RNA干扰沉默食管癌细胞MMP-2基因的实验研究
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摘要
背景及目的：食管癌是威胁人类健康的主要恶性肿瘤之一，其治疗效果不佳的主要原因是肿瘤的侵袭和转移，因此针对肿瘤侵袭转移相关的基因研究成为肿瘤研究的热点之一。基质金属蛋白酶-2是降解细胞外基质和基底膜的重要酶，与肿瘤侵袭、转移和血管生成密切相关，在多种肿瘤组织中有较高的表达。RNA干扰技术是近年来产生的新兴生物技术，可特异性地导致相应的mRNA降解，从而阻断特定基因的表达。利用RNA干扰特异性地抑制癌基因、癌相关基因或突变基因的过度表达，使这类基因保持在静默或休眠状态，从而有望用这种新的手段治疗肿瘤。我们采用RNA干扰的技术，特异性抑制食管癌细胞MMP-2基因的表达，观察食管癌细胞迁移和侵袭能力的变化，并使用基因芯片技术检测MMP-2表达下调前后食管癌细胞基因表达谱的差异。以期进一步研究食管癌细胞侵袭转移机制，探讨控制食管癌细胞的侵袭转移的策略以及探索食管癌基因治疗的靶点。
方法：1．对4种食管癌细胞系EC9706、KYSE510、KYSE150、TE13进行常规培养，采用荧光定量PCR和Western blot方法对食管癌细胞MMP-2基因在mRNA和蛋白水平的表达进行检测，分析其表达丰度与食管癌细胞恶性程度的关系，同时筛选出高表达MMP-2细胞。
2．根据siRNA设计原则，设计合成3对靶向MMP-2基因的siRNA(siRNA-1，2，3)，采用阳离子脂质体试剂瞬时转染食管癌细胞KYSE150,利用荧光定量PCR和Western blot检测RNA干扰后MMP-2基因的沉默效果，并筛选干扰效果较好的siRNA以及适宜的转染浓度，用于下一步实验。
3．采用阳离子脂质体试剂向食管癌细胞KYSE150转染靶向MMP-2基因的siRNA，下调MMP-2的表达。于转染siRNA后48小时，应用细胞划痕实验检测MMP-2基因表达下调后食管癌细胞迁移能力的变化；应用boyden chamber细胞侵袭实验检测MMP-2基因表达下调后，食管癌细胞KYSE150侵袭能力的改变。
4．采用阳离子脂质体试剂向食管癌细胞KYSE150转染靶向MMP-2基因的siRNA，下调MMP-2的表达。应用Affymetrix HU133 plus 2基因芯片检测食管癌细胞KYSE150MMP-2基因表达下调后，基因表达谱的差异，并应用生物信息学技术对相关基因和信号通路进行综合分析。
结果：1．4种食管癌细胞株均有MMP-2基因表达，其中低分化鳞癌细胞株KYSE150表达水平最高，与其它3种食管癌细胞株比较差异有统计学意义(P＜0．01)；食管癌细胞株MMP-2基因表达丰度与食管癌细胞恶性程度有关，随着恶性程度的上升MMP-2表达水平升高。
2．3对不同siRNA转染组MMP-2表达在mRNA和蛋白水平均有明显下调，其干扰效率分别为siRNA-1(97．7％)、siRNA-2(91．9％)、siRNA-3(94．9％)。其中siRNA-1干扰效率最高，与其它两组转染siRNA组和空白对照及阴性对照组比较，差异有显著性(P＜0．01)；浓度梯度实验结果，转染siRNA为50nM时，干扰效率最高。
3．转染siRNA下调食管癌细胞KYSE150MMP-2基因表达后，细胞划痕后24h后空白对照组和阴性对照组细胞划痕明显愈合，而siRNA转染组几乎没有愈合；48h后空白对照组和阴性对照组已经完全愈合，siRNA转染组仅有少量愈合，与其它两组比较差异显著(P＜0．01)；boyden chamber小室细胞侵袭实验结果siRNA转染组穿膜细胞数明显减少，与空白对照组和阴性对照组比较，差异有显著性(P＜0．05)。
4．干扰食管癌细胞KYSE150MMP-2基因表达后，基因芯片分析其表达谱的变化。结果发现330个探针表达明显改变，对应有明确名称的基因为235个，其中上调的127个，下调的108个；EST序列53个，其中上调17个，下调36个。共涉及87个信号通路，主要与细胞基质黏附、细胞迁移、细胞信号转导、细胞周期、细胞凋亡以及衰老等相关。
结论：1．食管癌细胞表达MMP-2与其恶性程度和临床病理改变相关，随恶性程度的升高其表达丰度升高；食管癌细胞KYSE150为MMP-2高表达细胞系，可作为进一步研究的实验材料。
2．RNA干扰技术可高效地下调食管癌细胞KYSE150MMP-2基因的表达，可作为一种基因治疗的策略深入研究。
3．食管癌细胞KYSE150MMP-2基因下调后，细胞迁移和侵袭能力显著被抑制，表明MMP-2可作为食管癌基因治疗的靶点。
4．食管癌细胞KYSE150MMP-2基因下调后，其细胞基因表达谱有明显变化，主要涉及细胞周期、凋亡、粘附、迁移、衰老等方面。表明MMP-2基因表达受很多相关基因及信号通路的调控，其分子机制有待进一步深入探讨。
Background and objective:Esophageal carcinoma is one of main malignancies inChina,and its invasion and metastasis are the leading cause of poor prognosis.Forthis reason,studying molecular mechanisms of invasion and metastasis in esophagealcarcinoma became one of the hotspots.Previous studies indicated that matrixmetalloproteinase(MMP)s,which can degrade and destruct the tissue around tumorsuch as basement