AIM: To study the difference in gene expression between solitary large hepatocellular carcinoma (SLHCC) and nodular hepatocellular carcinoma (NHCC).METHODS: Polymerase chain reaction (PCR) products of 8464 human genes...AIM: To study the difference in gene expression between solitary large hepatocellular carcinoma (SLHCC) and nodular hepatocellular carcinoma (NHCC).METHODS: Polymerase chain reaction (PCR) products of 8464 human genes were spotted on a chip in array. DNAs were then fixed on a glass plate. Total RNA was isolated from freshly excised human SLHCC (n = 7) and NHCC (n = 15) tissues, and was reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparation of hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing,cDNA microarray was scanned for the fluorescent signals to display the difference between the two kinds of HCC. In addition, the expression of RhoC and protocadherin LKC was also detected with the reverse transcriptase polymerase chain reaction (RT-PCR) method.RESULTS: Among the 8 464 human genes, 668 (7.89%) genes were expressed differentially at the mRNA levels between SLHCC and NHCC. Three hundred and fifty five (4.19%) genes, including protocadherin LKC, were upregulated, whereas 313 (3.70%) genes, including RhoC,were down-regulated. The mRNA expression levels of RhoC and protocadherin LKCwere confirmed by RT-PCR. Analysis of differentially expressed genes confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of SLHCC and NHCC.CONCLUSION: cDNA microarray is an effective technique in screening the difference in gene expression between SLHCC and NHCC. Many of these differentially expressed genes are involved in the invasion and metastasis of HCC.Further analysis of these genes will help to understand the different molecular mechanisms of SLHCC and NHCC.展开更多
目的探究休眠的多倍体巨大肿瘤细胞(polyploid giant cancer cells,PGCC)对鼻咽癌(nasopharyngeal carcinoma,NPC)复发的影响,明确抑制自噬在阻止NPC复发中的作用。方法利用紫杉醇(paclitaxel,PTX)诱导NPC细胞来源的PGCC(NPC-PGCC)形成...目的探究休眠的多倍体巨大肿瘤细胞(polyploid giant cancer cells,PGCC)对鼻咽癌(nasopharyngeal carcinoma,NPC)复发的影响,明确抑制自噬在阻止NPC复发中的作用。方法利用紫杉醇(paclitaxel,PTX)诱导NPC细胞来源的PGCC(NPC-PGCC)形成,并利用光学显微镜、细胞免疫荧光、活/死细胞双染色实验对PGCC形态、多倍体特性、细胞活性等进行鉴定。采用转录组测序(RNA-seq)检测NPC-PGCC和二倍体NPC细胞CNE2的差异表达基因。采用基因本体论(gene ontology,GO)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)对差异基因进行功能富集和通路注释分析。利用免疫蛋白印迹与细胞透射电镜实验评估NPC-PGCC细胞中的自噬水平。利用临床高度相关的裸鼠NPC复发模型研究自噬在NPC-PGCC形成中的作用及NPC-PGCC对NPC复发的影响。所有数据采用GraphPad Prism 6进行统计学分析,以P<0.05为差异具有统计学意义。结果紫杉醇诱导形成的NPC-PGCC具有休眠后爆炸性分裂的特征。NPC-PGCC和二倍体NPC细胞CNE2的差异基因GO富集、KEGG通路注释主要集中在自噬及其相关通路。NPC-PGCC细胞中的自噬水平显著增强。临床高度相关裸鼠NPC复发模型中,进行顺铂治疗的裸鼠原发肿瘤中PGCC数量高于其余各组;自噬抑制剂预处理后与顺铂联合治疗的裸鼠原发肿瘤中PGCC数量少,同时复发率显著低于其余各组。结论休眠多倍体巨大肿瘤细胞的形成机制与自噬有关,抑制自噬可通过抑制PGCC形成进而抑制NPC复发。展开更多
基金Supported by National Key Technologies RD Program of China during the 10~(th) Five-year plan period,No.2001BA703B04National Natural Science Foundation of China,No.30371595Hunan Provin e Developing Planning Committee,No.2001-907
文摘AIM: To study the difference in gene expression between solitary large hepatocellular carcinoma (SLHCC) and nodular hepatocellular carcinoma (NHCC).METHODS: Polymerase chain reaction (PCR) products of 8464 human genes were spotted on a chip in array. DNAs were then fixed on a glass plate. Total RNA was isolated from freshly excised human SLHCC (n = 7) and NHCC (n = 15) tissues, and was reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparation of hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing,cDNA microarray was scanned for the fluorescent signals to display the difference between the two kinds of HCC. In addition, the expression of RhoC and protocadherin LKC was also detected with the reverse transcriptase polymerase chain reaction (RT-PCR) method.RESULTS: Among the 8 464 human genes, 668 (7.89%) genes were expressed differentially at the mRNA levels between SLHCC and NHCC. Three hundred and fifty five (4.19%) genes, including protocadherin LKC, were upregulated, whereas 313 (3.70%) genes, including RhoC,were down-regulated. The mRNA expression levels of RhoC and protocadherin LKCwere confirmed by RT-PCR. Analysis of differentially expressed genes confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of SLHCC and NHCC.CONCLUSION: cDNA microarray is an effective technique in screening the difference in gene expression between SLHCC and NHCC. Many of these differentially expressed genes are involved in the invasion and metastasis of HCC.Further analysis of these genes will help to understand the different molecular mechanisms of SLHCC and NHCC.
文摘目的探究休眠的多倍体巨大肿瘤细胞(polyploid giant cancer cells,PGCC)对鼻咽癌(nasopharyngeal carcinoma,NPC)复发的影响,明确抑制自噬在阻止NPC复发中的作用。方法利用紫杉醇(paclitaxel,PTX)诱导NPC细胞来源的PGCC(NPC-PGCC)形成,并利用光学显微镜、细胞免疫荧光、活/死细胞双染色实验对PGCC形态、多倍体特性、细胞活性等进行鉴定。采用转录组测序(RNA-seq)检测NPC-PGCC和二倍体NPC细胞CNE2的差异表达基因。采用基因本体论(gene ontology,GO)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)对差异基因进行功能富集和通路注释分析。利用免疫蛋白印迹与细胞透射电镜实验评估NPC-PGCC细胞中的自噬水平。利用临床高度相关的裸鼠NPC复发模型研究自噬在NPC-PGCC形成中的作用及NPC-PGCC对NPC复发的影响。所有数据采用GraphPad Prism 6进行统计学分析,以P<0.05为差异具有统计学意义。结果紫杉醇诱导形成的NPC-PGCC具有休眠后爆炸性分裂的特征。NPC-PGCC和二倍体NPC细胞CNE2的差异基因GO富集、KEGG通路注释主要集中在自噬及其相关通路。NPC-PGCC细胞中的自噬水平显著增强。临床高度相关裸鼠NPC复发模型中,进行顺铂治疗的裸鼠原发肿瘤中PGCC数量高于其余各组;自噬抑制剂预处理后与顺铂联合治疗的裸鼠原发肿瘤中PGCC数量少,同时复发率显著低于其余各组。结论休眠多倍体巨大肿瘤细胞的形成机制与自噬有关,抑制自噬可通过抑制PGCC形成进而抑制NPC复发。
