【作者】 王玉兰；

【导师】 韦立新；

【作者基本信息】 中国人民解放军军医进修学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的分析肝细胞癌中miR-10b、RHOC及相关基因的表达,探讨这些基因参与肝细胞癌恶性生物学行为,及miR-10b可能参与调控RHOC转移浸润的机制。材料和方法选取2000年1月至2005年12月在我院行手术切除的肝细胞癌病例,共375例,获得随访信息185例。分析本组病例临床病理特征及与预后的关系。留取2007～2008年间手术切除的新鲜肝细胞癌及癌旁肝组织标本19例,western blot检测RHOC蛋白的表达,免疫组化检测cyclin D1、P21WAF1的表达,分析其相关性及与临床病理特征的关系;选取有随访资料的病例80例,免疫组化检测cyclin D1、P21WAF1、p53、ki-67及GPC3在肝细胞癌中的表达,分析各基因表达与临床病理特征的关系及各基因表达之间的相关性;Kaplan-Meier生存分析分析各基因表达的预后意义;COX风险模型分析各临床病理特征及基因表达的预后意义。提取新鲜肝细胞癌及癌旁肝组织中的RNA,实时定量PCR法检测miR-10b和RHOC mRNA基因的表达,分析二者相关性及与临床病理特征的关系。结果1.本组资料显示肝细胞癌发病年龄19～72岁,平均50.57±10.46岁,发病高峰年龄40～60岁,男女比例为10.7:1;HBsAg（+）87.4%,anti-HCV抗体（+）4.3%;术后3年生存率为52%,5年生存率为38%;肿瘤数目（p=0.000）、肿瘤大小（p=0.025）、组织学结构类型（p=0.000）、核分级（p=0.000）、分化程度（p=0.001）及血管浸润均（p=0.000）与预后明显相关;其中组织学结构类型中细梁状型生存时间与致密梁状型和腺样型无明显区别（p＞0.05）而明显高于粗梁状型、实性型和硬化型（p≤0.009）,硬化型生存时间与实性型之间无明显区（p＞0.05）而明显低于其余各型（p≤0.006）;核分级1级与2级生存时间无明显区别（p＞0.05）,核分级3级与4级生存时间无明显区别（p＞0.05）,而核分级3级生存时间明显低于2（p=0.000）;小细胞型、巨细胞型和梭形细胞型主要由实性型和粗梁状型组织学结构类型构成,明显不同于透明细胞型（主要由细梁状型和粗梁状型构成）（p≤1.006）。2.Western blot显示9/19例肝细胞癌中RHOC蛋白高表达,与血管浸润明显相关（p=0.020）。RHOC阳性表达与cyclin D1阳性表达明显正相关（P=0.010）;cyclin D1阳性表达与高核分级明显相关（p=0.041）;p21^WAF1及p53阳性表达均与分化程度（分别为p=0.032,p=0.031）和血管浸润（p=0.036,p=0.011）正相关;ki67阳性表达与分化程度（p=0.004）、核分级（p=0.045）和血管浸润（p=0.001）正相关;GPC3表达与肿瘤数目（p=0.038）和分化程度（p=0.013）正相关。生存分析显示cyclin D1及ki67有明显预后意义。ki67阳性表达与p53（p=0.000）及p21^WAF1（p=0.047）阳性表达有明显相关性,其他各基因之间表达无明显关系。3.COX回归模型分析显示肿瘤大小（p=0.042）、肿瘤数目（p=0.004）及血管浸润（p=0.000）为独立预后因素。4.与癌旁肝组织相比,miR-10b在肝细胞癌中的平均表达增高（p=0.049）,但miR-10b低表达组血管浸润明显高于高表达组（P=0.020）;与癌旁肝组织相比,RHOC mRNA在癌组织中的表达增高（P=0.032）,同时RHOCmRNA高表达组血管浸润明显高于低表达组（P=0.041）。miR-10b和RHOC mRNA的表达未发现有明显相关性。结论1.除肿瘤大小、肿瘤数量、分化程度、血管浸润以外,肝细胞癌组织学结构类型和核分级均有明显预后意义。2.miR-10b、RHOC、cyclin D1、ki67及GPC3均参了与肝细胞癌的生物学进程;RHOC可能参与了细胞周期调控。cyclin D1及ki67有明显预后意义。GPC3可作为肿瘤标记物。miR-10b与RHOC的表达无明显相关性。3.COX回归模型分析显示肿瘤大小、肿瘤数目及血管浸润为独立预后因素。更多还原

