AFB1诱发大鼠肝癌形成过程中差异表达基因筛选及Cullin7的功能研究
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摘要
肝细胞癌(hepatocellular carcinoma,HCC,以下简称肝癌)是最常见的恶性肿瘤之一,是一种早期诊断困难、高度恶性、预后凶险的肿瘤。肝癌的形成是一个多因素、多步骤、多基因参与的过程,传统的单因素、单基因相关性分析难以全面准确反映肝癌复杂的病理机制,近年来随着功能基因组学及蛋白质组学的迅猛发展,不断地推动了肝癌发生机制、诊断和治疗等方面的研究进展。
本研究用AFB1(Aflatoxin B1,AFB1)诱发大鼠肝癌形成,以γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,γ-GT)染色阳性的肝细胞增生灶(γ-GT阳性灶)为标志获取肝癌前病变的组织标本,运用基因芯片技术对大鼠肝癌组织、肝癌前病变组织和正常肝组织的全基因组表达谱进行差异分析,筛选出一批差异基因。本研究对筛选出来的差异表达基因CUL7(cullin7)进行表达水平改变的验证,再应用RNA干扰(RNA interfering,RNAi)技术对CUL7进行肿瘤相关生物学功能分析和机制探讨,以及应用实时定量PCR和免疫组化技术观察和分析CUL7在较大样本量的人肝癌组织中的表达及其临床意义。研究结果表明,CUL7在肝癌的发生发展过程中可能影响细胞的增殖、凋亡能力和细胞周期的调控,它编码的蛋白是E3泛素-蛋白连接酶复合物的组件,参与泛素依赖的蛋白质降解,CUL7在肝癌组织中高表达。这些结果提示CUL7可能是个潜在的肝癌诊断和预后评估的分子标志物。
研究分以下四部分进行。
第一部分AFB1诱发大鼠肝癌发生过程中差异表达基因研究
目的:通过建立AFB1诱发大鼠肝癌实验模型,应用基因芯片技术观察大鼠肝癌发生发展不同阶段的肝组织中基因表达谱的改变,从中筛选出在肝癌发生过程中起重要作用的基因。
方法:4周龄的雄性近交系Wistar大鼠随机分为实验组和对照组,实验组动物经腹腔注射AFB1诱发肝癌,对照组不予AFB1处理。分别于实验第14W、28W、42W、55W对全部动物进行肝活检,第64W处死全部动物。通过γ-GT组织化学染色并结合HE染色识别和获取肝癌前病变组织、肝癌组织和正常对照肝组织。三种组织标本各取3例,分别提取肝组织总RNA,然后按组织类别将3份总RNA等量混合,获得癌前、肝癌和正常三种组织RNA。经基因芯片技术筛选出癌前、肝癌和正常三种组织差异性表达基因。最后通过Gene Ontology、KEGG和NCBI等数据库分析差异表达基因分子功能、生物过程、细胞组分以及生物学通路。
结果:动物实验于64周结束,结果显示实验组共有19只动物发生肝癌,肝癌发生的最早时间为实验第51周;对照组无1例发生肝癌。γ-GT组织化学染色显示随着AFB1诱癌时间延长,γ-GT阳性灶数量增多、面积增大,癌前组织中γ-GT阳性灶总面积与肝组织切片面积比达35%～47%;肝癌组织γ-GT染色全为阳性;正常肝组织内无γ-GT阳性灶。应用基因芯片技术筛选出组间表达水平差异基因(以表达差异≥2.0或≤0.5倍为限),其中肝癌组织与正常组织相比,有3753个上调表达基因,有3042个下调表达基因;肝癌组织与癌前组织相比,有4885个上调表达基因,有3477个下调表达基因,癌前组织与正常组织相比,有1027个上调表达基因,有1117个下调表达基因。在正常肝组织、癌前病变肝组织、肝癌组织表达依次上调的基因30个,在正常肝组织、癌前病变肝组织、肝癌组织表达依次下调的基因85个。
经生物信息学分析,这些差异表达基因与细胞的增生、分化,细胞周期,细胞凋亡和细胞的信号传导密切相关。结论:通过γ-GT组织化学染色和HE染色相结合的方法有助于识别和获取大鼠肝癌前病变组织、肝癌组织及正常肝组织;大鼠肝癌发生发展不同阶段的基因表达谱存在明显差别;肝癌的发生发展可能与多种基因表达水平的改变有关。
第二部分差异表达基因CUL7在大鼠和人肝癌组织中的表达验证
目的:对筛选出来的感兴趣基因CUL7进行差异表达的验证。
方法:应用RT-PCR和Western blot方法检测CUL7在大鼠和人肝癌组织中的mRNA和蛋白质水平水平的表达情况。
