食管鳞癌发生过程中基因表达谱变化初步分析
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摘要
目的:
1.应用Agilent寡核苷酸芯片技术初步分析食管鳞癌发生过程中基因表达谱变化。
2.探讨CTHRC1在人食管鳞癌组织、细胞中的表达及临床意义。
3.观察抑制CTHRC1表达对人食管鳞癌Eca-109细胞增殖、侵袭及迁移的影响。
方法:
1.寡核苷酸芯片扫描食管鳞癌患者癌、癌旁及正常组织,挑选明显差异基因进行实时荧光定量RT-PCR验证。
2.免疫组化法检测CTHRC1在食管鳞癌组织中的表达,并探讨其与临床病理特征之间的关系。
3.免疫荧光、Western-blot观察CTHRC1在食管癌细胞株Eca-109及TE-13中的表达。
4.靶向CTHRC1的3对siRNA转染Eca-109细胞,RT-PCR及Western-blot检测CTHRC1受抑程度,挑选干扰效率最佳的siRNA。
5. siRNA转染Eca-109细胞,MTT检测细胞增殖;Transwell侵袭及迁移实验观察转染前后细胞侵袭迁移能力;Western-blot法检测MMP2和MMP9表达变化。
结果:
1.寡核苷酸芯片:配对组织总RNA质量高,反转录cRNA及荧光标记质量好;食管癌共同上调基因38个,下调基因61个。
2.实时荧光定量RT-PCR:芯片检测结果准确,CTHRC1差异最为显著。
3.免疫组织化学:CTHRC1在食管癌胞膜及胞浆中均见表达,阳性表达率为56.5%。淋巴结转移组高于未转移组(P﹤0.05),与年龄、性别、肿瘤大小、肿瘤分化程度等无明显相关性(P﹥0.05)。
4.细胞免疫荧光、Western-blot:在食管癌细胞株Eca-109及TE13中均见CTHRC1表达。
5. siRNA显著抑制Eca-109细胞CTHRC1表达,siRNA1干扰效率最佳。
6. CTHRC1基因沉默明显抑制细胞增殖及侵袭转移。
结论:
1.应用寡核苷酸芯片高通量、高效率分析食管鳞癌发生发展过程中基因表达差异,可以筛选新的治疗靶点。
2.食管鳞癌的发生发展是一个涉及多基因、多阶段的复杂过程。
3. CTHRC1在人食管鳞癌组织及细胞株Eca-109和TE-13中表达上调,且与淋巴结转移相关。
4. RNAi可有效沉默人食管鳞癌Eca-109细胞中CTHRC1表达。
5.沉默CTHRC1能够抑制Eca-109细胞增殖及侵袭转移,MMP2和MMP9表达下降。
Objectives:
1. To explore the differential expression genes preliminarily in ESCC by Oligomicroarray.
2. To investigate the expression of CTHRC1 in human ESCC tissue and cell lines, and to discuss its clinicopathological features in development of ESCC.
3. To observe the effects resulted from inhibition of CTHRC1 by RNAi on proliferation,invasion and migration of Eca-109 cells.
Methods:
1. The obviously differential expression genes among ESCC,PCT and NET were selected by Oligomicroarray,and was checked by Real-time Fluorescent Qantitation RT-PCR.
2. Expression of CTHRC1 was observed in ESCC tissure by Immunohistochemistry and their relationship with clinicopathological features was evaluated.
3. Expression of CTHRC1 in esophageal carcinoma cell lines Eca-109 and TE-13 was detected by Immunofluorescence and Western-blot.
4. Eca-109 cells were transfected with siRNAs targeting CTHRC1.Then expression of CTHRC1 was detected by RT-PCR and Western-blot in order to assess the interference efficiency,accordingly the optimal siRNA was choosed.
5. With RNA silencing of CTHRC1 in cell line Eca-109,the cellular proliferation was analyzed by MTT,the changes of invision and migration were assessed by Transwell and expression of MMP2 and MMP9 was detected by Western-blot.
Results:
1. Oligomicroarray:The total RNA,everse transcription product and fluorescence labeled cRNA were all of high quality;38 genes were up-regulated and 61 were down-regulated.
2. Real-time Fluorescent Qantitation RT-PCR:The most differential gene was CTHRC1.
3. Immunohistochemistry:The positive expression of CTHRC1 was existing in cellular membrane and cytoplasm of ESCC.The expression rate was 56.5%. There was significantly higher expression in the cases with metastasis than these with non-metastasis(P<0.05),and the expression of CTHRC1 was not related with age,sex,size of tumer and pathological type(P>0.05).
