【作者】 王飞；

【导师】 平季根；

【作者基本信息】 苏州大学 ， 泌尿外科学（专业学位）， 2016， 硕士

【摘要】 目的:研究Cullin1基因在肾癌中的表达情况及其在肾癌细胞增殖、迁移与侵袭中的作用,并深入研究Cullin1基因在肾癌发生发展过程中发挥上述作用的分子机制。方法:体外实验:首先利用组织微列阵技术及免疫组织化学法(IHC)检测Cullin1在307例肾癌组织与34正常肾组织中的表达情况,分析Cullin1(Cul1)表达与临床病理因素及肾癌患者生存预后的关系;然后用在体外化学合成的Cullin1 si RNA和Control si RNA转染人肾癌786-O及ACHN细胞,用Western Blot技术检测Cullin1si RNA干涉Cullin1蛋白表达的效果,用CCK-8及细胞周期流式分析检测Cullin1si RNA转染后肾癌细胞增殖情况的变化,用迁移及侵袭实验检测转染Cullin1 si RNA后肾癌细胞迁移与侵袭能力的变化,用Western Blot实验研究Cullin1 si RNA干涉肾癌786-O及ACHN细胞后,肾癌细胞中Cyclins、p21、p27、MMP-2、MMP-9、TIMP-1及TIMP-2蛋白表达的变化情况。体内实验:通过慢病毒重组技术将Cullin1 si RNA和Control si RNA序列整入慢病毒中,再用整合好的慢病毒转染786-O-Luc细胞,用嘌呤霉素筛选转染后的细胞一个月,之后选择BALB/c裸鼠,用PBS重悬786-O-Luc-Cullin1-sh RNA及786-O-Luc-Control-sh RNA细胞,细胞密度约为1×107个/200μL,每只老鼠皮下注射Control sh RNA及Cullin1 sh RNA细胞200μL,一月后用小动物活体成像系统检测皮下成瘤生物发光信号,观察其成瘤情况。结果:体外实验:1、免疫组织化学法(IHC)显示:Cullin1在肾癌组织的表达高于正常肾组织,差异有统计学意义(P<0.05),Cullin1的表达在T4期肾癌患者中明显高于T1-T3期患者,差异有统计学意义(P<0.05),Cullin1的表达情况与患者的疾病相关存活率及总生存率无关(P>0.05)。2、Western Blot实验显示:与Control si RNA组相比,转染Cullin1 si RNA后肾癌786-O和ACHN细胞中的Cullin1蛋白表达量下降;3、细胞增殖实验显示:转染Cullin1 si RNA的肾癌786-O和ACHN细胞增殖能力与Control si RNA组相比明显下降,差异有统计学意义(786-O组P<0.05,ACHN组P<0.05),流式分析显示在干涉Cullin1的表达后两种细胞G1期细胞数增多,S期减少;4、细胞迁移实验显示:Cullin1 si RNA转染肾癌786-O和ACHN细胞后,其穿过Transwell小室的细胞的数目与Control si RNA组相比分别下降了53%和79%,差异有统计学意义(786-O组P<0.05,ACHN组P<0.05);5、细胞侵袭实验显示:Cullin1si RNA转染肾癌786-O和ACHN细胞后,其穿过Transwell小室的细胞数目与Control si RNA组相比分别下降了95%和74%,差异有统计学意义(786-O组P<0.05,ACHN组P<0.05);6、Western Blot实验显示:肾癌786-O和ACHN细胞转染Cullin1 si RNA后,与对照组相比其p21、p27、TIMP-1蛋白表达量增多,Cyclin D1、Cyclin E2、MMP-9蛋白表达量下降,MMP-2及TIMP-2蛋白表达量无变化。体内实验:与Control sh RNA组相比,Cullin1 sh RNA组裸鼠皮下生物发光信号明显减弱,两组比较差异有统计学意义(P<0.05)。结论:Cullin1在肾癌组织中呈高表达,表明其可能是一个促癌基因,干涉Cullin1基因可以通过增加周期蛋白抑制p21及p27蛋白的表达来抑制周期蛋白Cyclin D1及Cyclin E2的表达从而抑制细胞增殖,此外干涉肾癌Cullin1基因可以通过增强基质金属蛋白酶抑制剂TIMP-1蛋白的表达来抑制MMP-9信号通路,从而降低肾癌细胞的迁移、侵袭能力。由实验结果推断,Cullin1可以作为肾癌患者预后监测指标,是一个潜在治疗靶点。更多还原