结果:①CUL7 mRNA表达水平和蛋白表达水平在大鼠正常肝、肝癌前病变、肝癌组织中依次上调;②CUL7蛋白表达水平在人正常肝、癌旁肝及肝癌组织中也依次上调。
结论: CUL7表达变化趋势与基因芯片筛选结果基本一致,在mRNA和蛋白质两个水平上,CUL7不仅在大鼠正常肝、肝癌前病变、肝癌组织中表达依次上调,并且在人正常肝、肝癌旁组织、肝癌组织中的表达水平也显著依次升高,提示CUL7可能参与人类肝癌的发生发展过程。
第三部分肝癌差异表达基因CUL7的功能研究
目的:为了进一步探讨CUL7在肝癌发生发展中的作用,本部分应用RNAi技术和肿瘤相关生物学功能鉴定方法,研究CUL7表达沉默对人肝癌细胞系SMMC-7721相关功能的影响。
方法:将化学合成的CUL7小干扰RNA( siRNA)瞬时转染SMMC-7721细胞后,用Real-Time PCR和Western blot检测证实SMMC-7721细胞中CUL7的mRNA和蛋白表达均明显受到抑制。设置CUL7 siRNA转染组(RNAi组)、转染无效用dsRNA组(阴性对照组,Mock)和未转染组(空白对照组,Con),观察三组细胞的凋亡、生长、粘附、运动和侵袭等肿瘤相关生物学功能的变化。
结果:流式细胞仪分析结果显示,凋亡细胞比例在RNAi组、Mock组、Con组分别为67.45±5.83%、13.74±4.25%、16.69±3.18%。MTT实验结果显示,RNAi组的OD490值在24h、48h、72h分别为0.941±0.04 , 0.924±0.13, 0.783±0.12。体外运动试验结果显示,穿过微孔滤膜到达下室面的细胞数在RNAi组、Mock组和Con组分别为26.35±1.55、68.33±3.55和65.77±3.18个, RNAi组与其余两组比较有显著差异(p<0.05)。体外侵袭试验结果显示,穿过Matrigel和微孔滤膜到达下室面的细胞数在RNAi组、Mock组和Con组分别为14.38±2.37、45.38±4.16、48.67±2.51个, RNAi组与其余两组比较有显著差异(p<0.05)。
结论: CUL7可能通过促进细胞增殖、抑制细胞凋亡,从而导致肝细胞的恶性转化。
第四部分CUL7在较大样本人肝癌组织中的表达及临床意义的研究
目的:分析人肝癌组织中CUL7的表达与患者的临床病理特征的关系。
方法:利用Real-Time PCR和免疫组化技术检测CUL7 mRNA和蛋白水平在62例人肝癌及其相应癌旁组织、12例正常人肝组织中的表达情况。
结果: CUL7 mRNA和CUL7蛋白表达水平在人肝癌中的表达明显高于癌旁及正常肝组织(均P <0.05),差异有统计学意义;而在正常组织的表达,与癌旁相比差别无统计学意义(P >0.05)。CUL7在肝癌组织的表达水平与该组织的Edmondson病理分级、复发和远处转移相关,(p<0.05)。
结论:CUL7 mRNA和CUL7蛋白表达水平在人肝癌中的表达明显高于癌旁及正常肝组织,差异有统计学意义;而在正常肝组织的表达,与癌旁相比差别无统计学意义,提示CUL7在肝癌的诊断和预后评估等方面具有潜在的临床应用价值。
Hepatocellular carcinoma (HCC) is one of the most common malignancies with difficulty in early diagnosis and extremely poor prognosis. As the formation of HCC is a multi-factor, multi-step and multi-gene process, the study on HCC with the methods that only focus on single factor or single gene can not accurately reflect the complexity of its pathological mechanism.In recent years, with the rapid development of the functional genomics and the proteomics,the research of HCC in pathogenesis , diagnosis and treatment have been greatly promoted.
