Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor invol...Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor involved in the development of CRC,we screened the upregulated genes in tumor tissues through four datasets from an online database.The expression of reticulocalbin 1(RCN1),a Ca2+-binding protein,was upregulated in the four datasets.Based on loss-offunction experiments,the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8(CCK-8)assay.The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide(PI)staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay in RKO and SW480 cells.Activation of endoplasmic reticulum(ER)stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase(PERK),inositol-requiring kinase-1(IRE1),transcription factor 6(ACT6),and CCAAT/enhancer-binding protein-homologous protein(CHOP).The intracellular Ca2+homeostasis regulated by RCN1 was determined through the detection of Ca2+concentration and mitochondrial membrane potential(MMP)measurement.Moreover,whether inositol 1,4,5-trisphosphate receptor type 1(IP3R1)was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.Results:Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells.Phosphorylation of PERK and IRE1,activation of ATF6,and upregulation of CHOP were induced by the absence of RCN1,suggesting that the unfolded protein response(UPR)was activated in CRC cells.The concentration of Ca2+in mitochondria was increased after RCN1 depletion,followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells.Moreover,it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells.The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca2+concentration.Conclusion:Taken together,our results confirmed that silencing of RCN1 disrupted intracellular Ca2+homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.展开更多
Background： Maternal obesity alters oocytes and subsequent fetal metabolism. An increasing number of studies have shown that the endoplasmic reticulums（ER） or mitochondria have important effects on oocyte quality, ...Background： Maternal obesity alters oocytes and subsequent fetal metabolism. An increasing number of studies have shown that the endoplasmic reticulums（ER） or mitochondria have important effects on oocyte quality, but there has been no study of the effect of mitochondria-associated ER membranes（MAMs） on oocyte quality. The present study was designed to assess whether the level of MAM and MAM-related proteins were different in oocytes from obese and control mice.Results： First, oocytes from mice with high-fat-diet（HFD）-induced obesity had higher levels（either greater numbers or a higher proportion for the same numbers） of MAM than oocytes from control mice. The abundance of MAM-related proteins in oocytes from obese mice was significantly greater at both the messenger RNA and protein levels, including inositol 1,4,5-trisphosphate receptor, type 1（IP3R1）, inositol 1,4,5-trisphosphate receptor,type 2（IP3 R2） and phosphofurin acidic cluster sorting protein 2（PACS-2）. Further, there was an increase in mitochondrial Ca^（2＋）（[Ca^（2＋）]_m） which was associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. Down-regulation of MAM-related protein IP3R1 in oocytes from obese mice decreased [Ca^2＋]_mand apoptosis and improved cytoplasmic maturation but did not reduce the overal MAM level. However, down-regulating MAM-related protein PACS-2 in oocytes from obese mice did reduce the level of MAM and [Ca^（2＋）]_m, which decreased the rate of apoptosis and improved cytoplasmic maturation of oocytes from obese mice.Conclusions： It is possible that enriched MAM could increase [Ca^（2＋）]_m, and this increase has been found to be associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. This finding suggests a novel therapeutic target for obesity-induced oocyte defects.展开更多
文摘Background:The incidence of colorectal cancer(CRC)has been increasing in recent years.Thus,the discovery of factors that can assist in alleviating CRC is urgently warranted.Methods:To identify a potential factor involved in the development of CRC,we screened the upregulated genes in tumor tissues through four datasets from an online database.The expression of reticulocalbin 1(RCN1),a Ca2+-binding protein,was upregulated in the four datasets.Based on loss-offunction experiments,the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8(CCK-8)assay.The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate(FITC)/propidium iodide(PI)staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay in RKO and SW480 cells.Activation of endoplasmic reticulum(ER)stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase(PERK),inositol-requiring kinase-1(IRE1),transcription factor 6(ACT6),and CCAAT/enhancer-binding protein-homologous protein(CHOP).The intracellular Ca2+homeostasis regulated by RCN1 was determined through the detection of Ca2+concentration and mitochondrial membrane potential(MMP)measurement.Moreover,whether inositol 1,4,5-trisphosphate receptor type 1(IP3R1)was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.Results:Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells.Phosphorylation of PERK and IRE1,activation of ATF6,and upregulation of CHOP were induced by the absence of RCN1,suggesting that the unfolded protein response(UPR)was activated in CRC cells.The concentration of Ca2+in mitochondria was increased after RCN1 depletion,followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells.Moreover,it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells.The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca2+concentration.Conclusion:Taken together,our results confirmed that silencing of RCN1 disrupted intracellular Ca2+homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.
基金supported by the National Basic Research Program of China[grant number 2014CB138500]
文摘Background： Maternal obesity alters oocytes and subsequent fetal metabolism. An increasing number of studies have shown that the endoplasmic reticulums（ER） or mitochondria have important effects on oocyte quality, but there has been no study of the effect of mitochondria-associated ER membranes（MAMs） on oocyte quality. The present study was designed to assess whether the level of MAM and MAM-related proteins were different in oocytes from obese and control mice.Results： First, oocytes from mice with high-fat-diet（HFD）-induced obesity had higher levels（either greater numbers or a higher proportion for the same numbers） of MAM than oocytes from control mice. The abundance of MAM-related proteins in oocytes from obese mice was significantly greater at both the messenger RNA and protein levels, including inositol 1,4,5-trisphosphate receptor, type 1（IP3R1）, inositol 1,4,5-trisphosphate receptor,type 2（IP3 R2） and phosphofurin acidic cluster sorting protein 2（PACS-2）. Further, there was an increase in mitochondrial Ca^（2＋）（[Ca^（2＋）]_m） which was associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. Down-regulation of MAM-related protein IP3R1 in oocytes from obese mice decreased [Ca^2＋]_mand apoptosis and improved cytoplasmic maturation but did not reduce the overal MAM level. However, down-regulating MAM-related protein PACS-2 in oocytes from obese mice did reduce the level of MAM and [Ca^（2＋）]_m, which decreased the rate of apoptosis and improved cytoplasmic maturation of oocytes from obese mice.Conclusions： It is possible that enriched MAM could increase [Ca^（2＋）]_m, and this increase has been found to be associated with increased apoptosis and compromised cytoplasmic maturation in oocytes from obese mice. This finding suggests a novel therapeutic target for obesity-induced oocyte defects.
