PiR-823 Inhibits Cell Apoptosis Via Modulating Mitophagy In Colorectal Cancer

Colorectal cancer is a serious threat to human beings with a high incidence and mortality rate globally and in China for its hidden onset and poor prognosis.Further insight into the molecular mechanisms underlying CRC is important to search for novel therapeutic targets and improve therapeutic efficiency.Piwi-interacting RNAs(piRNAs) are small ncRNAs and play important roles in the tumorigenesis and development of many types of tumors.Previous research from our team indicates that piR-823 regulated the expression of mitochondrial chaperonins HSP 60 and HSP 70 in colorectal cancer(CRC)cells.Mitophagy is the main way to regulate the mitochondrial quality and quantity.However,whether piR-823 participates in the regulation of mitophagy has not been investigated.In the classic mitophagy pathway of PINK1-Parkin-mediated mitophagy,damaged mitochondria are selectively cleared.Several studies have revealed that mitophagy is involved in the regulation of apoptosis.However,whether piR-823 inhibits CRC cell apoptosis via modulating mitophagy remains unclear.Digital gene expression analysis(DGE) was conducted to analyse the changes of differential expressed genes in combination with related databases.We then verified the mitophagy,mitochondrial dysfunction and mitochondrial loss induced by inhhibition of piR-823 in vitro and in vivo,uncovered the intrinsic relationships among piR-823,mitophagy and apoptosis in CRC cells,and to explore the specific mechanism of piR-823 in the regulation of mitophagy.The experiment mainly includes the following three parts:Part one: Effects of piR-823 in the regulation of mitophagy and mitochondrial function in colorectal cancer cellsObjectives: The aim of this part of the study was to evaluate the roles of piR-823 in the regulation of mitophagy and observe the effects of piR-823 in mitochondrial function and number in CRC cells.Methods: A piR-823 antagomir(Ant-823) was used to inhibit piR-823 expression.DGE was performed to explore the potential functions of piR-823 in combination with related databases.Mitophagy induced by Ant-823 was measured in vitro by fluorescence probe labeling technique and Western blot analysis.Changes in mitochondrial quality and number were detected by JC-1staining,ATP production,RT-q PCR,flow cytometry and Western blot analysis.Lentiviral vectors carrying the antisense sequence of piR-823(LV-sh-piR-823)or nonspecific scrambled RNA sequence(LV-sh-NC)were synthesized and infected the HCT116 cells,followed by the establishment of xenograft tumor implantation models.Tumor weights and volumes were detected.Mitophagy and mitochondrial number changes in HCT116 xenograft tumors were measured by immunofluorescence and Western blot analysis.Results: DGE and related databases analysis showed that Ant-823 led to the dysregulation of mitophagy-related genes,indicating that piR-823 might be involved in the regulation of mitophagy.The inhibition of piR-823 promoted the colocalization of mitochondria with lysosomes and indicating the activation of mitophagy in vitro.The repression of piR-823 by Ant-823 facilitated the phosphorylation of Parkin and ubiquitin at Ser 65 in HCT116 and DLD-1 cells and also increased the expression of PINK1 and Parkin as well as the the translocation of Parkin to mitochondria,indicating that PINK1-Parkin mediated mitophagy had been promoted.The inhibition of piR-823 decreased JC-1 red staining but increased JC-1 green staining,indicating a decline in mitochondrial membrane potential.Additionally,the ATP content was significantly decreased in Ant-823-transfected versus Ant-NC-transfected HCT116 and DLD-1 cells,suggesting the contribution of piR-823 in maintenance of mitochondrial function.The intensity of Mito Tracker Red,relative mt DNA copy number and expression of the mitochondrial components TOM 20 and COX IV were significantly reduced after piR-823 inhibition,suggesting the mitochondrial loss in HCT116 and DLD-1 cells.The inhibition of piR-823 suppressed the growth of subcutaneous HCT116 xenografts tumor in nude mice.Moreover,inhibition of piR-823 increased the colocalization of mitochondria with lysosomes and p-S65-Parkin expression levels but decreased TOM20 and COX IV protein expression levels in