This study applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1). The preneoplastic tissue was distinguished byγ-glutamyl transpeptidase (γ-GT) staining which marks the foci of liver cell proliferation positive. Among the candidates, the changes of Cullin7 expression were confirmed by Western blot and RT-PCR. The biological functions and mechanism of Cullin7 were further studied with RNA interference (RNAi) technique. Its expression in a larger scale of human HCC samples and the clinical significance were studied by RT-PCR and immunohistochemical techniques.The results indicated that Cullin7 probably affects cell proliferation and apoptosis by regulating the cell cycle, and thereby plays a role in the occurrence and development of HCC.Cullin7 codes the component of the E3 sumo-protein ligase,which involves in sumo depended protein degradation.The up-expression of Cullin7 in HCC indicates that Cullin7 could be a molecular marker for the early diagnosis and the prognosis analysis of HCC.
The entire study includes three parts.
Part One Study on differentially expressed genes in the rat hepatocarcinogenesis induced by AFB1
Objective: Part one applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1).
Methods: Male Wistar rats, 4 weeks old, were divided into AFB1 group and control group. Rats in AFB1 group were injected AFB1 in order to induce HCC, and the rats in control group were raised normally. The animal experiment lasted 64 weeks, during which all the animals from each group were biopsied periodically for collecting liver tissues samples(14W、28W、42W、55W、64W). The samples were stained withγ-GT and HE to distinguish the normal, preneoplastic and HCC tissue.3 samples were picked randomly from each kind of above tissues for extracting the total RNA.Gene array technology was applied to screen the differenetially expressed genes.The molecular function,biological process,cell component,and the biological pathways were analyzed by the Gene Ontology,KEGG and NCBI database.
Results: The animal experiment was end in 64 weeks. A total of 19 rats in AFB1 group developed HCC until the end of animal experiment. The earliest HCC occurred at the 51st week in the animals in AFB1 group, while none in control group. With the extension of time by AFB1-induced HCC, the number and size ofγ-GT foci in the liver of animals in AFB1 group increased, and the totalγ-GT positive area reached 29%～43% of the whole liver tissue area. HCC tissues wereγ-GT positive entirely. There was noγ-GT focus in control group.The results of gene array (limit differenece times≥2.0 or≤0.5)showed that 3753 genes expressed upwards while 3042 genes downwards in the HCC and normal group, 4885 genes expressed upwards while 3447 genes downwards in the HCC and preneoplastic group, 1027 genes expressed upwards while 1117 genes downwards in the normal and preneoplastic group. Though the bioinformatics, the conclusion suggested that such genes were relate to the cell proliferation and apoptosis or the signal pathway.
Conclusion: The results showed the methods thatγ-GT staining combined with HE were able to distinguish the normal, preneoplastic and HCC tissue.The significant difference of gene expression existed in the developing stage of HCC. The happening and development of HCC may be related to the change of gene expression.
Part two Validation on differential expression of Cullin7 in rat and human HCC
Objective: To verify the candidate Cullin7 expression in rat and human HCC.
Methods: The changes of Cullin7 expression were confirmed by Western blot and RT-PCR.
Results:①mRNA and protein expression of Cullin7 increased in the rat normal, preneoplastic and HCC tissue.②Cullin7 protein expression also turned up in the human normal, preneoplastic and HCC tissue. Conclusion: Above results from mRNA and protein levels were consistent with the gene microarray, which indicates Cullin7 may be involved in the development and progression of human HCC.
Part three Biological function and mechanism study of Cullin7 in hepatocarcinogenesis
Objective: To explore the role of Cullin7 in hepatocarcinogenesis, further study on its biological function and mechanism was carried on by RNAi technique on the HCC cell line of SMMC-7721.
