TRPM7对前列腺癌PC-3细胞凋亡的调控及其作用机制研究

发布时间：2018-06-05 06:39

本文选题：瞬时受体电位通道7(TRPM7) + 前列腺癌(PCa)　；参考：《安徽医科大学》2015年博士论文

【摘要】：前列腺癌(PCa)是老年男性最常见的恶性肿瘤,在美国已经多年占据新发恶性肿瘤病例首位,严重危害到男性的健康。由于PCa在早期阶段缺少特异性的临床症状,极易与前列腺增生相混淆,患者在因症状就诊时,往往已经进入晚期甚至转移。因此,早期诊断对于PCa治疗时机的把握尤为重要。目前公认PCa筛查标准仍然是直肠指诊联合血清前列腺特异抗原(prostate specific antigen, PS A)检测,但因特异性偏低,往往能导致过度诊断和过度治疗而受到广泛质疑(1)。近年来的影像学发展日新月异,新技术不断改进,如MRI波谱分析手段,仍然对PCa的诊断正确率提高仍十分有限。临床PCa的诊断“金标准”还是前列腺穿刺活检,但因其有创性、阳性率不高、较高感染率、以及潜在并发症风险,临床仍不是满意的PCa诊断方法,促使广大的科研工作者及临床医生努力寻求、探索新的筛选标准、办法。在PCa早期阶段的治疗以根治手术为主,其后伴随病程进展,可以有放疗和内分泌治疗以及化疗的推荐,尽管有多种治疗方式层出不穷,尤其新的化疗药物和二线内分泌治疗药物不断推出,大大延长了患者的生存时间并改善生活质量,但患者仍会不可避免地进入到去势抵抗性前列腺癌(Castration-resistant prostate cancer, CRPC)阶段,最终短期内发生死亡。因此,对于PCa的发生和发展的机制上的研究是探索新型PCa肿瘤标记物、前瞻性判断治疗以及靶向治疗的必要途径。在肿瘤的研究领域,离子通道的探索始终方兴未艾,并发现了一系列参与肿瘤形成的离子通道。文献报道,瞬时受体电位(transient receptor potential, TRP)作为新型的Ca2+通道,参与人体众多生理和病理过程。近年来TRP家族受到广泛重视,其家族成员之一的瞬时受体电位通道7(transient receptor potential melastatin 7, TRPM7)更是肿瘤研究的热点。多项研究表明,该通道蛋白在人甲状腺肿瘤、乳腺癌、胃癌、胰腺肿瘤、成视网膜细胞瘤、肺癌等组织或细胞上表达有显著升高,已经成为浸润性乳腺导管乳腺癌的预后指标,而具有靶向治疗的意义。进而,TRPM7是否也在PCa的发生发展中发挥作用,最近也有学者在尝试探究。本研究旨在于研究PCaPC-3细胞中,TRPM7在其中的调控及其作用机制,观察TRPM7对TRAIL诱导PC-3细胞凋亡的影响,并探讨其可能的作用机制。主要研究内容概括如下：1. TRPM7在前列腺癌组织中的表达为了检测PCa组织中TRPM7是否表达,通过Western blot检测PCa组织中TRPM7的蛋白表达情况。结果表明,癌变组织中TRPM7的蛋白表达明显高于正常组织。2. TRPM7非特异性阻断剂及TRPM7-siRNA对PC-3细胞中的TRPM7的抑制作用采用非特异性TRPM7阻断剂Gd3+或2-APB,阻断中TRPM7的表达,通过RT-PCR、 Western blot检测PC-3细胞TRPM7表达水平。结果发现,与正常组相比,Gd3+或2-APB都能够不同程度地降低PC-3细胞中T RPM7 mRNA和蛋白的表达。另一方面,利用LipofectamineTM2000将TRPM7-siRNA转染到PC-3细胞中,同样观察到TRPM7 mRNA和蛋白在TRPM7-siRNA转染PC-3细胞中的表达明显下调。3. TRPM7对PC-3细胞凋亡的影响采用RT-PCR、Western blot和流式细胞仪检测方法,检测TRPM7非特异性抑制剂及TRPM7-siRNA 对 PC-3细胞的凋亡的影响。结果发现,非特异性阻断剂Gd3+、2-APB 或 TRPM7-siRNA刺激PC-3细胞后,能显著促进其凋亡。另外,还发现DR4、DR5 和 cleaved Caspase-3蛋白明显增加。4. TRAIL诱导的PC-3细胞凋亡后对TRPM7的影响采用RT-PCR、Western blot和流式细胞仪检测方法,观察TRAIL诱导PC-3细胞凋亡后,观察TRPM7的影响。结果发现,TRAIL组的PC-3细胞中TRPM7的mRNA和蛋白表达明显高于正常组。表明TRAIL诱导后PC-3细胞中TRPM7表达水平上升。5. TRAIL诱导的PC-3细胞凋亡后,阻断TRPM7对PC-3细胞凋亡的影响采用RT-PCR、Western blot和流式细胞仪检测方法,观察非特异性阻断剂Gd3+、 2-APB 或 TRPM7-siRNA对TRAIL诱导PC-3细胞凋亡的影响。结果发现,TRPM7非特异性抑制剂Gd3+、2-APB 及 TRPM7-siRNA,可以明显促进TRAIL诱导PC-3细胞的凋亡活动。同时,还发现沉默TRPM7后,PC-3细胞中DR4、DR5 和 cleaved Caspase-3凋亡蛋白明显激活。6. TRPM7通过AKT通路对PC-3细胞凋亡的调节机制研究采用RT-PCR及Western blot的方法,检测TRPM7对PC-3细胞中AKT信号通路的调控作用。结果发现,沉默TRPM7后,磷酸化AKT水平降低,表明TRPM7沉默可以调节AKT信号通路。同时, AKT下游蛋白P70S6K的磷酸化水平在TRPM7沉默后降低,进一步提示TRPM7可以正向调控AKT信号通路。以上结果表明：下调TRPM7表达可以增加PC-3细胞的凋亡,其机制可能与AKT信号通路有关,这将为PCa的研究与治疗提供新的思路。
[Abstract]:Prostate cancer (PCa) is the most common malignant tumor in old men. It has been the first case of new malignant tumor in the United States for many years and seriously endangers the health of men. Because of the lack of specific clinical symptoms in the early stage, PCa is very easy to be confused with the hyperplasia of prostate. Therefore, early diagnosis is particularly important for the timing of PCa treatment. It is recognized that the PCa screening standard is still the detection of prostate specific antigen (PS A) of the combined rectal finger diagnosis (prostate), but it is widely questioned because of its low specificity, which often leads to excessive diagnosis and over treatment (1). With the development of new technology and new technology, such as MRI spectrum analysis, the diagnostic accuracy of PCa is still very limited. Clinical PCa diagnosis "gold standard" or prostate biopsy, but because of its invasive, low positive rate, high infection rate, and potential complications risk, clinical still is not satisfactory PCa diagnostic method, promoting the clinical The majority of researchers and clinicians seek to explore new screening criteria. In the early stages of the PCa treatment, radical surgery is the main procedure, followed by progress in the course of the disease, which can be recommended for radiotherapy, endocrine therapy and chemotherapy, although there are a variety of treatments, especially new chemotherapeutic drugs and second line endocrinology. The continuous release of therapeutic drugs greatly prolongs the patient's survival time and improves the quality of life, but the patient will still inevitably enter the Castration-resistant prostate cancer (CRPC) stage and eventually die in the short term. Therefore, research on the mechanism of the occurrence and development