4. Immunofluorescence and Western-blot:The expression of CTHRC1 was observed in esophageal carcinoma cell lines Eca-109 and TE-13.
5. siRNA inhibited expression of CTHRC1 in Eca-109 cells dramatically:siRNA1 was selected for its excellent interference efficiency of CTHRC1 which was used in subsequent experiment.
6. RNA silencing of CTHRC1 obviously inhibited proliferation and apoptosis of Eca-109 cells.
Conclusions:
1. The high throughput and effective Agilent oligomicroarray can screen novel therapy targeted genes by analyzing the differential expression genes in development and progression of ESCC.
2. The development and progression of ESCC is a complicated process involving multigenes and multiprocedures.
3. The expression of CTHRC1 was up-regulated in ESCC tissue and cell lines Eca-109,TE-13 and was associated with lymph-node metastasis.
4. RNA interference can effectively silence CTHRC1 in cell line Eca-109.
5. RNA silencing of CTHRC1 in cell line Eca-109 may lead to inhibition of cellular proliferation,migration and invision.It also leads to inhibition of MMP2 and MMP9 expression.
1.应用Agilent寡核苷酸芯片技术初步分析食管鳞癌发生过程中基因表达谱变化。
2.探讨CTHRC1在人食管鳞癌组织、细胞中的表达及临床意义。
3.观察抑制CTHRC1表达对人食管鳞癌Eca-109细胞增殖、侵袭及迁移的影响。
方法:
1.寡核苷酸芯片扫描食管鳞癌患者癌、癌旁及正常组织,挑选明显差异基因进行实时荧光定量RT-PCR验证。
2.免疫组化法检测CTHRC1在食管鳞癌组织中的表达,并探讨其与临床病理特征之间的关系。
3.免疫荧光、Western-blot观察CTHRC1在食管癌细胞株Eca-109及TE-13中的表达。
4.靶向CTHRC1的3对siRNA转染Eca-109细胞,RT-PCR及Western-blot检测CTHRC1受抑程度,挑选干扰效率最佳的siRNA。
5. siRNA转染Eca-109细胞,MTT检测细胞增殖;Transwell侵袭及迁移实验观察转染前后细胞侵袭迁移能力;Western-blot法检测MMP2和MMP9表达变化。
结果:
1.寡核苷酸芯片:配对组织总RNA质量高,反转录cRNA及荧光标记质量好;食管癌共同上调基因38个,下调基因61个。
2.实时荧光定量RT-PCR:芯片检测结果准确,CTHRC1差异最为显著。
3.免疫组织化学:CTHRC1在食管癌胞膜及胞浆中均见表达,阳性表达率为56.5%。淋巴结转移组高于未转移组(P﹤0.05),与年龄、性别、肿瘤大小、肿瘤分化程度等无明显相关性(P﹥0.05)。
4.细胞免疫荧光、Western-blot:在食管癌细胞株Eca-109及TE13中均见CTHRC1表达。
5. siRNA显著抑制Eca-109细胞CTHRC1表达,siRNA1干扰效率最佳。
6. CTHRC1基因沉默明显抑制细胞增殖及侵袭转移。
结论:
1.应用寡核苷酸芯片高通量、高效率分析食管鳞癌发生发展过程中基因表达差异,可以筛选新的治疗靶点。
2.食管鳞癌的发生发展是一个涉及多基因、多阶段的复杂过程。
3. CTHRC1在人食管鳞癌组织及细胞株Eca-109和TE-13中表达上调,且与淋巴结转移相关。
4. RNAi可有效沉默人食管鳞癌Eca-109细胞中CTHRC1表达。
5.沉默CTHRC1能够抑制Eca-109细胞增殖及侵袭转移,MMP2和MMP9表达下降。
Objectives:
1. To explore the differential expression genes preliminarily in ESCC by Oligomicroarray.
2. To investigate the expression of CTHRC1 in human ESCC tissue and cell lines, and to discuss its clinicopathological features in development of ESCC.
3. To observe the effects resulted from inhibition of CTHRC1 by RNAi on proliferation,invasion and migration of Eca-109 cells.