Methods: Chemically synthesized small interfering RNA (siRNA) targeted on Cullin7 were transfected into SMMC-7721 cells. mRNA and protein levels of the Cullin7 in HCC cell lines SMMC-7721 were examinated by RT- PCR and Western blot.Establishing the RNAi,negtive and control group,the function and mechanism study were designed to analyze the change of apoptosis, proliferation, adhesion, and invasion of the above three cell groups.
Results: Flow cytometry analysis showed that the proportion of apoptotic cells in the RNAi , Mock and Con group were 67.45±5.83%, 13.74±4.25%, 16.69±3.18% respectively. MTT results showed that the value of OD490 RNAi group in 24h, 48h, and 72h were 0.941±0.04, 0.924±0.13, 0.783±0.12 respectively. Movement test in vitro shows that the porous cells in the RNAi, negtive and control group were 26.35±1.55, 68.33±3.55 and 65.77±3.18. Cell invasion assay showed the average invading cell numbers perfield in the group RNAi, mock and control were 14.38±2.37、45.38±4.16、and 48.67±2.51 respectively. There was siginificant difference between RNAi and the other two groups.
Conclusion: By promoting cell proliferation, inhibiting cell apoptosis, Cullin7 may be lead to the transformation from normal cells to the malignant.
Part four The expression and clinical significance of Cullin7 in a larger scale of human HCC samples
Objective: To analyze the clinical relationship between Cullin7 expression and human hepatocellular carcinoma .
Methods: To explore the clinical significance of Cullin7 expression in HCC and evaluate the potential value of Cullin7 as a new molecular marker for diagnosis early HCC, the Cullin7 protein expression level was examinated in 62 cases of human HCC and the adjacent liver tissues, as well as in 12 cases of normal human liver tissue, by Real-Time PCR and immunohistochemical techniques.
Results: The results showed the expression level of Cullin7 was up-regulated in all kinds of tissues. There was siginificant difference between human HCC and the adjacent liver tissues (p<0.05), and no siginificant difference between the adjacent liver tissues and normal liver tissues. The Cullin7 level had significant correlation with Edmondson stage, recurrence and metastasisof the HCC cases.(p<0.05).
Conclusions: Cullin7 expression turned up in the HCC tissue,which indicates that Cullin7 could be a molecular marker for the diagnosis and the prognosis analysis of HCC.
