内质网蛋白Reticulocalbin-1表达下调诱导前列腺癌细胞发生凋亡和坏死性凋亡的研究

发布时间：2018-08-09 20:41

【摘要】：研究背景内质网是细胞重要的细胞器,在蛋白质的折叠、质量控制、维持钙稳态等方面都极其重要,直接参与并影响细胞的各种生命活动。内质网的各种定位蛋白,如分子伴侣、酶等通过对新合成的蛋白肽链进行加工、折叠修饰,帮助蛋白质形成特定的空间构象并定向转运。Ca2+结合蛋白、二硫键异构酶等内质网定位蛋白不仅可以发挥分子伴侣作用,还可定位于细胞胞液、细胞外,参与细胞的增殖、迁移及恶性疾病如肿瘤的发生发展。Reticulocalbin 1(RCN1)具有钙离子结合蛋白的特点,是CREC家族成员之一。人源RCN1蛋白由位于染色体11p13的RCN1基因编码,全长331个氨基酸残基,其结构特点是:N端是一段信号肽,由29个氨基酸残基组成;中间区域包含6个EF-hand结构域;在羧基末端是一段内质网驻留信号HDEL。RCN1的定位与分布:RCN1是内质网定位蛋白,但也发现RCN1可定位干骨内皮细胞、前列腺癌细胞的表面,但机制不详;而且RCN1的C端的HDEL与典型的内质网定位信号KDEL有别,因此,RCN1也可分泌至胞外。RCN1主要分布于·分泌器官,不同器官的组成细胞中RCN1的表达高低不同。RCN1的功能:RCN1功能研究不多。研究显示,鼠源RCN1基因的纯合缺失可导致胚胎致死;另外,分泌的RCN1可作为配体,介导凋亡的神经细胞被吞噬。RCN1的表达异常与疾病、特别是肿瘤的关系引起关注,RCN1在高侵袭性的乳腺癌细胞、结直肠癌细胞、胃癌细胞、肝癌细胞中均呈高表达。而在前列腺癌中,RCN1在侵袭力较高的细胞中呈低表达。基于RCN1功能不详,相关研究初露端倪,尚需详尽探讨。本论文首先从临床样本、数据库对其表达进行分析,并通过细胞和动物实验初步探讨了 RCN1在前列腺癌中的作用。研究结果第一部分RCN1在前列腺癌组织中高表达1.RCN1在前列腺癌组织中高表达根据肿瘤基因数据库TCGA,oncomine已有的数据分析前列腺癌中RCN1的mRNA水平高,但是生存分析却显示高水平的mRNA与病人的生存率无显著相关性。本论文分别收集了前列腺癌组织样本11例,良性前列腺增生组织样本15例,利用免疫组化检测RCN1在癌组织和增生组织中的蛋白表达水平。结果发现前列腺癌组织中RCN1显著高表达。为了确定RCN1是否与前列腺癌的发生有关,利用免疫组化检测周期蛋白CyclinD1,临床样本检测结果显示,CyclinD1不仅在前列腺癌组织中低表达,在良性增生组织中也呈现低表达,且与RCN1的表达无相关性。2.RCN1表达与细胞增殖无相关性细胞水平的检测结果显示,RCN1的表达水平在激素非依赖、侵袭性较强的PC3细胞中RCN1的表达最低,低于RWPE1,即前列腺正常上皮细胞。前列腺癌LNCaP细胞中RCN1表达最高,DU145细胞中RCN1表达介于PC3和LNCaP细胞之间。因此,选用RCN1低表达的PC3过表达RCN1,细胞增殖结果却显示过表达RCN1对细胞增殖无显著影响。因此,RCN1可能与细胞增殖无关。第二部分下调RCN1的表达可促进不同前列腺癌细胞凋亡或坏死性凋亡DU145和LNCaP细胞RCN1表达高,我们利用流式细胞术分析降低RCN1表达对细胞周期、细胞存活的影响。1.下调RCN1阻滞DU145细胞于S期下调RCN1可影响细胞周期。下调DU145细胞中的RCN1,发现细胞周期阻滞于S期。下调LNCaP细胞中的RCN1可使周期阻滞在G2/M期。检测S期和G2/M期关键调控蛋白Cyclin B在前列腺癌组织和良性增生组织的表达,结果显示,前列腺癌组织中CyclinB显著高表达,且与RCN1具有一定正相关性。2.下调RCN1诱导DU145细胞凋亡DU145细胞中下调RCN1可以抑制细胞活力,引起细胞死亡。下调RCN1后DU145细胞凋亡现象明显,Caspase-3酶活性升高,WB结果显示PARP剪切带明显。Z-VAD-FMK是Caspase抑制剂,在下调RCN1后加入,可以逆转细胞凋亡。为了进一步验证,将DU145细胞注入裸鼠皮下成瘤,瘤内注射siRCN1。结果显示,瘤内注射siRCN1后肿瘤重量和体积显著低于NC组。结果证明,瘤内注射siRCN1可以下调瘤内细胞RCN1的表达,促进肿瘤细胞凋亡。所以,下调RCN1使DU145发生细胞凋亡。3.下调RCN1促进LNCaP细胞发生坏死性凋亡下调RCN1可引起LNCaP细胞死亡。下调RCN1后,LNCaP细胞坏死明显。但是,Caspase-3无明显变化,同时其底物PARP剪切无差异,Z-VAD-FMK不能逆转下调RCN1造成的LNCaP死亡。然而,Necrostatin-1,坏死性凋亡抑制剂,可以部分逆转LNCaP死亡。这说明下调RCN1能引起LNCaP细胞坏死性凋亡。第三部分下调RCN1引起DU145和LNCaP凋亡和坏死性凋亡的机制研究1.RCN1 下调引起内质网应激参与细胞凋亡和坏死性凋亡(1)下调RCN1诱导内质网应激下调RCN1可增强分子伴侣GRP78活性,同时PERK活性增强,eIF2α活性降低,表明内质网应激产生。下调RCN1可使DU145细胞CHOP被激活,诱导凋亡产生。另外,内质网应激抑制剂4-PBA 可逆转下调RCN1造成的DU145和LNCaP细胞死亡,说明内质网应激参与了 RCN1下调引起的DU145细胞凋亡和LNCaP细胞的坏死性凋亡。(2)Ca2+释放诱导细胞凋亡和坏死性凋亡下调RCN1后,CaMKⅡ被激活,说明胞液中的Ca2+增加。加入内质网钙库释放通道受体蛋白IP3R的抑制剂XestosponginC,可以部分逆转细胞死亡。而兰尼碱受体RyR抑制剂Ryanodine对下调RCN1造成的细胞死亡却无明显逆转作用。说明下调RCN1导致内质网内Ca2+通过IP3R通道释放到胞液中,Ca2+信号通路被激活,导致细胞凋亡和坏死性凋亡发生。2.PTEN参与下调RCN1诱导的细胞凋亡(1)PTEN参与下调RCN1诱导的DU145细胞凋亡通过前期实验数据初步推测,RCN1可能更多的参与维持细胞存活,而不是促进细胞增殖。结果证实,下调DU145细胞中的RCN1可降低AKT活性,LNCaP细胞无变化。这说明AKT通路参与了 RCN1下调诱导的DU145细胞的凋亡。通过遗传学背景分析,DU145是PTEN野生型前列腺癌细胞,在DU145细胞中,下调RCN1可使PTEN活性升高,AKT活性降低。而同时下调RCN1和PTEN,DU145细胞凋亡缓解,AKT活性水平升高,说明下调RCN1影响细胞的存活是通过PTEN/AKT通路。(2)AR和TP53可能参与下调RCN1诱导的LNCaP细胞坏死性凋亡雄激素依赖的LNCaP癌细胞,其增殖很大程度上依赖于雄激素/雄激素受体(AR),但实验结果证明,下调AR,RCN1略有变化,说明其可能参与介导RCN1下调引起的LNCaP死亡。LNCaP中TP53可以发挥正常功能,下调RCN1,TP53表达略有降低,说明AR和TP53可能参与了下调RCN1诱导的LNCaP细胞坏死性凋亡过程。结论:1.前列腺癌组织中RCN1显著高表达,但与病人生存率无相关性。周期蛋白Cyclin D1表达水平较低,与RCN1无显著相关性。过表达RCN1对细胞增殖无显著影响。2.下调RCN1后,DU145阻滞于S期,诱导DU145细胞凋亡。