Effect And Mechanism Of IMPA2 On Mitophagy In Cervical Cancer Cells

Objective:To investigate the relationship between the inositol monophosphatase 2(IMPA2)gene and mitophagy in cervical cancer cells and elucidate its mechanism.Methods:1.Construct stable IMPA2 knockdown cervical cancer cell lines.Western Blotting,immunofluorescence,MDC staining,electron microscopy,sens GFP-stub RFP-LC3 dual-fluorescence indicator system,and lysotracker-mitotracker probe labeling were used to investigate the effect of IMPA2 knockdown on mitophagy in cervical cancer cells.2.Perform transcriptome sequencing and label-free proteome analysis in IMPA2-knockdown cells to determine the mitophagy-related signaling pathways that IMPA2 may regulate.Subsequently,the co-expressed differentially expressed molecules were screened through transcriptome and proteome combined analysis,and their GO function and KEGG pathway were annotated,enriched,and analyzed.Ultimately,search the candidate pathways based on literature and omics results.3.Verify the omics results by Western Blotting and RT-qPCR.Treat IMPA2-knockdown cells with pathway inhibitors to confirm that IMPA2 regulates mitophagy of cervical cancer cells through this pathway.4.Determine potential upstream micro RNA(miRNA)of IMPA2 through bioinformatic analysis and RT-qPCR.A dual-luciferase reporter assay and expression assay were used to validate their regulatory relationship.Then,over-express the potential miRNA in cervical cancer cells and observe whether it regulates mitophagy.Finally,design a rescue experiment to confirm that this miRNA indeed modulates mitophagy in cervical cancer cells through IMPA2.Results:1.IMPA2 knockdown inhibits the generation of autophagosomes and autophagolysosomes and inhibits autophagy in cervical cancer cells: IMPA2 knockdown significantly reduced the levels of autophagy marker molecules p62 and LC3-II in cervical cancer cells.After pretreatment with Baf-A1,the expression of LC3-II in the IMPA2-knockdown group was still significantly reduced,indicating that IMPA2 knockdown inhibited the formation of autophagosomes in cervical cancer cells.MDC staining and transmission electron microscopy showed that IMPA2 knockdown reduced the number of autophagolysosomes in cervical cancer cells.Western Blotting results suggested that IMPA2 knockdown inhibited the expression of lysosomal membrane-associated proteins LAMP1 and LAMP2,indicating the decrease of autophagolysosomes.Fluorescence expression in the co-transfected cells of sens GFP-stub RFP-LC3 and sh-IMPA2 was observed by the confocal laser microscopy.It was found that IMPA2 knockdown blocked the autophagy flux in cervical cancer cells.2.IMPA2 knockdown inhibits mitophagy in cervical cancer cells:Western Blotting results showed that IMPA2 knockdown in cervical cancer cells resulted in the down-regulation of mito-p62,mito-LC3,PINK1 and BNIP3,but the level of mitochondrial outer membrane protein TOMM20 increased.Compared with the control group,the co-localization relationship between mitochondria and lysosome was poor in the IMPA2-knockdown group,indicating inhibition of mitophagy.3.IMPA2 potentially regulates NF-κB signaling: This study identified 2201 differential transcripts and 735 differential proteins by transcriptome sequencing and proteomic mass spectrometry in IMPA2-knockdown cells.A combined analysis of transcriptome and proteome data revealed 285 co-differentially expressed molecules(differentially expressed in both transcriptome and proteome with the same trend).Further GO enrichment indicated that the co-expressed differential molecules were located in lysosome and mitochondria and affected autophagy,mitophagy,autophagosome assembly,mitochondrial cytochrome c release,and mitochondrial apoptosis.KEGG analysis suggested that IMPA2 knockdown may potentially regulate NOD-like receptor,TNF,and NF-κB signaling pathways in cervical cancer cells.NOD-like receptor and TNF signaling pathways are essential upstream of NF-κB activation.Therefore,NF-κB was selected as a candidate signaling pathway for mechanism verification.4.IMPA2 knockdown inhibits mitophagy in cervical cancer cells by activating NF-κB: Western Blotting showed that the phosphorylation level of p65 in cervical cancer cells increased significantly after IMPA2 knockdown.According to sequencing results,RT-qPCR and Western Blotting,knockdown IMPA2 activated the transcription of the IKBKE gene,a new member of the IKK kinase family,and up-regulated the expression of IKKε.In addition,NF-κB inhibitor BAY 11-7082 successfully reversed IMPA2-mediated mitophagy inhibition in cervical cancer cells.5.MiR-625-5p targeting IMPA2 inhibits mitophagy in cervical cancer cells: A total of 11 potential upstream regulatory miRNAs of IMPA2 were predicted by Target Scan,miRDB,and Starbase.Further screening by RT-qPCR revealed that miR-625-5p might be involved in regulating IMPA2 in cervical cancer cells.Subsequently,dual-luciferase reporter group assay results showed that miR-625-5p targeted IMPA2 m RNA 306-312 at 3’UTR.Then,miR-625-5p mimics and inhibitors were transfected into cervical cancer cells,and the m RNA and protein levels of IMPA2 were detected.It was found that miR-625-5p stably inhibited the protein expression of IMPA2 but did not affect the m RNA.Western Blotting and transmission electron microscopy results showed that miR-625-5p also inhibited mitophagy in cervical cancer cells.The rescue experiment results suggested that miR-625-5p affected mitophagy in cervical cancer cells through IMPA2.Conclusion:IMPA2 knockdown inhibits mitophagy in cervical cancer cells by activating the NF-κB signaling pathway.MiR-625-5p is an upstream regulatory molecule of the IMPA2-mitophagy axis.This study provides a valuable experimental basis for further elucidating the cancer-promoting mechanism of IMPA2 and provides a theoretical basis for the molecular mechanism research of cervical cancer and the development of targeted drugs.