本研究用AFB1(Aflatoxin B1,AFB1)诱发大鼠肝癌形成,以γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,γ-GT)染色阳性的肝细胞增生灶(γ-GT阳性灶)为标志获取肝癌前病变的组织标本,运用基因芯片技术对大鼠肝癌组织、肝癌前病变组织和正常肝组织的全基因组表达谱进行差异分析,筛选出一批差异基因。本研究对筛选出来的差异表达基因CUL7(cullin7)进行表达水平改变的验证,再应用RNA干扰(RNA interfering,RNAi)技术对CUL7进行肿瘤相关生物学功能分析和机制探讨,以及应用实时定量PCR和免疫组化技术观察和分析CUL7在较大样本量的人肝癌组织中的表达及其临床意义。研究结果表明,CUL7在肝癌的发生发展过程中可能影响细胞的增殖、凋亡能力和细胞周期的调控,它编码的蛋白是E3泛素-蛋白连接酶复合物的组件,参与泛素依赖的蛋白质降解,CUL7在肝癌组织中高表达。这些结果提示CUL7可能是个潜在的肝癌诊断和预后评估的分子标志物。
研究分以下四部分进行。
第一部分AFB1诱发大鼠肝癌发生过程中差异表达基因研究
目的:通过建立AFB1诱发大鼠肝癌实验模型,应用基因芯片技术观察大鼠肝癌发生发展不同阶段的肝组织中基因表达谱的改变,从中筛选出在肝癌发生过程中起重要作用的基因。
方法:4周龄的雄性近交系Wistar大鼠随机分为实验组和对照组,实验组动物经腹腔注射AFB1诱发肝癌,对照组不予AFB1处理。分别于实验第14W、28W、42W、55W对全部动物进行肝活检,第64W处死全部动物。通过γ-GT组织化学染色并结合HE染色识别和获取肝癌前病变组织、肝癌组织和正常对照肝组织。三种组织标本各取3例,分别提取肝组织总RNA,然后按组织类别将3份总RNA等量混合,获得癌前、肝癌和正常三种组织RNA。经基因芯片技术筛选出癌前、肝癌和正常三种组织差异性表达基因。最后通过Gene Ontology、KEGG和NCBI等数据库分析差异表达基因分子功能、生物过程、细胞组分以及生物学通路。
结果:动物实验于64周结束,结果显示实验组共有19只动物发生肝癌,肝癌发生的最早时间为实验第51周;对照组无1例发生肝癌。γ-GT组织化学染色显示随着AFB1诱癌时间延长,γ-GT阳性灶数量增多、面积增大,癌前组织中γ-GT阳性灶总面积与肝组织切片面积比达35%～47%;肝癌组织γ-GT染色全为阳性;正常肝组织内无γ-GT阳性灶。应用基因芯片技术筛选出组间表达水平差异基因(以表达差异≥2.0或≤0.5倍为限),其中肝癌组织与正常组织相比,有3753个上调表达基因,有3042个下调表达基因;肝癌组织与癌前组织相比,有4885个上调表达基因,有3477个下调表达基因,癌前组织与正常组织相比,有1027个上调表达基因,有1117个下调表达基因。在正常肝组织、癌前病变肝组织、肝癌组织表达依次上调的基因30个,在正常肝组织、癌前病变肝组织、肝癌组织表达依次下调的基因85个。
经生物信息学分析,这些差异表达基因与细胞的增生、分化,细胞周期,细胞凋亡和细胞的信号传导密切相关。结论:通过γ-GT组织化学染色和HE染色相结合的方法有助于识别和获取大鼠肝癌前病变组织、肝癌组织及正常肝组织;大鼠肝癌发生发展不同阶段的基因表达谱存在明显差别;肝癌的发生发展可能与多种基因表达水平的改变有关。
第二部分差异表达基因CUL7在大鼠和人肝癌组织中的表达验证
目的:对筛选出来的感兴趣基因CUL7进行差异表达的验证。
方法:应用RT-PCR和Western blot方法检测CUL7在大鼠和人肝癌组织中的mRNA和蛋白质水平水平的表达情况。
结果:①CUL7 mRNA表达水平和蛋白表达水平在大鼠正常肝、肝癌前病变、肝癌组织中依次上调;②CUL7蛋白表达水平在人正常肝、癌旁肝及肝癌组织中也依次上调。
结论: CUL7表达变化趋势与基因芯片筛选结果基本一致,在mRNA和蛋白质两个水平上,CUL7不仅在大鼠正常肝、肝癌前病变、肝癌组织中表达依次上调,并且在人正常肝、肝癌旁组织、肝癌组织中的表达水平也显著依次升高,提示CUL7可能参与人类肝癌的发生发展过程。
第三部分肝癌差异表达基因CUL7的功能研究
目的:为了进一步探讨CUL7在肝癌发生发展中的作用,本部分应用RNAi技术和肿瘤相关生物学功能鉴定方法,研究CUL7表达沉默对人肝癌细胞系SMMC-7721相关功能的影响。
方法:将化学合成的CUL7小干扰RNA( siRNA)瞬时转染SMMC-7721细胞后,用Real-Time PCR和Western blot检测证实SMMC-7721细胞中CUL7的mRNA和蛋白表达均明显受到抑制。设置CUL7 siRNA转染组(RNAi组)、转染无效用dsRNA组(阴性对照组,Mock)和未转染组(空白对照组,Con),观察三组细胞的凋亡、生长、粘附、运动和侵袭等肿瘤相关生物学功能的变化。
结果:流式细胞仪分析结果显示,凋亡细胞比例在RNAi组、Mock组、Con组分别为67.45±5.83%、13.74±4.25%、16.69±3.18%。MTT实验结果显示,RNAi组的OD490值在24h、48h、72h分别为0.941±0.04 , 0.924±0.13, 0.783±0.12。体外运动试验结果显示,穿过微孔滤膜到达下室面的细胞数在RNAi组、Mock组和Con组分别为26.35±1.55、68.33±3.55和65.77±3.18个, RNAi组与其余两组比较有显著差异(p<0.05)。体外侵袭试验结果显示,穿过Matrigel和微孔滤膜到达下室面的细胞数在RNAi组、Mock组和Con组分别为14.38±2.37、45.38±4.16、48.67±2.51个, RNAi组与其余两组比较有显著差异(p<0.05)。
结论: CUL7可能通过促进细胞增殖、抑制细胞凋亡,从而导致肝细胞的恶性转化。
第四部分CUL7在较大样本人肝癌组织中的表达及临床意义的研究
目的:分析人肝癌组织中CUL7的表达与患者的临床病理特征的关系。
方法:利用Real-Time PCR和免疫组化技术检测CUL7 mRNA和蛋白水平在62例人肝癌及其相应癌旁组织、12例正常人肝组织中的表达情况。
结果: CUL7 mRNA和CUL7蛋白表达水平在人肝癌中的表达明显高于癌旁及正常肝组织(均P <0.05),差异有统计学意义;而在正常组织的表达,与癌旁相比差别无统计学意义(P >0.05)。CUL7在肝癌组织的表达水平与该组织的Edmondson病理分级、复发和远处转移相关,(p<0.05)。
