【作者】 杨开焰；

【导师】 陈道瑾；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 结直肠癌是最常见的恶性肿瘤之一,也是全球癌症相关性的发病率和死亡率的一个重要原因。手术切除是首选治疗方式。高达50%的患者由于初诊时即存在隐性转移灶最终死于肿瘤的复发。转移是恶性肿瘤的基本特点,其分子机制至今尚未明了。肿瘤发生和发展是多基因改变协同作用的过程,癌基因的激活与抑癌基因的失活是肿瘤发生发展的分子基础。Rho小GTP酶家族属Ras超家族成员,通过激活下游的信号传导通路,参与调节细胞的多种生命过程,如细胞增殖、细胞运动和细胞凋亡等。RhoA是Rho GTP酶家族的重要成员,近年来研究发现在多种恶性肿瘤中高表达,并和肿瘤的发生、侵袭和转移密切相关。然而,RhoA在结直肠癌的发生发展中所起的作用还不是很清楚。国内外尚未见RhoA的shRNA结直肠癌细胞系统的实验研究。因此,我们构建针对RhoA的shRNA并转染至人结肠癌细胞株LoVo,特异性敲除其内源性RhoA的表达,分析结直肠癌侵袭和转移RhoA的作用,为探索结直肠癌基因治疗提供一定的理论基础。实验分如下四部分进行：目的：为研究靶向RhoA基因的shRNA对人结直肠癌细胞株体外生物学行为的影响,根据基因库中的人RhoA基因序列,构建针对RhoA基因的小干扰RNA (siRNA)及其表达载体。方法：设计RhoA靶向的发夹状siRNA, BLAST同源分析证实其特异性,合成2条互补的寡核苷酸链共2对,退火后连接入pGPU6/GFP/Neo载体,转化后进行酶切和序列鉴定。结果：设计出2条针对RhoA的siRNA,将退火后的双链寡核苷酸片段克隆到pGPU6/GFP/Neo载体,经过阳性菌落酶切鉴定与测序,结果正确。结论：成功构建了包含靶向RhoA基因的shRNA的重组表达质粒.目的：研究将RhoA shRNA转染入人结肠癌细胞株LoVo观察其对细胞RhoA表达的沉默效应。方法：设计制备2对针对RhoA基因的shRNA,转染结肠癌细胞株LoVo,荧光显微镜观察绿色荧光蛋白的表达；采用real-time RT-PCR法测定RhoA mRNA的表达,Western Blot测定RhoA蛋白的表达。结果：与未转染的LoVo比较,LoVo-P1细胞RhoA mRNA与RhoA蛋白的表达强度分别为56.33%±2.18%,33.22%±2.27%, Western blot结果与real-time RT-PCR结果一致,和对照组比较有显著性差异(P<0.05)。结论：构建的RhoA shRNA能有效抑制LoVo细胞中RhoA基因的表达；其中RhoA shRNA1干扰效果最佳。目的：研究RhoAshRNA对结直肠癌细胞株LoVo体外增殖、凋亡、迁移侵袭能力的影响,并初步探讨其可能的机理。方法：以成功构建的稳定表达shRNA的LoVo-P1为研究对象,以未转染的LoVo细胞、LoVo-P0为对照,绘制三组细胞的生长曲线比较生长差异,计算细胞倍增时间；MTT法比较三组细胞存活率的差异；细胞粘附实验、划痕实验、Transwell小室检测三组细胞体外侵袭力的变化。结果：未转染的LoVo细胞、LoVo-P0组细胞的各项指标比较无明显差异。与对照两组的细胞比较,LoVo-P1组的细胞生长明显缓慢,从第四天开始生长速度明显低于另外两组,至对数生长期更明显,生长曲线较平缓；LoVo-P1组的细胞的倍增时间31.24小时左右,延长约10小时。LoVo-P1组的细胞存活率明显下降(P<0.05)。LoVo-P1组的细胞的粘附力明显下降(P<0.05)。LoVo-P1迁移到划痕区的细胞数明显低于对照组(P<0.05)。LoVo-P1穿过Matrigel滤膜的细胞数明显低于对照组(P<0.05)。结论：RhoA干扰质粒P1稳定转染可抑制结肠癌细胞株LoVo的体外生长,使细胞倍增时间延长,抑制癌细胞的体外粘附、迁移侵袭能力。目的：建立裸鼠皮下人结直肠癌种植瘤模型,研究RhoA基因沉默对结肠癌细胞株LoVo裸鼠成瘤的影响。方法：实验细胞分为3组,即稳定转染RhoA shRNA的LoVo-P1组、未转染的LoVo细胞组和转染空载体的LoVo-PO组。将各组结肠癌细胞直接接种于裸鼠皮下,观察种植瘤的生长情况,4周后测量瘤体组织的体积和重量；免疫组化检测肿瘤组织RhoA、VEGF、CD34(观测微血管密度的变化)和MMP-2蛋白表达。结果:：LoVo-P1组的肿瘤生长速度慢于其他两组(P<0.05)。实验结束时瘤体组织的体积与瘤重明显低于其他两组(P<0.05)。免疫组化证实LoVo-P1组的肿瘤组织RhoA. VEGF和MMP-2蛋白表达降低(P<0.05),微血管密度降低(P<0.05)。结论：沉默RhoA可以抑制结肠癌细胞移植瘤的生长。更多还原

【Abstract】 Colorectal cancer is one of the most common malignancies and one important cause of cancer-related morbidity and mortality worldwide. Primary treatment is surgical. However, disease relapse because of undetected metastatic disease occurs in up to 50% of patients. Metastasis formation is an essential aspect of cancer, for which the molecular underpinning has long been subject to debate.Multiple genetic aberrations are required for tumor initiation and progression. Gain-of-function mutations of proto-oncogenes or loss-of-function mutations of tumor suppressor genes play an important role in the invasion and metastasis. Metastasis genes encode homing receptors, their ligands,and extracellular matrix-degrading proteinases, which jointly cause invasion.The Rho small GTPases, a subgroup of the Ras superfamily, Rho GTPases play a major role in a variety of cellular signalling processes involved in cell proliferation, differentiation, cell mobility, apoptosis and membrane trafficking .As one of the Rho small GTPases, GTP-bound active RhoA specifically induces the formation of focal adhesions and the assembly of stress fibers. RhoA was found to be increased in some human cancers and played an important role in the invasion and metastasis of the tumor.But what kind of role RhoA playing was not yet very clear in the development of colorectal cancer. Experimental study on siRNA of RhoA transfecting colorectal cancer cell lines was not yet reported at home and abroad. The first time in the world,we transfected shRNA to human colon cancer cell line LoVo to specifically knockdown endogenous RhoA expression, to analyze the functional role of RhoA in colorectal cancer invasion and metastasis, then to explore a new way