membrane and extracellular matrix,play important roles in theprocess of tumor invasion and metastasis in several types of the solid tumors.Among all of the MMPs discovered recently,MMP-2 is one of MMPs closelyassociated with tumor invasion.The recent studies have demonstrated that MMP-2overexpersses in many kinds of malignant tumor,and the expression levels of MMP-2associated with the degree of tumor invasion and poor prognosis.RNAinterference(RNAi)is the process of sequence-specific post transcriptional genesilencing(PTGS)triggered by double stranded RNA(dsRNA).Now,RNAi is veryuseful technique and has been applied in gene function studies.In this study,wesuppressed MMP-2 expression in esophageal carcinoma cell line KYSE150 withRNA interference and then observed the behavior of KYSE150 in invasion andmigration after MMP-2 gene expression was silenced.We further analysized thealteration of downstream genes of MMP-2.
Methods:1.Four esophageal carcinoma cell lines(EC9706,KYSE510,KYSE150and TE13)were cultured using common methods.Western blot and real-timequantitative PCR were used to verify the expression level of MMP-2.Therelationship between MMP-2 expression level and the malignant degree ofesophageal carcinoma cell was analyzed.The MMP-2 over-expressed cell line wasselected.
2.Three MMP-2-targeting siRNAs were designed and synthesized.ThesesiRNAs were transiently transfected into KYSE150 via cationic liposomeLipofectamine 2000.Western Blot and real-time quantitative PCR were used to verifythe interference efficiency of MMP-2 protein and mRNA expression levels.ThesiRNA with high interference efficiency and the eligible siRNA density were selected.
3.After MMP-2 was down-regulated in esophageal carcinoma cell lineKYSE150 by siRNA,the ability of cell invasion was measured by Boyden Chamber,the ability of cell migration was evaluated by wound healing assay.
4.After silencing MMP-2 expression,the alteration of gene expression profiles was investigated through Affymetrix HU133 plus 2 gene chip,and the bioinformaticswas used to analysize data.
Results:1.MMP-2 expressed in all 4 esophageal carcinoma cell lines,the expressionlevel has a correlation with cell malignant degree.The MMP-2 was significantlyexpressed in KYSE150 as compared to the other cell lines(P＜0.01).
2.The levels MMP-2 mRNA and protein in KYSE150 were significantlydecreased by RNA interference(P＜0.01).The siRNA-1 is the most effective siRNA,and 50nM is the most eligible density.
