【作者】 刘斌；

【导师】 赵莉；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:我们用生物信息学分析筛选得到一个在多种癌组织中高表达,而在癌旁正常组织低表达的新基因CXorf36(chromosome X open reading frame 36),并用RT-PCR方法在组织和细胞系中加以验证.以了解CXorf36在多种癌组织的阳性表达情况,从而希望得到一种筛查癌症的新方法。材料与方法:1材料:标本选自河北医科大学第二医院外科2006年1月至2007年12月的癌症手术治疗患者。786-O、A498、Caki三种肾癌细胞系为北京大学人民医院泌尿研究所提供。宫颈癌Hela、肝癌HepG2、结肠癌Lovo、肺腺癌A549、前列腺癌PC3细胞系为北京大学医学部生化实验室提供。2方法:分别取肝癌,胆管癌,胃癌,结直肠癌,肾癌的病人(均术后病理检查证实)手术切除的肿瘤标本癌组织和癌旁组织(位置距离癌组织至少5厘米)作对照。分为5组,分别为肝癌10例,胆管癌5例,胃癌15例,大肠癌10例,肾癌10例。组织标本行RT-PCR方法检测CXorf36mRNA在各组的表达情况。同时行RT-PCR方法检测CXorf36mRNA在宫颈癌Hela、肝癌HepG2、结肠癌Lovo、肺腺癌A549、前列腺癌PC3细胞系和786-O、A498、Caki三种肾癌细胞系的表达情况。3数据处理:所得结果运用SPSS10.0统计软件包处理,每组计量资料均数比较采用t检验,五组计量资料间均数比较采用方差分析。以α=0.05为显著性检验水准。结果:1癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.27士0.87、0.47士0.23。癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。CXorf36 mRNA在各组癌组织(肝癌组、胆管癌组、胃癌组、结直肠癌组、肾癌组)表达情况,以均数±标准差表示,依次为:4.39士0.81、4.10士0.97、4.28士0.69、4.46士0.64、4.09士1.26,差异无统计学意义(P>0.05)。CXorf36 mRNA在各组癌旁正常组织(肝癌组、胆管癌组、胃癌组、结直肠癌组、肾癌组)的表达情况以均数±标准差表示,依次为:0.49士0.24、0.49士0.29、0.47士0.23、0.46士0.20、0.45士0.28,差异无统计学意义(P>0.05)。2肝癌癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在肝癌癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.39士0.81、0.49士0.24。肝癌癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。3胆管癌癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在胆管癌癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.10士0.97、0.49士0.29。胆管癌癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。4结直肠癌癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在结直肠癌癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.28士0.69、0.47士0.23。结直肠癌癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。5胃癌癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在胃癌癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.46士0.64、0.46士0.20。胃癌癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。6肾癌癌组织和癌旁正常组织中CXorf36 mRNA表达情况CXorf36 mRNA在肾癌癌组织和癌旁正常组织表达情况,以均数±标准差表示,依次为:4.09士1.26、0.45士0.28。肾癌癌组织CXorf36 mRNA表达明显高于癌旁正常组织,差异有统计学意义(P<0.05)。7 CXorf36基因mRNA在肿瘤细胞系中的表达情况CXorf36基因mRNA在3种肾癌细胞系(786-O、A498、Caki)以及前列腺癌细胞(PC3)、结肠癌细胞(LOVO)、宫颈癌细胞(Hela)、肺腺癌细胞(A549)、肝癌细胞(HepG2)中的表达丰度很低。8 CXorf36基因克隆和氨基酸序列分析:CXorf36基因序列全长由http://www.genome.ucsc.edu/获得,编码区序列全长用RT-PCR方法从癌组织总RNA扩增得到.推译的蛋白含有182个氨基酸,预测分子量20.7kDa。氨基酸序列分析显示CXorf36蛋白有六个丝氨酸磷酸化位点和两个苏氨酸磷酸化位点,没有酪氨酸磷酸化位点。比对分析显示CXorf36蛋白与小鼠来源的猜测蛋白LOC75905氨基端有79%的同源性,而该小鼠猜测蛋白可能是一种水解酶。结论:1 CXorf36 mRNA在恶性肿瘤中肿瘤组织有明显高表达,说明Cxorf36基因可能参与恶性肿瘤的发生、发展及转移侵袭过程,并在其中发挥重要作用。2 CXorf36 mRNA在肝癌、胆管癌、结直肠癌、胃癌和肾癌的肿瘤组织明显高表达,而在相对应的正常组织中表达明显降低,说明肝癌、胆管癌、结直肠癌、胃癌和肾癌均为表达癌相关基因CXorf36的肿瘤谱。癌相关基因CXorf36可能作为肿瘤筛查监测、检测肿瘤是否侵袭转移、以及判断预后、了解早期复发的有效指标。同时癌相关基因CXorf36也为抗肿瘤侵袭转移治疗提供了一个新的靶点。3 CXorf36 mRNA肿瘤细胞系中的低表达提示我们该基因可能属于间质表达的蛋白,但也不排除肿瘤细胞建系后体外培养逐渐失去该基因的表达.因此,肿瘤细胞及间质细胞的原代培养以及免疫组化实验对于探讨该基因在肿瘤组织中的定位和功能研究将有重要意义。更多还原