the LV-sh-piR-823 group versus the LV-sh-NC group,indicating the activation of mitophagy and mitochondrial loss in vivo.Conclusions: These data reveals that the inhibition of piR-823 leads to the dysregulation of mitophagy-related genes,promotes the Parkin activation and mitophagy in vitro and in vivo,as well as the induction of mitochondrial dysfunction and loss.Part two: piR-823 helps the maintenance of mitochondrial function and inhibits colorectal cancer cell apoptosisby modulating mitophagyObjectives: This part of the study aimed to evaluate the regulation of mitophagy induced by piR-823 inhibition on mitochondrial function,number and apoptosis in CRC cells.Methods: siRNAs target Parkin were used to downregulate Parkin,thus repressing mitophagy in piR-823-knockdown CRC cells.JC-1 staining and Western blot analysis were used to measure changes in mitochondrial function and number.TUNEL staining and Annexin V/ PI staining was used to detect the proportion of apoptotic CRC cells.Meanwhile,the relative expression of apoptotic proteins of cleaved PARP,cleaved caspase 9 and cleaved caspase 3were measured by Western blot analysis to evaluate the effects of mitophagy induced by piR-823-inhibition on apoptosis and verify whether piR-823 regulates colorectal cell apoptosis by PINK1-Parkin mediated mitophagy.Results: Compared with the Ant-823 plus si NC treatment groups,the Ant-823 plus si Parkin group showed increased JC-1 red staining but decreased JC-1 green staining,indicating an increase in the mitochondrial membrane potential.Consistently,the addition of si Parkin to Ant-823-treated cells partially restored the mitochondrial number,as evidenced by increased TOM20 and COX IV expression levels.These data suggest that inhibition of mitophagy can partly restore the mitochondrial dysfunction and mitochondrial loss in HCT116 and DLD-1 cells.The inhibition of mitophagy partly rescued the Ant-823-dependent apoptosis in CRC cells,as evidenced by a decreased proportion of TUNEL-positive cells and Annexin V-positive cells.Furthermore,the introduction of si Parkin led to a decrease in the protein levels of cleaved PARP,cleaved caspase 9 and cleaved caspase 3 in piR-823-inhibited cells relative to the control group.These results suggest that inhibition of mitophagy can rescue the apoptosis induced by Ant-823 in HCT116 and DLD-1 cells.Conclusions: piR-823 contributes to the maintenance of mitochondrial function and inhibits cell apoptosis in HCT116 and DLD-1 cells by regulating mitophagy.Part three: piR-823 suppressed CRC cells mitophagy by inhibiting the degradation of PINK1Objectives: The aim of this part was to elucidate the mechanism by which piR-823 regulates mitophagy.Methods: RNA-protein pull-down and RNA-binding protein immunoprecipitation(RIP)was conducted to confirm the proteins interacting with piR-823 in combination with the analysis of DGE.The molecular mechanism of target proteins by regulation of piR-823 was explored at cellular levels.Results: By DGE analysis,piR-823 was speculated to interact with PINK1 or Parkin.The results indicated that piR-823 interacted with cleaved PINK1 but not Parkin.Consistently,RIP assay showed an enrichment of piR-823 by anti-PINK1.These data suggest the interaction between piR-823 and PINK1.Furthermore,the addition of si PINK1 to Ant-823-treated cells partially restored the inhibition of mitophagy and mitochondrial loss,indicating the crucial roles of PINK1 in the Ant-823-induced mitophagy.The inhibition of piR-823 increased the protein levels of PINK1,however,there was no significant difference in PINK1 m RNA levels between the groups transfected with Ant-NC or Ant-823,indicating that piR-823 could modulate PINK1 in a transcriptional-independent manner.Additionally,the half-life of the PINK1 protein was prolonged upon the inhibition of de novo PINK1 protein synthesis using CHX.Immunoprecipitation for ubiquitinated PINK1 in HCT116 cells in the presence of MG132 also showed that the inhibition of piR-823 decreased the polyubiquitination of PINK1.Collectively,these data demonstrated that piR-823 promoted the proteasome-mediated degradation of PINK1.Conclusions: piR-823 interacts with PINK1 to promote the proteasome-mediated degradation by promotion of the polyubiquitination of PINK1,thus suppressing mitophagy.