DU145裸鼠实验同样表明,在体内干扰RCN1也可以促进细胞凋亡。3.下调RCN1可引起前列腺癌LNCaP细胞发生细胞坏死性凋亡。4.下调RCN1引起的细胞凋亡和坏死性凋亡均依赖于内质网应激以及Ca2+释放。但不同的是,下调RCN1激活了 DU145细胞内的PTEN,抑制AKT活性,细胞凋亡产生。而AR和TP53可能参与了下调RCN1引起的LNCaP细胞的坏死性凋亡。创新点与不足之处1.创新点(1)首次报道内质网蛋白Reticulocalbin-1(RCN1)在前列腺癌组织中高表达,并通过肿瘤基因数据库分析RCN1表达水平与病人生存率无显著相关性。(2)首次报道RCN1可能参与调控细胞周期,但对TP53野生的LNCaP细胞作用并不显著;且与促进细胞增殖相比,RCN1可能更多的参与维持细胞存活。(3)首次报道RCN1下调可通过内质网应激、CaMKⅡ的活化、PTEN活化、抑制AKT活性参与DU145细胞凋亡。对TP53野生的LNCaP细胞,下调RCN1可通过内质网应激、CaMKⅡ的活化调控诱导LNCaP细胞坏死性凋亡。2.不足(1)文献报道,CaMKⅡ活化可介导坏死性凋亡,因此本论文中对于LNCaP的坏死性凋亡并未做过多研究。(2)本论文只探讨了与细胞增殖、存活相关的AKT信号通路,而与细胞增殖相关的ERK通路是否也参与了 RCN1的作用需要进一步探讨和讨论。
[Abstract]:Background endoplasmic reticulum (endoplasmic reticulum) is an important cell organelle, which is extremely important in protein folding, quality control, and calcium homeostasis. It directly participates in and affects the various life activities of the cells. The various localization proteins of the endoplasmic reticulum, such as molecular chaperones, enzymes, etc., are processed, folded, and helped to help the protein peptide chain. The formation of specific space conformation and directional transport of.Ca2+ binding proteins, two sulfur bond isomerase and other endoplasmic reticulum localizing proteins can not only play the role of molecular chaperone, but also be located in cell fluid, extracellular, cell proliferation, migration and malignant diseases such as the occurrence of tumor development.Reticulocalbin 1 (RCN1) with calcium binding protein It is one of the members of the CREC family. The human RCN1 protein is encoded by the RCN1 gene located on the chromosome 11p13 and has a full length of 331 amino acid residues. The structure of the protein is that the N end is a signal peptide and consists of 29 amino acid residues; the middle region contains 6 EF-hand domains; the end of the carboxyl group is the localization of a segment of the endoplasmic reticulum signal HDEL.RCN1. And distribution: RCN1 is a endoplasmic reticulum localizing protein, but it is also found that RCN1 can locate the stem bone endothelial cells, the surface of the prostate cancer cells, but the mechanism of the HDEL at the C end of RCN1 is different from the typical endoplasmic reticulum location signal KDEL, so RCN1 can also be secreted to the extracellular.RCN1, mainly distributed in the secretory organs, and RCN1 in the constituent cells of different organs. The function of high and low.RCN1 is expressed as the function of the RCN1 function. The study shows that the homozygous deletion of the mouse RCN1 gene can lead to the death of the embryo; in addition, the secreted RCN1 can be used as a ligand to mediate the abnormal expression of the apoptotic neurons by phagocytic.RCN1 and the relationship between the disease, especially the tumor, and RCN1 in highly invasive breast cancer cells, Colorectal cancer cells, gastric cancer cells and hepatoma cells are highly expressed. In the prostate cancer, RCN1 is low in the aggressive cells. Based on the unknown RCN1 function, the related research has been revealed. This paper first analyzed the expression from the