Downregulation Of ER-resident Reticulocalbin-1 Differentially Facilitates Apoptosis And Necroptosis In Human Prostate Cancer Cells

BackgroundEndoplasmic reticulum is an important organelle in the cell.It plays an important role in protein folding,quality control and maintenance of calcium homeostasis.All kinds of located protein of the endoplasmic reticulum,such as molecular chaperones and enzymes participating in folding processing,modification of newly synthesized protein peptide,help proteins to form a spatial structure and directional transport,to maintain homeostasis.In recent years,the study found that some endoplasmic reticulum localization protein,such as two disulfide isomerase,Ca2+ binding protein not only can reside in the endoplasmic reticulum as molecular chaperones,also can be located in the cytosol,extracellular,promoting cell proliferation and migration.involving in the occurrence and development of diseases such as malignant tumor.Reticulocalbinl(RCN1)is a member of the CREC family,and has the characteristics of calcium binding protein.The RCN1 protein is encoding by RCN1 gene in the chromosome 11p13.containing 331 amino acid residues.which is characterized in that the N terminal 29 amino acid residues signal peptide;intermediate region containing 6 EF-hand domains can bind with Ca2+:carboxyl terminal HDEL mammalian signal.Localization and distribution of RCN1:RCN1 located in the endoplasmic reticulum of cells,but also found that RCN1 may be located in the surface of both bone endothelial cells and prostate cancer cells,but the mechanism is unknown;and HDEL in C side is different with the endoplasmic reticulum localization signal KDEL.Therefore,RCN1 can also be secreted into the extracellular.RCN1 is widely distributed in various tissues and organs of human body,which is mainly distributed to secreted organs.In non-epithelial organs,such as germ cells,nerve cells,RCN1 has high expression.The functions of RCN1:RCN1 is widely expressed in human tissues,but its functions are limited.Studies have shown that homozygous deletion of RCN1 gene in the mice can lead to embryonic lethality.In addition,the secreted RCN1 can be as a ligand,specifically mediated apoptosis of the nerve cells are engulfed.The expression of RCN1 is associated with diseases.especially tumors,and RCN1 is highly expressed in highly invasive breast cancer cells,colorectal cancer cells,gastric cancer cells and liver cancer cells.In prostate cancer,RCN1 expression was lower in the invasive cells,and the expression in breast cancer cells was opposite.Based on the expression of RCN1 its function in the occurrence and development of tumors,but the research horizon,still need a detailed discussion.This paper in prostate cancer as the research object,first carries on the analysis of clinical samples,the expression of the database,cell and animal experiments of RCN1 in prostate cancer.Results:Part Ⅰ RCN1 is highly expressed in prostate cancer tissues1.RCN1 is highly expressed in prostate cancerFirst of all,according to data from the tumor gene database TCGA and oncomine.RCN1 mRNA levels in prostate cancer were high,but survival analysis showed no association with patient survival rates.In order to know the protein level of RCN1 in prostate cancer,we collected 11 prostate cancer tissues and 15 benign prostatic hyperplasia tissues,and detected the expression levels of RCN1 in different tissues by immunohistochemistry.The results showed that the expression level of RCN1 in prostate cancer tissue was higher than that in BPH tissue.In order to determine whether RCN1 is associated with the occurrence of prostate cancer,cyclin D1 is detected.The results of clinical samples show that Cyclin D1 showed low expression in prostate cancer and benign prostatic hyperplasia tissues,and no significant correlation with RCN1 expression.2.RCN1 was not correlated with cell proliferationThe expression level of RCN1 was the lowest in hormone-independent and aggressive PC3 cells,even lower than the normal prostate epithelial cells RWPE1.The basic level of RCN1 in hormone dependent LNCaP cells is highest,and the expression level of RCN1 in hormone-independent DU 145 cells is between PC3 and LNCaP