【Abstract】 Objective: We find a new cancer-related gene by bioinformatics analysis, CXorf36 （chromosome X open reading frame 36）, which is upregurated in many kinds cancer tissue.We testified this phenomenon with RT-PCR in many kinds of tissues and cell lines,but to our surprising ,the mRNA level of CXorf36 in several human cancer cell lines,including three kinds of renal cell carcinoma cell lines is very low. We tried to find out what kinds of cancer which CXorf36 is upregurated.So we can find out a new method of cancer screening .Materials and method:1 Materials: Specimens from the second Hospital of Hebei Medical University tumor surgery from January 2006 to December 2007 in patients with cancer who had been treated with surgery. Hela cervical cancer, liver, HepG2, colon Lovo, A549 lung adenocarcinoma, prostate cancer cell line PC3 form the medical department of Peking University. 786 - O, A498, Caki three renal cancer cell lines form People’s Hospital of Beijing University Institute for urinary tract.2 Method: Admitted patients surgical resection of tumor specimens cancer and adjacent normal tissues as control. Divided into five groups, namely 10 cases of liver cancer, cholangiocarcinoma five cases, 15 cases of gastric cancer, colorectal cancer 10 cases, 10 cases of renal cell carcinoma. Tissue specimens by RT-PCR method to detect CXorf36 in each group expression. At the same time specimens by RT-PCR method to detect CXorf36 in Hela cervical cancer, liver, HepG2, colon Lovo, A549 lung adenocarcinoma, prostate cancer cell line PC3 and 786 - O, A498, Caki three renal carcinoma cell line expression.3 Data Processing : The results were analyzed with T-test and chi-square test by SPSS10.0,and we established the standard of statistic significance asα=0.05.Results:1 Expression of CXorf36 mRNA in cancer tissues and normal tissuesThe average expression level of CXorf 36 mRNA in cancer tissues and normal tissues are 4.27士0.87,0.47士0.23. The average expression level of CXorf 36 mRNA in cancer tissues is significantly higher than that in normal tissues（P<0.05）. There is significant difference between cancer tissues and normal tissues. The average expression level of CXorf 36 mRNA in each group of tissues are 4.39士0.81,4.10士0.97,4.28士0.69,4.46士0.64,4.09士1.26.There are no significant difference （P> 0.05）. The average expression level of CXorf 36 mRNA in each group of normal tissues are 0.49士0.24,0.49士0.29,0.47士0.23,0.46士0.20,0.45士0.28.There are no significant difference （P> 0.05）. 2 Expression of CXorf36 mRNA in liver cancer tissues and normal liver tissuesThe average expression of CXorf36 mRNA in liver cancer tissues and normal liver tissues are 4.39士0.81,0.49士0.24. The average expression level of CXorf 36 mRNA in liver cancer tissues is significantly higher than that in normal liver tissues（P<0.05）. There is significant difference between liver cancer tissues and normal liver tissues.3 Expression of CXorf36 mRNA in cholangiocarcinoma tissues and normal bile duct tissuesThe average expression of CXorf36 mRNA in cholangiocarci- noma tissues and normal bile duct tissues are 4.10士0.97,0.49士0.29.The average expression level of CXorf 36 mRNA in chola- ngiocarcinoma tissues is significantly higher than that in normal bile duct tissues （P<0.05） . There is significant difference betwe- en cholangiocarcinoma tissues and normal normal bile tissues.4 Expression of CXorf36 mRNA in colorectal cancer tissues and normal large intestine tissuesThe