clinical samples, and through the initial cell and animal experiments. The role of RCN1 in prostate cancer is discussed. Part 1, the high expression of RCN1 in prostate cancer tissue is high in prostate cancer tissue based on the tumor gene database TCGA, and oncomine has been used to analyze the high mRNA level of RCN1 in prostate cancer, but the survival analysis shows the high level of mRNA and the patients in the prostate cancer. There was no significant correlation between survival rate and survival rate. 11 samples of prostate cancer tissue and 15 samples of benign prostatic hyperplasia were collected in this paper. The protein expression levels of RCN1 in cancer tissues and proliferative tissues were detected by immunohistochemistry. The results showed that RCN1 was significantly high in prostate cancer tissue. To determine whether RCN1 was associated with prostate cancer. The results of detection of cyclin CyclinD1 by immunohistochemistry showed that CyclinD1 not only expressed low expression in the prostate cancer tissue, but also showed low expression in benign hyperplasia tissue, and there was no correlation between the expression of.2.RCN1 expression and cell proliferation without correlation with RCN1 expression, and the expression of RCN1 expressed water. The expression of RCN1 is the lowest in PC3 cells with strong invasiveness, which is lower than RWPE1, that is, normal prostate epithelial cells. The expression of RCN1 in prostate cancer LNCaP cells is the highest, and the expression of RCN1 in DU145 cells is between PC3 and LNCaP cells. Therefore, PC3 overexpression RCN1 is used for RCN1 low expression, but the cell proliferation results show excessive expression. There is no significant effect on cell proliferation. Therefore, RCN1 may not be related to cell proliferation. Second down regulation of RCN1 expression can promote the apoptosis of different prostate cancer cells or necrotic apoptosis of DU145 and LNCaP cells, RCN1 expression is high. We use flow cytometry to reduce RCN1 expression to cell cycle, cell survival effect.1. down RCN1 block DU1 The down-regulation of RCN1 in the S phase of 45 cells could affect the cell cycle. Down regulation of RCN1 in DU145 cells, the cell cycle was blocked at S stage. The RCN1 in the down-regulation of LNCaP cells could block the cycle at G2/M stage. The expression of Cyclin B in the prostate cancer tissue and benign hyperplasia tissues was detected in S and G2/M phase. The results showed that the prostate cancer tissues were expressed. The expression of inB was significantly higher, and it had a positive correlation with RCN1..2. down regulated the apoptosis of DU145 cells induced by RCN1. The downregulation of RCN1 could inhibit cell viability and cause cell death. The apoptotic phenomenon of DU145 cells was obvious, the Caspase-3 enzyme activity increased after the downregulation of RCN1. WB results showed that the PARP shear zone was obviously a inhibitor. The addition of N1 could reverse the apoptosis of cells. In order to further verify, the DU145 cells were injected into the