NKAIN2通过钠钾泵β1亚基抑制前列腺癌增殖和进展的实验研究

发布时间：2018-05-13 17:24

本文选题：前列腺癌 + 钠钾泵　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景前列腺癌占西方发达国家男性恶性肿瘤发病率首位,近年来在中国的发病率也逐年上升。大部分的前列腺癌属于相对惰性的肿瘤,然而部分前列腺癌进展迅速,并很快发生转移威胁病人生命,如何区分这部分进展迅速的病人,研究前列腺癌增殖进展的分子机制,对提高前列腺癌病人的生存率意义重大。早期研究者将NKAIN2命名为T细胞淋巴瘤断裂位点相关靶点1(T-cell lymphoma breakpoint associated target 1(TCBA1)),基于能与钠钾泵β1 亚基相互作用(Na,K-ATPase)而被命名为钠钾泵相互作用因子2,属于NKAIN跨膜蛋白超家族的一员。课题组前期通过高通量SNP芯片分析发现NKAIN2在前列腺癌标本中发生断裂及缺失,推测NKAIN2可能是抑癌基因。进一步通过分析大量前列腺癌样本,发现前列腺癌中,尤其是中国的病例中,NKAIN2经常通过缺失及断裂发生失活,并且通过体外细胞实验证明NKAIN2能够抑制肿瘤细胞的增殖、迁移和侵袭,并促进肿瘤细胞凋亡。钠钾泵在肿瘤中的作用已经引起了广泛关注,很多研究发现其在多种肿瘤中活性增强,而钠钾泵抑制剂如强心苷等,已经被证实对多种肿瘤包括前列腺癌有抑制作用,并且有些抑制剂已经进入临床试验。课题组前期发现NKAIN2在前列腺癌中发挥抑癌作用,而作为钠钾泵相互作用因子,我们推测其可能通过抑制钠钾泵发挥抑癌作用,并且在细胞水平上进行探索和验证。方法基于以上认识,本研究首先通过western blotting及钠钾泵活性试剂盒检测前列腺癌细胞系中钠钾泵α1和β1亚基的表达及钠钾泵活性,并通过免疫组化在前列腺癌组织标本中检测钠钾泵α1和β1亚基的表达;运用免疫共沉淀验证了 NKAIN2与钠钾泵β1亚基的相互作用;运用CRISPR/CAS9基因敲除技术,构建Cas9/SgRNA质粒慢病毒载体转染22RV1细胞系(NKAIN2高表达),并通过Western blotting和Cruiser酶切敲除及突变检测试剂盒检测敲除效率;进一步通过裸鼠成瘤实验在体内验证NKAIN2的抑癌作用,同时通过Western blotting检测敲除NKAIN2后PI3K/Akt通路激活情况;接着在过表达、沉默NKAIN2的前列腺癌细胞系22RV1、PC-3中通过Western blotting及无机磷试剂盒检测钠钾泵β1亚基的表达及钠钾泵活性,分析NKAIN2对钠钾泵的影响;并在前列腺癌细胞系22RV1、PC-3中沉默钠钾泵β1亚基,分析钠钾泵对前列腺癌生物学功能的影响。结果1.本研究通过western blotting检测发现前列腺癌细胞系中钠钾泵α1和β1亚基的表达较前列腺永生化上皮细胞系增高;并通过免疫组化检测了 30对前列腺癌标本,发现钠钾泵α1和β1亚基在前列腺癌组织中较癌旁组织表达增高;钠钾泵活性检测试剂盒同样发现前列腺癌细胞系钠钾泵活性较前列腺永生化上皮细胞系增强。2.Western blotting和Cruiser酶检测结果鉴定22RV1 NKAIN2敲除细胞株构建成功,进一步裸鼠成瘤实验提示NKAIN2敲除细胞系成瘤体积明显较对照组增大,并且发现敲除NKAIN2后激活PI3K/Akt通路。3.在PC-3及22RV1细胞中,过表达NKAIN2能抑制钠钾泵β1亚基的表达,相反沉默NKAIN2能增强钠钾泵β1亚基的表达。4.在PC-3及22RV1细胞中,过表达NKAIN2能抑制钠钾泵活性,相反沉默NKAIN2能增强钠钾泵的活性。5.在PC-3及22RV1细胞中,沉默钠钾泵β1亚基的表达,抑制细胞增殖、迁移和侵袭,促进细胞凋亡。结论本研究通过体内实验证实NKAIN2对前列腺癌生长有抑制作用,而钠钾泵α1和β1亚基的表达在前列腺癌细胞系及标本中增高,钠钾泵活性在前列腺癌细胞系同样增强。NKAIN2可能通过抑制钠钾泵的表达及活性,进一步抑制前列腺癌的增殖和进展。这一发现可能为前列腺癌诊断治疗和预后评估提供新的理论依据和研究方向。
[Abstract]:Background prostate cancer accounts for the highest incidence of male malignant tumors in developed countries in western countries. In recent years, the incidence of prostate cancer in China has also increased year by year. Most of the prostate cancer is a relatively inert tumor. However, some prostate cancers are progressing rapidly, and the metastasis threatens the patient's life quickly, and how to distinguish this part of the rapid progress of the patients, The molecular mechanism of the progression of prostate cancer is of great significance for improving the survival rate of prostate cancer patients. Early researchers named NKAIN2 as the target of T cell lymphoma at the target point 1 (T-cell lymphoma breakpoint associated target 1 (TCBA1)), based on the ability to interact with the sodium and potassium pump beta 1 subunit (Na, K-ATPase) and be named sodium potassium The pump interaction factor 2 is a member of the NKAIN transmembrane protein superfamily. In the earlier period, we found that the NKAIN2 was broken and missing in the specimens of prostate cancer by high throughput SNP chip analysis, and that NKAIN2 might be a tumor suppressor gene. Further analysis of a large number of prostate cancer samples, especially in Chinese cases, N KAIN2 is often inactivated by deletion and fracture, and it has been proved that NKAIN2 can inhibit the proliferation, migration and invasion of tumor cells and promote tumor cell apoptosis through in vitro cell experiment. The role of sodium potassium pump in tumor has attracted wide attention. Many studies have found that the activity of the sodium potassium pump is enhanced in many kinds of tumors, and the sodium potassium pump inhibitors such as Strong glycosides have been proved to have inhibitory effects on a variety of tumors, including prostate cancer, and some of the inhibitors have already entered clinical trials. We have found that NKAIN2 plays a role in inhibiting cancer in prostate cancer. As a sodium potassium