Effect of Circ_ZFYVE1 on the cell cycle and autophagy of pancreatic islet β cells
Objective:To investigate the effect of Circ_ZFYVE1(cZFYVE1)on the cell cycle and autophagy of mouse pan-creatic β-cell(MIN6).Methods:Sanger sequencing analysis was used to verify the cyclisation site of cZFYVE1;MIN6 cells were divided into the normal group(NC,containing glucose concentration of 5.5 mmol/L),the high glucose 1 group(HG1,containing glucose concentration of 15 mmol/L)and the high glucose 2 group(HG2,containing glucose concentration of 25 mmol/L)for culture for 48 h.The expression levels of cZFYVE1 in NC group,HG1 group,and HG2 group were detected using qRT-PCR;Lentiviral transfection experiments were performed to construct the empty vector group(sh-NC),to stablize the overexpression of cZFYVE1 group(circ-ZFYVE1)and the knockdown of cZFYVE1 group(si-cZFYVE1).qRT-PCR detection of cZFYVE1 gene expression was used in various groups of MIN6 cells after transfection.Distribution of different cell cycles in different groups of MIN6 cells was detected by flow cytometry.Changes in autophagic vesicles and organelles in various groups of MIN6 cells were analysed by transmission electron microscopic viewing.Detection of Beclin-1 fluorescence intensity of autophagy-related protein in various groups of cells by were analyzed immunofluorescence assay.Western Blot method was used to detect the expression levels of cell cycle regulatory protein Cyclin D1,autophagy-related proteins Beclin-1,P62 and LC3-II in each group.Results:Sanger se-quencing confirmed the presence of a cyclized splice site in cZFYVE1.The qRT-PCR results showed that the cZFYVE1 expres-sion level was reduced in the HG1 and HG2 groups compared with the NC group,and the cZFYVE1 level was reduced in the HG2 group compared with that in the HG1 group(P<0.01).cZFYVE1 expression was elevated in the circ-ZFYVE1 group and decreased in the si-cZFYVE1 group compared with the sh-NC group(P<0.01).Flow cytometry results showed that compared with the sh-NC group,the circ-ZFYVE1 group had a lower percentage of cells in the G1 phase,a higher percentage of cells in the S phase,and a higher percentage of cells in the G2 phase,and the si-cZFYVE1 group had a higher percentage of cells in the G1 phase,a lower percentage of cells in the S phase,and a lower percentage of cells in the G2 phase(P<0.01).Transmission elec-tron microscopy results showed that autophagosomes were reduced in the si-cZFYVE1 group compared with the sh-NC group.Im-munofluorescence results showed that the intensity of autophagy-associated protein Beclin-1 immunofluorescence was elevated in the circ-ZFYVE1 group,and Beclin-1 immunofluorescence intensity was decreased in the si-cZFYVE1 group,compared with the sh-NC group(P<0.01).WB results showed that compared with the sh-NC group,the circ-ZFYVE1 group had increased expres-sion levels of the cell cycle regulatory protein Cyclin D1,increased expression levels of the autophagy-related proteins Beclin-1 and LC3-II,and decreased expression levels of P62,while the si-cZFYVE1 group had decreased expression levels of the Cyclin D1 protein,decreased expression levels of the Beclin-1 and LC3-Ⅱ protein expression levels were reduced,and P62 protein expres-sion levels were elevated(P<0.05).Conclusion:cZFYVE1 may promote autophagy and affect the cell cycle in MIN6 pancreatic β-cells.
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维普
