siRNA沉默DAD1基因表达对肾癌细胞A498增殖与侵袭能力的影响

发布时间：2018-03-29 02:35

本文选题：DAD1　切入点：肾癌　出处：《南华大学》2015年硕士论文

【摘要】：背景：肾癌又称肾细胞癌(renal cell carcinooma RCC)是成人肾脏最常见的恶性肿瘤,占成人恶性肿瘤2%-3%,在男性和女性恶性肿瘤中分别排第7位和第9位。在全球范围内,每年大约新发病例27万,其中12万死亡,在我国,肾癌的发病率在泌尿系肿瘤中仅次于膀胱癌,并在过去20年中以每年6%速度递增。病理上肾癌可分为：透明细胞癌、肾乳头状癌、嫌色细胞癌、集合管癌及未分化癌。其中透明细胞癌(clear cell renal cell carcinoma ccRCC)是最常见的病理类型,起源于近曲小管上皮细胞,且易发生转移,占转移性肾癌的80%-85%。肾癌对放疗及化疗均不敏感,手术切除仍是目前最佳的治疗手段,但术后复发率约为20%-40%。由于肾癌的化疗及放疗抵抗性,因此临床上没有有效的术后治疗手段。肾癌作为泌尿科常见疾病,尤其是早期肾癌无特殊临床表现,待出现血尿、腰痛或腹部包块时多已是晚期。肾癌对放疗、化疗均不敏感,临床上一般予行肾癌根治性切除术,但是一旦出现淋巴结转移,即使行根治性淋巴结清扫术,患者生存期也极少超过5年,若出现肝、肺转移或临近器官浸润则预后更差,因此摸索出一条新的治疗方案变的尤为重要。目前靶向沉默目的基因来治疗肿瘤正在被广大研究者所关注,相关研究显示沉默肿瘤细胞中抗凋亡的基因可以促使肿瘤细胞大幅度凋亡,起到治疗肿瘤的作用。目的：合成寡糖基转移酶亚基DAD1的siRNA序列,观察其对人A498肾癌细胞增殖和侵袭能力的影响。方法：设计合成三条DAD1-siRNA干扰片段,采用脂质体法转染人A498肾癌细胞后采用定量PCR和免疫印迹法检测转染前后人A498肾癌细胞中DAD1 mRNA和蛋白的表达变化；MTT检测转染前后A498细胞增殖能力变化；AV-PI流式细胞术检测各组人A498肾癌细胞转染48h后的凋亡率变化；Transwell检测细胞侵袭能力的变化。结果：定量PCR及免疫印迹法检测显示3条siRNA均能有效抑制DAD1 mRNA及蛋白表达(P0.05),以DAD1-siRNA 1作用效果最显著(P0.05)。 MTT检测显示DAD1-siRNA转染24、48和72h后的人A498肾癌细胞的增殖受到显著抑制(P0.05)；流式细胞术检测结果显不DAD1-siRNA转染人A498肾癌细胞后凋亡率增加(p0.05)。Transwell检测结果显示沉默人A498肾癌细胞的DAD1 mRNA表达后,其侵袭能力显著降低(p0.05)。结论：DAD1-siRNA可明显抑制人A498肾癌细胞的增殖,促进人A498肾癌细胞的凋亡,降低其侵袭能力。
[Abstract]:Background: renal cell carcinoma (RCC), also known as renal cell carcinoma (RCC), is the most common malignant tumor in adult kidney, accounting for 2-3% of adult malignant tumors. It ranks 7th and 9th in male and female malignant tumors respectively. Globally, about 270000 new cases occur every year. Among them, 120000 died. In our country, the incidence of renal cell carcinoma is second only to that of bladder cancer, and has been increasing at an annual rate of 6% in the past 20 years. Pathologically, renal cell carcinoma can be classified as clear cell carcinoma, papillary carcinoma of kidney, chromophobe cell carcinoma. Clear cell renal cell carcinoma ccRCC is the most common pathological type, originated from proximal convoluted tubule epithelial cells, and easily metastasized, accounting for 80% -85% of metastatic RCC. RCC is not sensitive to radiotherapy and chemotherapy. Surgical resection is still the best treatment method at present, but the recurrence rate is about 20% -40%. Because of the resistance to chemotherapy and radiotherapy of renal cell carcinoma, there is no effective postoperative treatment in clinic. Renal cell carcinoma is a common disease in urology. In particular, early renal cell carcinoma has no special clinical manifestation, and it is usually late when hematuria, low back pain or abdominal mass appears. Renal cell carcinoma is not sensitive to radiotherapy or chemotherapy, and is usually treated with radical resection of renal cell carcinoma clinically, but once lymph node metastasis occurs, Even with radical lymphadenectomy, the survival time of the patient is rarely more than 5 years. If liver, lung metastasis or adjacent organ infiltration occur, the prognosis is even worse. Therefore, it is very important to explore a new therapeutic scheme. At present, the target silencing target gene to treat tumor is being paid attention to by many researchers. Related studies have shown that silencing the anti-apoptotic genes in tumor cells can promote the large apoptosis of tumor cells and play a role in the treatment of tumor. Objective: to synthesize the siRNA sequence of oligosyltransferase subunit DAD1. To observe its effect on proliferation and invasion of human renal carcinoma cell A498. Methods: three interfering fragments of DAD1-siRNA were designed and synthesized. The expression of DAD1 mRNA and protein in human A498 renal carcinoma cells before and after transfection were detected by quantitative PCR and Western blotting after transfection with liposome. The apoptotic rate of human A498 renal cancer cell line was detected by RT-PCR. Transwell was used to detect the cell invasion ability. Results: quantitative PCR and Western blot analysis showed that three siRNA could effectively inhibit the expression of DAD1 mRNA and protein, and DAD1-siRNA 1 was used to detect the invasion ability of A498 renal cancer cells. MTT assay showed that the proliferation of human A498 renal cancer cells transfected with DAD1-siRNA for 24 h and 72 h was significantly inhibited, and flow cytometry showed that the apoptosis rate increased after transfection of DAD1-siRNA into human A498 renal cancer cells by flow cytometry. After silencing the expression of DAD1 mRNA in human A498 renal cancer cells, Conclusion the cell proliferation of human renal cancer cell line A498 can be inhibited, the apoptosis of human renal cancer cell line A498 can be promoted, and the invasiveness of human renal cancer cell line A498 can be decreased.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.11

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