levels of micro RNAs and m RNA expression levels of SET7/9 and LSD1 in each group.Hepatocytes（LO2,WRL-68）were transiently transfected with antisense or overexpressed micro RNAs to silence or overexpress micro RNAs.Chromatin immunoprecipitation assays（Ch IP）was used to detect ER stress-marker proteins and the related proteins of PERK/CHOP signaling pathway.Results:RTCA results showed that compared with the control group,the inhibition rate of cell proliferation in arsenic treatment group was significantly increased（P<0.05）;Compared with the arsenic+negative transfection group,the proliferation inhibition rate of the arsenic+mi R-153 up-regulated group was significantly decreased（P<0.05）,the proliferation inhibition rate of arsenic+mi R-153 down-regulation group was significantly increased（P<0.05）.Compared with the arsenic+negative transfection group,the inhibiton rate of cell proliferation was significantly increased in the arsenic+upregulated mi R-137 group（P<0.05）;Compared with the arsenic+negative transfection group,the proliferation inhibition rate of the arsenic+mi R-137 down-regulation group was significantly decreased after the liver cells were transfected with the mi R-137 inhibition sequence by LO2（P<0.05）.The results of flow cytometry showed that compared with the control group,the total apoptosis rate and the proportion of G1 phase cells in arsenic treatment group were significantly increased（P<0.05）;Compared with the arsenic+negative transfection group,the total apoptosis rate and the proportion of G1 phase cells in the arsenic+mi R-153upregulated group were significantly decreased（P<0.05）,the total apoptosis rate and the proportion of G1 phase cells in arsenic+mi R-153 down-regulation group were significantly increased（P<0.05）.Compared with the arsenic+negative transfection group,the total apoptosis rate and the proportion of G1 phase cells in the arsenic+mi R-137 up-regulated group were significantly increased（P<0.05）;Compared with the arsenic+negative transfection group,the total apoptosis rate and the proportion of G1 phase cells in the arsenic+mi R-137 down-regulation group were significantly decreased after the hepatocyte LO2 transfection with mi R-137 inhibition sequence（P<0.05）.RT-q PCR results indicated that mi R-153 expression was significantly decreased and mi R-137 expression was significantly increased in the arsenic treatment group compared with the control group（P<0.05）;Compared with the arsenic+negative transfection group,the expression levels of mi R-153 and mi R-137in the arsenic+mi R-153 up-regulated group and the arsenic+mi R-137 up-regulated group were significantly increased（P<0.05）,the expression levels of mi R-153 and mi R-137 in arsenic+mi R-153 down-regulation group and arsenic+mi R-137down-regulation group were significantly decreased（P<0.05）.Compared with the control group,the m RNA expression level of SET7/9 in the arsenic treated group was significantly increased,and the m RNA expression level of SET7/9 in the arsenic+mi R-153 up-regulated group was significantly decreased compared with the arsenic+negative transfection group（P<0.05）,the m RNA expression level of SET7/9 in arsenic+mi R-153 down-regulation group was significantly increased（P<0.05）.Compared with the control group,the expression level of LSD1 m RNA in arsenic treatment group was significantly decreased（P<0.05）.Compared with the arsenic+negative transfection group,the expression level of LSD1 m RNA in the arsenic+mi R-137 up-regulated group was significantly decreased（P<0.05）,the expression level of LSD1 m RNA in arsenic+mi R-137 down-regulation group was significantly increased（P<0.05）.Western Blot analysis showed that compared with the control group,the expression levels of SET7/9,GRP78 and H3K4me1/me2 in arsenic treatment group were significantly increased,and the expression level of LSD1 was significantly decreased（P<0.05）;Compared with arsenic+negative transfection group,the protein expression levels of SET7/9,GRP78 and H3K4me1 in arsenic+mi R-153 up-regulated group were significantly decreased（P<0.05）;The protein expression levels of SET7/9,GRP78 and H3K4me1 in arsenic+mi R-153down-regulation group were significantly increased（P<0.05）.Compared with the negative transfection group,the protein expression levels of GRP78 and H3K4me1/me2 were significantly increased and LSD1 was significantly decreased in the arsenic+mi R-137 up-regulated group（P<0.05）;The expression level of H3K4me1/me2 protein in arsenic+mi R-137 down-regulation group was significantly decreased,and the expression of LSD1 was significantly increased（P<0.05）;Further verification by histone chromatin immunoprecipitation（Ch IP）assay,compared with the normal group,the enrichment of GRP78 and CHOP gene promoter H3K4me1/2levels was significantly increased in the arsenic treatment group,and the difference was statistically significant（P<0.05）.Conclusions:In the endoplasmic reticulum stress-related hepatocyte apoptosis induced by arsenic,mi R-153 and mi R-137 can regulate H3K4 methyltransferase SET7/9 and histone H3K4 demethyl transferase LSD1 to mediate H3K4me1/2 level changes,and then mediate apoptosis by activating key genes GRP78 and CHOP in the PERK/CHOP signaling pathway of endoplasmic reticulum stress. |
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