cell.Therefore,overexpression of RCN1 in PC3 cells,but the proliferation of cells showed that had no significant effect on cell proliferation.Therefore,RCN1 may be not related to cell proliferation,and may be negatively related to cell invasiveness and malignancy.Part II Downregulation of RCN1 expression may promotes apoptosis or necroptosis in different prostate cancer cellsBecause of the high expression of RCN1 in prostate cancer cells DU 145 and LNCaP.we used flow cytometry to analysis RCN1 knockdown on cell cycle and cell survival.1.Downregulation of RCN1 blocked DU 145 cells in S phaseDownregulation of RCN1 can affect cell cycle.Knockdown of RCN1 in DU 145 cells can block cell cycle in S phase.Knockdown of RCN1 in LNCaP cells.cycle arrest in the G2/M phase,but the effect is not significant.The expression of Cyclin B,vital regulatory proteins in S and G2/M phase,was detected in prostate cancer tissues and benign hyperplasia tissues.The results showed that Cyclin B was significantly higher in prostate cancer tissues and had a positive correlation with RCN1.2.Downregulation of RCN1 induced DU 145 cells apoptosisMTT and cell morphology results showed that downregulation of RCN1 could inhibit cell proliferation,and microscopy results showed that downregulation of RCN1 could cause DU 145 cells death.Downregulation of RCN1,flow cytometry results showed DU 145 apoptosis,increased the activity of Caspase-3 enzyme,its substrate PARP shear obviously,and Caspase inhibitor pan cysteine protease inhibitor Z-VAD-FMK can significantly reverse the apoptosis induced by downregulation of RCN1.To further validate,DU 145 cells were injected subcutaneously into nude mice,and siRCN1 was injected into the tumor and detect the tumor growth and proliferation status and the expression of related genes.The results showed that the weight and volume of tumor in the interference group were significantly lower than those in the control group.Western blotting and immunohistochemistry showed that intratumoral injection of siRCN1 could down regulate the expression of RCN1 in tumor cells and promote the apoptosis of tumor cells.Therefore,downregulation of RCN1 could induce Caspase dependent apoptosis in DU 145 cells.3.Downregulation of RCN1 promoted necropotosis of LNCaP cellsCell morphology and MTT results also showed that downregulation of RCN1 could cause LNCaP cell death.The results of flow cytometry showed that the necrosis of LNCaP cells was obvious after downregulation of RCN1.However,there was no obvious change in Caspase-3 enzyme activity and no significant difference in substrate PARP shear.Z-VAD-FMK could not reverse the death of LNCaP.However,Necrostatin-1,an inhibitor of necropotosis,significantly reversed LNCaP death caused by downregulation of RCN1.This indicated that downregulation of RCN1 could cause necroptosis of LNCaP cells.Part III The mechanism of DU145 apoptosis and LNCaP necroptosis induced by downregulation of RCN11.Downregulation of RCN1 caused endoplasmic reticulum stress to participate in apoptosis and necroptosis(1)Downregulation of RCN1 induced endoplasmic reticulum stressWestern blotting showed that downregulation of RCN1 could upregulate the expression of GRP78,and the activity of protein kinase-like endoplasmic reticulum kinase(PERK)increased,and the activity of protein initiation factor eIF2α was decreased,indicating that endoplasmic reticulum stress was produced.DU 145 cells CHOP activated,the apoptotic pathway generated which was induced by endoplasmic reticulum stress.In addition,endoplasmic reticulum stress suppressor 4-PBA could reverse the DU 145 and LNCaP cell death caused by downregulation of RCN1,which indicated that endoplasmic reticulum stress was not only involved in the apoptosis of DU 145 cells,but also involved in necroptosis of LNCaP cells.