pump interaction factor, we speculate that it may inhibit cancer by inhibiting the sodium potassium pump and is fine. In this study, the expression of sodium potassium pump alpha 1 and beta 1 subunits in prostate cancer cell lines and the activity of sodium potassium pump were detected by Western blotting and sodium potassium pump active kits. The interaction between NKAIN2 and sodium potassium pump beta 1 subunit was verified by immunoprecipitation; CRISPR/CAS9 gene knockout technique was used to construct the Cas9/SgRNA plasmid lentivirus vector transfected to 22RV1 cell line (NKAIN2 high expression), and the knockout efficiency was detected by Western blotting and Cruiser enzyme knockout and mutation detection kit, and the nude mice were further formed into a tumor. The experiment was used to verify the tumor suppressor effect of NKAIN2 in vivo, and the activation of PI3K/Akt pathway after knockout NKAIN2 was detected by Western blotting; then the prostate cancer cell line of NKAIN2 was expressed in 22RV1, and the expression of sodium potassium pump beta 1 subunit and sodium potassium pump activity were detected by Western blotting and inorganic phosphorus kit in PC-3, and the NKAIN2 pair was analyzed. The effect of sodium potassium pump, and the effect of sodium potassium pump beta 1 subunit in prostate cancer cell line 22RV1, PC-3, analysis of the effect of sodium potassium pump on the biological function of prostate cancer. Results 1. this study found that the expression of sodium potassium pump alpha 1 and beta 1 subunits in prostate cancer cell lines increased by Western blotting detection; and the results were higher than that of the prostatic immortalized epithelial cell line. 30 pairs of prostate cancer specimens were detected by immunohistochemistry. The expression of sodium potassium pump alpha 1 and beta 1 subunits in the prostate cancer tissues was higher than that of the para cancerous tissue. The sodium potassium pump activity detection kit also found that the activity of sodium potassium pump in the prostate cancer cell line was more than that of the enhanced.2.Western blotting and Cruiser enzyme of the prostatic immortalized epithelial cell line. The 2RV1 NKAIN2 knockout cell line was successfully constructed. Further tumor formation experiments in nude mice suggest that the tumor volume of NKAIN2 knockout cell line is obviously larger than that of the control group, and the expression of NKAIN2 can inhibit the expression of the sodium potassium pump beta 1 subunit in the PC-3 and 22RV1 cells after the knockout of NKAIN2 after knockout NKAIN2, and the reverse silencing NKAIN2 can enhance the sodium potassium pump beta 1 subunit. The expression of.4. in PC-3 and 22RV1 cells inhibits the activity of sodium and potassium pumps by overexpression of NKAIN2. On the contrary, silent NKAIN2 can enhance the activity.5. of the sodium potassium pump in PC-3 and 22RV1 cells, silence the expression of the sodium potassium pump beta 1 subunit, inhibit cell proliferation, migration and invasion, and promote cell apoptosis. Conclusion this study confirmed the actual NKAIN2 against the prostate in vivo. The expression of the sodium potassium pump alpha 1 and beta 1 subunits increased in the prostate cancer cell lines and specimens. The sodium potassium pump activity in the prostate cancer cell line also enhanced.NKAIN2 may further inhibit the proliferation and progression of prostate cancer by inhibiting the expression and activity of the sodium potassium pump. This discovery may be a diagnostic treatment for prostate cancer. And provide a new theoretical basis and research direction for prognosis evaluation.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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