Methods:
1. The obviously differential expression genes among ESCC,PCT and NET were selected by Oligomicroarray,and was checked by Real-time Fluorescent Qantitation RT-PCR.
2. Expression of CTHRC1 was observed in ESCC tissure by Immunohistochemistry and their relationship with clinicopathological features was evaluated.
3. Expression of CTHRC1 in esophageal carcinoma cell lines Eca-109 and TE-13 was detected by Immunofluorescence and Western-blot.
4. Eca-109 cells were transfected with siRNAs targeting CTHRC1.Then expression of CTHRC1 was detected by RT-PCR and Western-blot in order to assess the interference efficiency,accordingly the optimal siRNA was choosed.
5. With RNA silencing of CTHRC1 in cell line Eca-109,the cellular proliferation was analyzed by MTT,the changes of invision and migration were assessed by Transwell and expression of MMP2 and MMP9 was detected by Western-blot.
Results:
1. Oligomicroarray:The total RNA,everse transcription product and fluorescence labeled cRNA were all of high quality;38 genes were up-regulated and 61 were down-regulated.
2. Real-time Fluorescent Qantitation RT-PCR:The most differential gene was CTHRC1.
3. Immunohistochemistry:The positive expression of CTHRC1 was existing in cellular membrane and cytoplasm of ESCC.The expression rate was 56.5%. There was significantly higher expression in the cases with metastasis than these with non-metastasis(P<0.05),and the expression of CTHRC1 was not related with age,sex,size of tumer and pathological type(P>0.05).
4. Immunofluorescence and Western-blot:The expression of CTHRC1 was observed in esophageal carcinoma cell lines Eca-109 and TE-13.
5. siRNA inhibited expression of CTHRC1 in Eca-109 cells dramatically:siRNA1 was selected for its excellent interference efficiency of CTHRC1 which was used in subsequent experiment.
6. RNA silencing of CTHRC1 obviously inhibited proliferation and apoptosis of Eca-109 cells.
Conclusions:
1. The high throughput and effective Agilent oligomicroarray can screen novel therapy targeted genes by analyzing the differential expression genes in development and progression of ESCC.
2. The development and progression of ESCC is a complicated process involving multigenes and multiprocedures.
3. The expression of CTHRC1 was up-regulated in ESCC tissue and cell lines Eca-109,TE-13 and was associated with lymph-node metastasis.
4. RNA interference can effectively silence CTHRC1 in cell line Eca-109.
5. RNA silencing of CTHRC1 in cell line Eca-109 may lead to inhibition of cellular proliferation,migration and invision.It also leads to inhibition of MMP2 and MMP9 expression.
引文
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[2] Kwong KF.Molecular biology of esophageal cancer in the genomics era[J].Surg Clin North Am,2005,85(3):539-553.
[3] Chattopadhyay I,Kapur S,Purkayastha J,et al.Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis[J].World J Gastroenterol 2007,13(9):1438-1444.
[4] Peter Pyagay, Mélanie Heroult, Qiaozeng Wang,et al.Collagen Triple Helix Repeat Containing 1, a Novel Secreted Protein in Injured and Diseased Arteries, Inhibits Collagen Expression and Promotes Cell Migration[J].Circ Res 2005,96:261-268.
[5] Wang Y,Xu A,Knight C,et al. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity[J].J Biol Chem,2002,277:19521-19529.
[6] Liren Tang,Derek L Dai,Mingwan,et al.Aberrant Expression of Collagen Triple Helix Repeat Containing1in Human Solid Cancers[J].Clin Cancer Res 2006,12:3716–3722.
[7] Knupfer H,Preiss R.Significance of interleukin-6 (IL-6) in breast cancer[J].Brest Cancer Res Treat,2007,102(2):129-135
[8] Ziqler M,Villares GJ,Lev DC,et al.Tumor immunotherapy in melanoma: strategies for overcoming mechanisms of resistance and escape[J].Am J Clin Dermatol, 2008,9(5):307-311
[9] Knupfer H,Preiss R.Significance of interleukin-6 (IL-6) in breast cancer. Breast Cancer Res Treat.2007,102(2):129-135.
[10] Rosenkilde MM,Schwartz TW.The chemokine system-a major regulator of angiogenesis in health and disease. 2004,112(7-8):481-495.
[11] Rosenkilde MM,Schwartz TW.The chemokine system-a major regulator of angiogenesis in health and disease[J]. APMIS,2004,112(7-8):481-495
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