【Abstract】 Objective The purpose of our study is to evaluate the expression of Cullin1 in renal cell carcinoma and normal renal tissue and to detect the effects of Cullin1 gene on renal cancer cell proliferation, migration, invasion and the mechanisms underlying these effects.Methods In vitro: First,Immunohistochemistry was used to assess the expression of Cullin1 in 307 renal cancer tissues and 34 normal renal tissues, and its correlation with clinicopathological features and prognosis of renal cancer were also analyzed; Using Cullin1 small interfering RNA(si RNA) which was synthesized chemically in vitro to transfect 786-O and ACHN renal cancer cells and to investigate the effects of Cullin1 si RNA on Cullin1 expression in 786-O and ACHN cells by Western Blot. Flow cytometry was used to detect the cell cycle and CCK-8 assay was used to measure the proliferation abilities of 786-O and ACHN cells after Cullin1 knockdown. Cell migration assay was used to detect the migration abilities of 786-O and ACHN cells after Cullin1 knockdown. Cell invasion assay was used to detect the migration abilities of 786-O and ACHN cells after Cullin1 knockdown. After knockdown of Cullin1 gene in renal cancer 786-O and ACHN cells, we detect the p21,p27,Cyclin D1, Cyclin E2, MMP-2, MMP-9, TIMP-1 and TIMP-2 activities of the two renal cancer cells by Western Blot; 786-O-Luc was used to establish a xenograft model in immunodeficient BALB/c nude mice. Prior to implantation, The Cullin1 knockdown 786-O-Luc cell lines(Cullin1KD-786-O-Luc) and control 786-O-Luc cell lines(Control-786-O-Luc) were established by infecting with lentivirus packing Cullin1 sh RNA expression vector and control vector respectively. Target cells were infected with lentivirus for 48 hours then selected with puromycin for 4 weeks..In vitro:Female BALB/c nude mice(4 weeks of age) were used. Subconfluent 786-O-Luc-lenti-Ctrl and 786-O-Luc-lenti-sh Cullin1 cells were harvested and resuspended in matrigel which was dilution with RPMI 1640 medium in 1:1 ratio to a density of 5×107 cells/m L. 200μl cell suspension of 786-O-Luc-lenti-Control were injected subcutaneously into right flank of each animal, and 200μl cell suspension of 786-O-Luc-lenti-sh Cullin1 were injected subcutaneously into left flank of each animal. One month after injection, the pictures were captured using bioluminescence imaging with a Night OWL Imaging System.Results In vitro,(1) IHC showed that the expression of Cullin1 was increased significantly in RCC tissue compared with NRT(P<0.05) and it was correlated with renal carcinoma p T status(P<0.05),Cullin1 expression has no correlation with the survival of patients.(2) Western Blot showed that the Cullin1 expression is decreased after Cullin1 knockdown in both 786-O and ACHN renal carcinoma cells.(3) The CCK-8 assay shows that Cullin1 interference could drastically decreased the ability of cell proliferation in both renal cancer cells and arrest cell cycle at G1 phase after Cullin1 knockdown.(4) In cell migration assay, our data revealed that Cullin1 knockdown decreased cell migration ability of 786-O and ACHN by 53% and 79% when compared with the corresponding control. In cell invasion assay, cell invasion ability of 786-O and ACHN decreased95% and 44%.(5)Western Blot analysis showed that the expression of Cyclin D1、Cyclin E2、MMP-9 is significantly inhibited while p21、p27 and TIMP-1 is increased after the silencing of Cullin1 in 786-O and ACHN cells and no significant changes in MMP-2 and TIMP-2 protein expression were found.In vivo:Thirty days after implantation, in vivo imaging analysis of the mice revealed that the growth of tumors was significantly inhibited by decreased expression of Cullin1.Conclusion We founded that Cullin1 staining was increased significantly in RCC tissue compared with NRT. Knockdown of Cullin1 leads to inhibition of renal cancer cell proliferation in both vivo and vitro and arrested cell cycle at G1 phase by upregulating p21 and p27 levels which could down regulating Cyclin D1 and Cyclin E2 protein levels. Knockdown of Cullin1 inhibits the RCC cells migration and invasion abilities by up-regulating the expression of TIMP-1 that inhibit the expression of the MMP-9 which caused the ability of renal cancer cells for degrading components of ECM and basement membrane was decreased.更多还原