Role Of Sig-1R In Islet Beta Cells Proliferation And Damage Repair

Objective To investigate the role of Sig-1R in islet beta cell proliferation and study the effect of Sig-1R on islet beta cell injury under lipotoxic conditions.Methods MIN6 cell lines with stable knockdown and overexpressing Sig-1R were established using lentiviral vector transfection techniques.MIN6 cell proliferation rate and cell cycle was examined using flow cytometry.RNA sequencing was used to screen the differentially expressed genes between Sig-1R knockdown MIN6 cells and control cells.The enrichment of differentially expressed genes in functional entries and biological pathways was also analyzed by GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Gene and Genomes)analysis.QT-PCR was used to verify possible target genes of Sig-1R regulating MIN6 cell proliferation among these differentially expressed genes.MIN6 cells with stable knockdown and overexpressing Sig-1R were subjected to0.5 mmol/L palmitic acid intervention.The effect of Sig-1R on islet β cell survival,apoptosis rate and insulin secretion under lipotoxic conditions was separately determined by CCK8 kit,flow cytometry and enzyme-linked immunosorbent assay.Western blot analysis was used to determine the protein expression level of Pdx1(pancreatic and duodenal homeobox 1),ER stress marker GRP78(glucose regulated protein 78)and CHOP(C/EBP homologous protein),mitochondrial apoptotic proteins Bax(Bcl2-Associated X),Bcl-2(B-cell lymphoma-2)and Cyt-c(cytochrome c)in islet β cells.The effects of Sig-1R gene alterations on ATP(adenosine triphosphate),ROS(reactive oxygen species)level and mitochondrial membrane potential were examined by microplate reader and flow cytomeometer.MAM(mitochondria associated endoplasmic reticulum membrane)structure of MIN6 cells was detected by transmission electron microscopy.The colocalization of IP3 R and VDAC1 was then examined by immunofluorescence.The regulatory effect of Sig-1R on cytoplasmic calcium levels and calpain-1 protein expression was determined by flow cytomeometer and western blot analysis.Results Sig-1R knockdown in MIN6 cells inhibited cell proliferation,causing an increased cell cycle G0 / G1 phase cell ratio and a decreased S phase cell ratio to inhibit cell cycle progression;Sig-1R overexpression in MIN6 cells promoted cell proliferation,causing a decreased cell cycle G0 / G1 phase cell ratio and an increased S phase cell ratio to promote cell cycle progression.GO and KEGG analysis both showed that Sig-1R knockdown inhibited cell cycle.QT-PCR revealed that the regulatory pathway of Sig-1R knockdown on cell cycle might be the Fox M1/Plk1/Cenpa pathway.Sig-1R knockdown reduced cell survival,aggravated apoptosis and reduced insulin secretion under lipotoxic conditions.On the other hand,Sig-1R knockdown also increased ROS level and GRP78,CHOP,Bax,Cyt-c protein expression,and decreased Bcl-2 expression,ATP and mitochondrial membrane potential levels.Sig-1R overexpression could play the opposite role.Sig-1R could regulate mitochondrial associated ER membrane(MAM)structure in MIN6 cells.Sig-1R knockdown reduced contact sites between mitochondria and ER while Sig-1R overexpression increased contact sites.The expression level of the MAM key proteins IP3 R and VDAC1 decreased in Sig-1R knockdown MIN6 cells while Sig-1R overexpression increased VDAC1 expression level.Furthermore,the cytoplasmic calcium level increased in Sig-1R knockdown cells and decreased in Sig-1R overexpression cells.Conclusion Sig-1R knockdown can inhibit islet β cell proliferation while Sig-1R overexpression promote it and the possible mechanism is that Sig-1R can affect cell cycle by regulating the Fox M1/Plk1/Cenpa pathway.Sig-1R knockdown can aggravate islet β cell damage while Sig-1R overexpression improve it under lipotoxic conditions.Sig-1R can regulate MAM structure to affect cytoplasmic calcium level to further regulate ER stress and mitochondrial function,which might be a possible mechanism.