of PCa is to explore new P Ca tumor markers, prospective treatment and the necessary approach to target therapy. In the field of cancer research, the exploration of ion channels is always in the ascendant, and a series of ion channels involved in the formation of tumors have been discovered. It is reported that the transient receptor potential (transient receptor potential, TRP) is a new Ca2+ channel and participates in the human body. Many physiological and pathological processes. The TRP family has been widely recognized in recent years. The transient receptor potential channel 7 (transient receptor potential melastatin 7, TRPM7) is one of the hotspots of cancer research. A number of studies have shown that this channel protein is in human goiter, breast, gastric, pancreatic, and retinoblastoma. There is a significant increase in tissue or cell expression in lung cancer, which has become a prognostic indicator of invasive mammary ductal carcinoma and has the significance of targeting therapy. Further, TRPM7 has also played a role in the development of PCa. Recently, some scholars have been trying to explore. The purpose of this study is to study the regulation and regulation of TRPM7 in PCaPC-3 cells. Its mechanism is to observe the effect of TRPM7 on the apoptosis of PC-3 cells induced by TRAIL and explore its possible mechanism. The main contents are summarized as follows: 1. TRPM7 expression in the prostate cancer tissue was used to detect the expression of TRPM7 in PCa tissue and the protein expression of TRPM7 in PCa group by Western blot. The results showed that the cancer was cancerous. The protein expression of TRPM7 in the tissue was significantly higher than that of normal tissue.2. TRPM7 non specific blocker and TRPM7-siRNA in the inhibition of TRPM7 in PC-3 cells by using non-specific TRPM7 blocker Gd3+ or 2-APB, blocking the expression of TRPM7, and detecting the expression level by RT-PCR, and found that it was compared with the normal group. Both 3+ or 2-APB can reduce the expression of T RPM7 mRNA and protein in PC-3 cells to varying degrees. On the other hand, TRPM7-siRNA is transfected into PC-3 cells by LipofectamineTM2000, and the expression of TRPM7 mRNA and protein in TRPM7-siRNA cells is obviously down regulated. Tern blot and flow cytometry were used to detect the effect of TRPM7 non specific inhibitors and TRPM7-siRNA on the apoptosis of PC-3 cells. The results showed that the non specific blocker Gd3+, 2-APB or TRPM7-siRNA stimulated the PC-3 cells to promote apoptosis significantly. Furthermore, the DR4, DR5, and cleaved proteins were significantly increased. The effect of induced PC-3 cell apoptosis on TRPM7 was detected by RT-PCR, Western blot and flow cytometry. The effect of TRPM7 was observed after TRAIL induced apoptosis of PC-3 cells. The results showed that TRPM7 mRNA and protein expression in PC-3 cells of TRAIL group was obviously higher than that of normal group. After the apoptosis of PC-3 cells induced by.5. TRAIL, the effect of blocking TRPM7 on the apoptosis of PC-3 cells was detected by RT-PCR, Western blot and flow cytometry. The effect of the non specific blocker Gd3+, 2-APB, or TRPM7-siRNA on the apoptosis of the TRAIL induced cells was observed. The apoptotic activity of PC-3 cells was obviously promoted by TRAIL. Meanwhile, after the silence of TRPM7, the apoptotic proteins of DR4, DR5 and cleaved Caspase-3 in PC-3 cells obviously activated the regulation mechanism of.6. TRPM7 through AKT pathway. The results showed that after the silence of TRPM7, the level of phosphorylated AKT decreased, indicating that the TRPM7 silence could regulate the AKT signaling pathway. At the same time, the phosphorylation level of the downstream protein P70S6K of AKT decreased after TRPM7 silencing, further suggesting that TRPM7 could positively regulate the AKT signaling pathway. The above results showed that the downregulation of TRPM7 expression could increase the decline of PC-3 cells. Its mechanism may be related to the AKT signaling pathway, which will provide new ideas for the research and treatment of PCa.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

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