of gene therapy in colorectal cancer. The experiment are to be excuted by four sections as follows:Objective:According to RhoA gene sequence of GenBank, to construct small interfering RNA (siRNA) targeting RhoA and its expression vector.Methods:siRNA targeting RhoA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector,which was then restrictive enzyme digested and sequenced.Results:Two pairs of siRNA targeting RhoA were designed,then were inserted into pGPU6/GFP/Neo vector after annealing. Restrictive enzyme digestion and sequencingconfirmed the vector containing siRNA was what we wanted.Conclusion:The siRNA targeting RhoA and its vector are successfully constructed.Objective:To investigate the inhibitory effect of shRNA targeting RhoA on RhoA gene expression in Human colonic cancer cell Line LoVo transfected by the vector.Methods:The RhoA shRNA was transfected into LoVo cells by the liposome complexmethod and then fluo-rescence photographs were taken。RhoA gene expression was assessed by real-time RT-PCR, and RhoA protein expressionwas tested by Western blot.Results:The expression GFP was successfully observed under fluorescent microscope in Human colonic cancer cell Line LoVo transfected. Compared with untransfected LoVo cells,the expression rates of RhoA mRNA and the expression rates of RhoA protein were(47.66 %),(36.93%),respectively in LoVo.RhoA shRNA-transfected cells,significantly lower than those of control cells (P<0.05).Conclusion:The expression of RhoA gene could be effectively reduced by shRNA in LoVo cells. The RhoA shRNA targeting human RhoA is considered to be possible tool against colorectal cancer. Objective:To study the effect of knockdown of RhoA expression by recombinant plasmid pGPU6/GFP/Neo-RhoA shRNA on the characteristics of human colonic carcinoma cell line LoVo in vitro.Methods:The positive transfected LoVo cell clones were screened with G418. The cell growth curve was obtained by cell counter and the cell doubling time was computed. The cell survival rate was measured by MTT assay.The cell motility and invasion ability in vitro were obtained by cell adhere assay、scratch assay and Transwell experiment.Results:There were no significant difference between untransfected LoVo cells and the LoVo cells transfected with PO in all indicators. Compared with the cells in other two groups, the LoVo cells transfected with P1 grew significantly more slowly (P<0.05), having significantly lower survival rate (P<0.05)、also significantly lower in cell adhesion rate、cell motility and cell invasion ability (P<0.05).Conclusion:RhoA shRNA can inhibit the growth、adhesiveness and invasiveness of LoVo cells through intervening the expression of RhoA.Objective:To investigate the effect of silencing RhoA gene with RNA interference (RNAi) technique on colonic cancer LoVo cell proliferation in vivo.Methods:short hairpin RNA (shRNA) eukaryotic expression vector against RhoA gene was constructed, named as plasmid pGPU6/GFP/ Neo-RhoA, and transfected into LoVo cells. Then, the stable transfected and untransfected LoVo cells were injected subcutaneously into nude mice. During a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of RhoA、VEGF、CD34 (to observe changes of micro vessel density) and MMP-2 protein protein was observed by immunohistochemical technique.Results:Inhibition of tumor growth was demounstrated after treatment with RhoA shRNA. The volume and weight of tumors were significantly decreased in comparison with those of other control groups(P<0.05). Compared with those of other control groups, The expression of RhoA、VEGF、MVD and MMP-2 in LoVo-P1 group was showed significant decrease.Conclusions:Our results suggest that RhoA gene can act as a crucial therapeutic target for slowing colon cancer growth.更多还原