【Abstract】 Object To evaluate the invasion and metastasis caused by miR-10b,RHOC and related genes in hepatocellular carcionoma（HCC）.Materials and methods The clinical and pathological characteristics of 375 cases of HCC were analysised,which were collected from The general hospital of the People’s Liberation Army between 2000 and 2005.Of which 185 case’s follow up information had been obtained.And 19 fresh HCC specimens and corresponding pericarcinomatous liver tissues were obtained by surgical resection in The general hospital of the People’s Liberation Army between 2007 and 2008. Western blotting was used to investigate the expression of RHOC protein.80 cases with follow up information were examined by immunohistochemically staining to investigate the expression of cyclin D1、P21WAF1、p53、ki-67 and GPC3 genes.The relationship between the genes expression and clinicopathologic characteristics of HCC were evaluated,the clinicopathologic factors and the genes were analyzed by Kaplan-Meier and multivariate COX regression analysis. Of the 19 fresh HCC specimens and corresponding pericarcinomatous liver tissues, Real time quantitate PCR was performed to investigate the expression of miR-10b and RHOC mRNA.Results 1.These 375 patients had a median age of 50.57±10.46（range,19-72） with 87.4%HBsAg positive and 4.3%anti-HCV antibody positive;The apparent peak incidence age was 40～60 years old,and the ratio of male to female was 10.7:1;The 3 and 5 year postoperational survival rate were 52%and 38%;The tumour numbers（p=0.000）,tumor size（p=0.025）,histological pattern （p=0.000）,nuclear features（p=0.000）,differentiation（p=0.001） and vascular invasion（p=0.000） were significantly correlated with prognosis.The postoperational survival time of thin trabeculae pattern,compact pattern and pseudoglandular pattern were significantly longer than that of thick trabeculae, scirrhous pattern,and solid pattern（p＜0.009）;the postoperational survival time of 1 and 2 grade based on nuclear features were significantly longer than that of 3 and 4 grades（p=0.000）;The small cell variant,osteoclast-like giant cell variant, and spindle cell variant were mainly composed of thick trabeculae pattern and solid pattern,which were significantly different from that of clear cell variant.（p≤0.006）.2.RHOC protein expression was higher in HCC than pericarcinomatous liver tissues in 9/19 cases,and its positive expression was correlated with vien invasion（p=0.020）.RHOC expression was positively correlated with cyclin D1 （P=0.023）.Increased expression of cyclin D1 was significantly correlated with increasing nuclear grade（p=0.041）;High expression of p21^WAF1 and p53 were significantly correlated with poor tumor differentiation（p=0.032,p=0.031） and vascular invasion（p=0.036,p=0.011）;High expression of ki67 was significantly correlated with poor tumor differentiation（p=0.004）,increasing nuclear grade（p=0.045） and vascular invasion（p=0.001）.Expression of GPC3 was positively correlated with multi-tumour numbers（p=0.038） and poor tumor differentiation（p=0.013）.3.On COX regression analysis,tumour size（p=0.042）,tuomr numbers （p=0.004） and vascular invasion（p=0.000） were independent predictors of overall survival.4.miR-10b and RHOC mRNA expression levels in HCC were higher than that of in pericarcinomatous liver tissues,but lower expression of miR-10b was correlated with vien invasion（p=0.020）,contrastly,higher expression of RHOC mRNA was correlated with vien invasion（P=0.041）.But the significantly correlation was not found between the expression of miR-10b and RHOC mRNA（p＞0.05）.Conclusion 1.As well as tumor size,tumor numbers,tumor differentiation and vascular invasion of HCC,The histological pattern and nuclear features have prognostic significance. 2.miR-10b,RHOC,cyclin D1,ki67 and GPC3 associate with aggressive of HCC;RHOC may also regulate cell cycle;Cyclin D1 and ki67 have prognostic significance;GPC3 can be a tumour marker in HCC;The expression of miR-10b and RHOC mRNA has no significantly correlation.3.On COX regression analysis,tumour size,tuomur numbers and vascular invasion were independent predictors of overall survival.更多还原