nude mice subcutaneously into a tumor. The results of intratumoral injection of siRCN1. showed that the weight and volume of the tumor were significantly lower than that of the NC group. The results showed that the intra tumor injection of siRCN1 could downregulate the expression of RCN1 in the tumor cells and promote the apoptosis of the tumor cells. So, down regulation of RC. N1 makes DU145 apoptosis.3. down-regulation and RCN1 promotes necrotizing apoptosis of LNCaP cells and leads to LNCaP cell death. The LNCaP cell necrosis is obvious after RCN1 down regulation. However, Caspase-3 does not change obviously, and its substrate PARP shear no difference, Z-VAD-FMK can not reverse the death caused by the downward adjustment. The inhibitor of death apoptosis can partly reverse LNCaP death. This indicates that down regulation of RCN1 can cause necrotic apoptosis of LNCaP cells. Third the mechanism of down regulation of RCN1 induced apoptosis and necrotic apoptosis of DU145 and LNCaP, 1.RCN1 downregulation causes endoplasmic reticulum stress to participate in cell apoptosis and necrotic death (1) down regulation of RCN1 induced endoplasmic reticulum stress down regulation N1 enhanced the activity of molecular chaperone GRP78, while PERK activity increased and eIF2 alpha activity decreased, indicating that endoplasmic reticulum stress was produced. Down regulation of RCN1 could activate CHOP in DU145 cells and induce apoptosis. In addition, endoplasmic reticulum stress inhibitor 4-PBA can reverse DU145 and LNCaP cell death caused by RCN1, indicating that endoplasmic reticulum stress is involved in RCN1 down. Induced apoptosis of DU145 cells and necrotic apoptosis of LNCaP cells. (2) after Ca2+ release induced apoptosis and necrotic apoptosis, CaMK II was activated to indicate an increase in Ca2+ in the cytoplasm. XestosponginC, a inhibitor of the receptor protein IP3R added to the endoplasmic reticulum calcium pool, could partly reverse the cell death. The preparation of Ryanodine has no obvious reversal effect on the cell death caused by the downregulation of RCN1. It is suggested that the down-regulation of RCN1 leads to the release of Ca2+ in the endoplasmic reticulum through the IP3R channel to the cytoplasm, and the Ca2+ signaling pathway is activated, leading to the apoptosis and necrotic apoptosis of the.2.PTEN involved in the down-regulation of RCN1 induced apoptosis (1) PTEN involved in DU145 fine RCN1 induced RCN1 Apoptosis was preliminarily hypothesized that RCN1 might be more involved in maintaining cell survival, rather than promoting cell proliferation. The results showed that the downregulation of RCN1 in DU145 cells could reduce AKT activity and LNCaP cells were not changed. This indicates that the AKT pathway participates in the apoptosis of DU145 cells induced by RCN1 downregulation. By genetic background analysis, DU145 It is a PTEN wild type prostate cancer cell. In DU145 cells, the downregulation of RCN1 can increase the activity of PTEN and decrease the activity of AKT. While down regulation of RCN1 and PTEN, the apoptosis of