结论:CUL7 mRNA和CUL7蛋白表达水平在人肝癌中的表达明显高于癌旁及正常肝组织,差异有统计学意义;而在正常肝组织的表达,与癌旁相比差别无统计学意义,提示CUL7在肝癌的诊断和预后评估等方面具有潜在的临床应用价值。
Hepatocellular carcinoma (HCC) is one of the most common malignancies with difficulty in early diagnosis and extremely poor prognosis. As the formation of HCC is a multi-factor, multi-step and multi-gene process, the study on HCC with the methods that only focus on single factor or single gene can not accurately reflect the complexity of its pathological mechanism.In recent years, with the rapid development of the functional genomics and the proteomics,the research of HCC in pathogenesis , diagnosis and treatment have been greatly promoted.
This study applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1). The preneoplastic tissue was distinguished byγ-glutamyl transpeptidase (γ-GT) staining which marks the foci of liver cell proliferation positive. Among the candidates, the changes of Cullin7 expression were confirmed by Western blot and RT-PCR. The biological functions and mechanism of Cullin7 were further studied with RNA interference (RNAi) technique. Its expression in a larger scale of human HCC samples and the clinical significance were studied by RT-PCR and immunohistochemical techniques.The results indicated that Cullin7 probably affects cell proliferation and apoptosis by regulating the cell cycle, and thereby plays a role in the occurrence and development of HCC.Cullin7 codes the component of the E3 sumo-protein ligase,which involves in sumo depended protein degradation.The up-expression of Cullin7 in HCC indicates that Cullin7 could be a molecular marker for the early diagnosis and the prognosis analysis of HCC.
The entire study includes three parts.
Part One Study on differentially expressed genes in the rat hepatocarcinogenesis induced by AFB1
Objective: Part one applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1).
Methods: Male Wistar rats, 4 weeks old, were divided into AFB1 group and control group. Rats in AFB1 group were injected AFB1 in order to induce HCC, and the rats in control group were raised normally. The animal experiment lasted 64 weeks, during which all the animals from each group were biopsied periodically for collecting liver tissues samples(14W、28W、42W、55W、64W). The samples were stained withγ-GT and HE to distinguish the normal, preneoplastic and HCC tissue.3 samples were picked randomly from each kind of above tissues for extracting the total RNA.Gene array technology was applied to screen the differenetially expressed genes.The molecular function,biological process,cell component,and the biological pathways were analyzed by the Gene Ontology,KEGG and NCBI database.