average expression of CXorf36 mRNA in colorectal cancer tissues and normal large intestine tissues are 4.28士0.69,0.47士0.23.The average expression level of CXorf 36 mRNA in colorectal cancer tissues is significantly higher than that in normal large intestine tissues（P<0.05）. There is significant difference between colorectal cancer tissues and normal large intestine tissues.5 Expression of CXorf36 mRNA in gastric cancer tissues and normal stomach tissuesThe average expression of CXorf36 mRNA in gastric cancer tissues and normal stomach tissues are 4.46士0.64,0.46士0.20.The average expression level of CXorf 36 mRNA in gastric cancer tissues is significantly higher than that in normal stomach tissues（P<0.05）. There is significant difference between gastric cancer tissues and normal stomach tissues.6 Expression of CXorf36 mRNA in renal cancer tissues and normal renal tissuesThe average expression of CXorf36 mRNA in renal cancer tissues and normal renal tissues are 4.09士1.26,0.45士0.28. The average expression level of CXorf 36 mRNA in renal cancer tissues is significantly higher than that in normal renal tissues（P<0.05）. There is significant difference between renal cancer tissues and normal renal tissues.7 Expression of CXorf36 mRNA in eight kinds of cancer cell linesThe mRNA level of CXorf36 in several human cancer cell lines,including PC3、LOVO、Hela、A549、HepG2、786-O、A498、Caki cell lines is very low.8 CXorf36 gene cloning and analysis of amino acid sequenceswe got the CXorf36 full-length gene sequences form http://www.genome.ucsc.edu/.we got the Full-length coding sequences by RT-PCR. We forecast the protein containing 182 amino acid and Molecular weight of 20.7 kDa .The genome structure of CXorf36, which contains six serine phosphorylation sites and two threonine phosphorylation sites,have not tyrosine phosphorylation sites.Amino acid sequence alignment indicated that CXorf36 shares 79% identity with mouse hypothetical protein, NP₇80437,which is a putative hydrolase.Conclusion:1 CXorf36 mRNA overexpress in cancer tissues. The over expression maybe contribute to the occurrence of cancer.2 CXorf36 mRNA overexpress in liver cancer, cholangiocarcinoma, colorectal cancer, gastric cancer, renal cancer tissues. Tumor spectrum in CXorf36 are liver cancer, cholangiocarcinoma, colorectal cancer, gastric cancer, renal cancer. CXorf36 maybe the screening as a tumor surveillance, detection tumor invasion and metastasis, as well as predicting the prognosis, early recurrence understanding of the effective index. At the same time CXorf36 for the treatment of tumor invasion and metastasis provides a new target .3 The mRNA level of CXorf36 in several human cancer cell lines,including PC3、LOVO、Hela、A549、HepG2、786-O、A498、Caki cell lines is very low. The gene may belong to the protein expression of interstitial . After the tumor cells in vitro construction human cancer cell lines loss of the gene expression . Tumor cells and stromal cells in primary culture and immunohistochemistry experiments for the study of the gene in tumor tissues and function of the position will be important.更多还原