(2)Ca2+ release induced apoptosis and necroptosisAfter downregulation of RCN1,Ca2+/CaM dependent protein kinase CaMKII was activated,indicating an increase of Ca2+ in the cytosol.Addition of 1,4·5-triphosphate inositol receptor IP3R(endoplasmic reticulum calcium sink release receptor protein)inhibitor Xestospongin C,can significantly reverse the cell death induced by downregulation of RCN1.While the Ranidine RyRine receptor Ryodine had no significant reversal effect on cell death caused by downregulation of RCN1.Indicating that downregulation of RCN1 led to the release of Ca2+ in the endoplasmic reticulum through the IP3R channel into the cytosol,activating CaMKII-related Ca2+signaling pathway and leading to apoptosis and necroptosis.2.PTEN was involved in the apoptosis induced by downregulation of RCN1.(1)PTEN was involved in the apoptosis of DU 145 cells induced by RCN1knockdownEarly experimental data suggested that RCN1 might be more involved in maintaining cell survival rather than promoting cell proliferation.The results showed that,after downregulation of RCN1,the level of AKT activity decreased in DU 145 cells.LNCaP cells did not change.This suggested that the AKT pathway was involved in the apoptosis of DU 145 cells induced by RCN1 downregulation.Through the background analysis.DU 145 was PTEN wild-type cells,in DU145 cells.downregulation of RCN 1,PTEN activity increased,inhibiting of PI3K/AKT pathway,resulting in AKT activity decreased.While downregulation of RCN1 and PTEN could significantly reduce the occurrence of apoptosis in DU 145 cells and increase the level of AKT activity,indicating that RCN1 could affect cell survival through PTEN/AKT pathway.(2)AR and TP53 might be involved in LNCaP necroptosis induced by downregulation of RCN1LNCaP was a typical androgen-dependent prostate cancer cell,its proliferation was largely dependent on androgen/androgen receptor(AR),but the experimental results showed that downregulation of AR,RCN1 slightly changed,indicating that it might participate in mediating the LNCaP death caused by RCN1 deletion.LNCaP was TP53 wild type prostate cancer cells,and the expression of TP53 decreased slightly after downregulation of RCN1,which indicated that AR and TP53 may be involved in the LNCaP necroptosis induced by downregulation of RCN1.Conclusion:1.RCN1 was highly expressed in prostate cancer tissues,but had no association with patient survival.Cyclin D1 showed low expression in prostate cancer tissues and benign hyperplasia tissues,and had no significant correlation with the expression of RCN1.Overexpression of RCN1 had no significant effect on cell proliferation.2.Downregulation of RCN1 could cause prostate cancer cell DU 145 arrest in S phase.and Caspase dependent apoptosis occured.The DU 145 xenograft tumor in nude mice also showed that downregulation of RCN1 could induce apoptosis of tumor cells.3.Downregulation of RCN1 might cause necroptosis of LNCaP cells in prostate cancer cells.4.Downregulation of RCN1 induced apoptosis and necroptosis were dependent on ER stress and Ca2+ release.But the differences were that downregulation of RCN1 activated PTEN in DU145 cells,which could inhibit AKT activity and participated in mediating apoptosis.AR and TP53 might be involved in necroptosis of LNCaP cells induced by RCN1 deletion.Innovation and deficiency1 Innovation points(1)We first reported the expression of endoplasmic reticulum protein Reticulocalbin-1(RCN1)was highly expressed in prostate cancer tissues,and there was no significant correlation between the expression level of RCN1 and the survival rate of cancer patients.(2)We first reported RCN1 might be involved in the regulation of cell cycle,but it had no significant effect on TP53 wild LNCaP cells,and RCN1 may be more involved in the maintenance of cell survival than promotion cell proliferation.(3)We first reported RCN1 knockdown might be involved in the apoptosis of DU 145 cells through endoplasmic reticulum stress,CaMKII activation,PTEN activation and AKT inhibition.Knockdown of RCN1 could induce necroptosis of LNCaP cells through endoplasmic reticulum stress,CaMKII activation.2 Deficiency(1)It was reported that the activation of CaMKII could induce the necroptosis of the cells.Therefore,there is not much research on the necroptosis of LNCaP in this paper.The effect of AR and TP53 needed to be further studied.(2)This paper only researched the AKT signaling pathway related to cell proliferation and survival,and weather ERK pathway related to cell proliferation involved in the death induced by RCN1 knockdown needed research next.