Results: The animal experiment was end in 64 weeks. A total of 19 rats in AFB1 group developed HCC until the end of animal experiment. The earliest HCC occurred at the 51st week in the animals in AFB1 group, while none in control group. With the extension of time by AFB1-induced HCC, the number and size ofγ-GT foci in the liver of animals in AFB1 group increased, and the totalγ-GT positive area reached 29%～43% of the whole liver tissue area. HCC tissues wereγ-GT positive entirely. There was noγ-GT focus in control group.The results of gene array (limit differenece times≥2.0 or≤0.5)showed that 3753 genes expressed upwards while 3042 genes downwards in the HCC and normal group, 4885 genes expressed upwards while 3447 genes downwards in the HCC and preneoplastic group, 1027 genes expressed upwards while 1117 genes downwards in the normal and preneoplastic group. Though the bioinformatics, the conclusion suggested that such genes were relate to the cell proliferation and apoptosis or the signal pathway.
Conclusion: The results showed the methods thatγ-GT staining combined with HE were able to distinguish the normal, preneoplastic and HCC tissue.The significant difference of gene expression existed in the developing stage of HCC. The happening and development of HCC may be related to the change of gene expression.
Part two Validation on differential expression of Cullin7 in rat and human HCC
Objective: To verify the candidate Cullin7 expression in rat and human HCC.
Methods: The changes of Cullin7 expression were confirmed by Western blot and RT-PCR.
Results:①mRNA and protein expression of Cullin7 increased in the rat normal, preneoplastic and HCC tissue.②Cullin7 protein expression also turned up in the human normal, preneoplastic and HCC tissue. Conclusion: Above results from mRNA and protein levels were consistent with the gene microarray, which indicates Cullin7 may be involved in the development and progression of human HCC.
Part three Biological function and mechanism study of Cullin7 in hepatocarcinogenesis
Objective: To explore the role of Cullin7 in hepatocarcinogenesis, further study on its biological function and mechanism was carried on by RNAi technique on the HCC cell line of SMMC-7721.
Methods: Chemically synthesized small interfering RNA (siRNA) targeted on Cullin7 were transfected into SMMC-7721 cells. mRNA and protein levels of the Cullin7 in HCC cell lines SMMC-7721 were examinated by RT- PCR and Western blot.Establishing the RNAi,negtive and control group,the function and mechanism study were designed to analyze the change of apoptosis, proliferation, adhesion, and invasion of the above three cell groups.
Results: Flow cytometry analysis showed that the proportion of apoptotic cells in the RNAi , Mock and Con group were 67.45±5.83%, 13.74±4.25%, 16.69±3.18% respectively. MTT results showed that the value of OD490 RNAi group in 24h, 48h, and 72h were 0.941±0.04, 0.924±0.13, 0.783±0.12 respectively. Movement test in vitro shows that the porous cells in the RNAi, negtive and control group were 26.35±1.55, 68.33±3.55 and 65.77±3.18. Cell invasion assay showed the average invading cell numbers perfield in the group RNAi, mock and control were 14.38±2.37、45.38±4.16、and 48.67±2.51 respectively. There was siginificant difference between RNAi and the other two groups.
Conclusion: By promoting cell proliferation, inhibiting cell apoptosis, Cullin7 may be lead to the transformation from normal cells to the malignant.
Part four The expression and clinical significance of Cullin7 in a larger scale of human HCC samples
Objective: To analyze the clinical relationship between Cullin7 expression and human hepatocellular carcinoma .
Methods: To explore the clinical significance of Cullin7 expression in HCC and evaluate the potential value of Cullin7 as a new molecular marker for diagnosis early HCC, the Cullin7 protein expression level was examinated in 62 cases of human HCC and the adjacent liver tissues, as well as in 12 cases of normal human liver tissue, by Real-Time PCR and immunohistochemical techniques.
Results: The results showed the expression level of Cullin7 was up-regulated in all kinds of tissues. There was siginificant difference between human HCC and the adjacent liver tissues (p<0.05), and no siginificant difference between the adjacent liver tissues and normal liver tissues. The Cullin7 level had significant correlation with Edmondson stage, recurrence and metastasisof the HCC cases.(p<0.05).
Conclusions: Cullin7 expression turned up in the HCC tissue,which indicates that Cullin7 could be a molecular marker for the diagnosis and the prognosis analysis of HCC.
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