DU145 cells is relieved and the level of AKT activity increases. It shows that the down regulation of RCN1 affects the survival of cells through PTEN/AKT pathway. (2) AR and may participate in the necrosis induced cell necrosis induced by down regulation. The proliferation of androgen dependent LNCaP cells depends largely on androgen / androgen receptor (AR), but the experimental results show that the downregulation of AR and RCN1 changes slightly, indicating that it may participate in the LNCaP death induced by RCN1 down.LNCaP and TP53 can play normal work energy, down RCN1, TP53 expression a slight decrease, indicating AR and TP53. Can participate in the necrosis of apoptosis induced by RCN1 induced LNCaP cells. Conclusion: 1. the expression of RCN1 in the prostate cancer tissues is highly expressed, but there is no correlation with the survival rate of the patients. The expression level of cyclin Cyclin D1 is low, and there is no significant correlation with RCN1. The overexpression of RCN1 has no significant effect on the proliferation of.2. in.2., and DU145 is blocked in S stage. The apoptosis of DU145 cells in nude mice also showed that the interference of RCN1 in vivo could also promote apoptosis and.3. down regulation of RCN1, which could induce apoptosis and apoptosis of LNCaP cells in prostate cancer cells,.4. down regulation and necrotizing apoptosis were dependent on endoplasmic reticulum stress and Ca2+ release. However, down regulated RCN1 activates D. PTEN in U145 cells inhibit AKT activity and apoptosis, and AR and TP53 may be involved in necrotizing apoptosis of LNCaP cells induced by RCN1. Innovation and deficiency 1. innovation point (1) first reported the high expression of endoplasmic reticulum Reticulocalbin-1 (RCN1) in prostate cancer tissue, and the expression of RCN1 expression through a tumor gene database There was no significant correlation between the level and the survival rate of patients. (2) it was reported that RCN1 might participate in the regulation of cell cycle for the first time, but it did not play a significant role in the TP53 wild LNCaP cells; and RCN1 may be more involved in maintaining cell survival compared with the promotion of cell proliferation. (3) it is the first time to report that RCN1 can be regulated by endoplasmic reticulum stress, activation of CaMK II, activation of PTEN, and inhibition. AKT activity is involved in DU145 cell apoptosis. Down regulation of RCN1 in TP53 wild LNCaP cells can be regulated by endoplasmic reticulum stress, and CaMK II activation induces necrotizing apoptosis of LNCaP cells (1), and CaMK II activation can mediate necrotic apoptosis. Therefore, the necrotic apoptosis of LNCaP has not been much studied in this paper. (2) this paper The AKT signaling pathway related to cell proliferation and survival is discussed only, and whether the ERK pathway associated with cell proliferation is also involved in the role of RCN1 needs further discussion and discussion.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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