3.After MMP-2 expression was down-regulated in KYSE150 by siRNA,bothcell invasion and migration ability were significantly inhibited(P＜0.01).
4.Microarray assay revealed that expression of 330 probes were altered inresponse to MMP-2 gene silencing,including 235 genes and 53 ESTs.Among the 235genes,127 genes were up-regulated and 108 were down-regulated.These genes areinvolved in cell cycle,apoptosis,cell signal transduction,cell adhesion,cell migrationand aging.
Conclusion:1.The expression level of MMP-2 in esophageal carcinoma cell lineshas a correlation with cell malignant degree.As the MMP-2 overexpression cell line,KYSE150 is an ideal material for follow-up research.
2.siRNA can efficiently down-regulate the MMP-2 gene in esophagealcarcinoma cell line KYSE150,siRNA should be a method for gene therapy.
3.The ability of migration and invasion of esophageal carcinoma cell lineKYSE150 can be inhibited by MMP-2 silencing.MMP-2 may be considered as atarget gene for therapy of esophageal carcinoma.
4.MMP-2 is an important gene to regulate adhesion and invasion in tumorcells.Silencing MMP-2 gene led to a significant regulation in the downstream genes.These genes are involved in cell cycle,apoptosis,cell signal transduction,celladhesion,cell migration and aging.However,the present study still can notcompletely reveal the MMP-2 signal pathways and the molecular mechanism.Furtherstudies are needed to be carried out.
方法：1．对4种食管癌细胞系EC9706、KYSE510、KYSE150、TE13进行常规培养，采用荧光定量PCR和Western blot方法对食管癌细胞MMP-2基因在mRNA和蛋白水平的表达进行检测，分析其表达丰度与食管癌细胞恶性程度的关系，同时筛选出高表达MMP-2细胞。
2．根据siRNA设计原则，设计合成3对靶向MMP-2基因的siRNA(siRNA-1，2，3)，采用阳离子脂质体试剂瞬时转染食管癌细胞KYSE150,利用荧光定量PCR和Western blot检测RNA干扰后MMP-2基因的沉默效果，并筛选干扰效果较好的siRNA以及适宜的转染浓度，用于下一步实验。
3．采用阳离子脂质体试剂向食管癌细胞KYSE150转染靶向MMP-2基因的siRNA，下调MMP-2的表达。于转染siRNA后48小时，应用细胞划痕实验检测MMP-2基因表达下调后食管癌细胞迁移能力的变化；应用boyden chamber细胞侵袭实验检测MMP-2基因表达下调后，食管癌细胞KYSE150侵袭能力的改变。
4．采用阳离子脂质体试剂向食管癌细胞KYSE150转染靶向MMP-2基因的siRNA，下调MMP-2的表达。应用Affymetrix HU133 plus 2基因芯片检测食管癌细胞KYSE150MMP-2基因表达下调后，基因表达谱的差异，并应用生物信息学技术对相关基因和信号通路进行综合分析。
结果：1．4种食管癌细胞株均有MMP-2基因表达，其中低分化鳞癌细胞株KYSE150表达水平最高，与其它3种食管癌细胞株比较差异有统计学意义(P＜0．01)；食管癌细胞株MMP-2基因表达丰度与食管癌细胞恶性程度有关，随着恶性程度的上升MMP-2表达水平升高。
2．3对不同siRNA转染组MMP-2表达在mRNA和蛋白水平均有明显下调，其干扰效率分别为siRNA-1(97．7％)、siRNA-2(91．9％)、siRNA-3(94．9％)。其中siRNA-1干扰效率最高，与其它两组转染siRNA组和空白对照及阴性对照组比较，差异有显著性(P＜0．01)；浓度梯度实验结果，转染siRNA为50nM时，干扰效率最高。
3．转染siRNA下调食管癌细胞KYSE150MMP-2基因表达后，细胞划痕后24h后空白对照组和阴性对照组细胞划痕明显愈合，而siRNA转染组几乎没有愈合；48h后空白对照组和阴性对照组已经完全愈合，siRNA转染组仅有少量愈合，与其它两组比较差异显著(P＜0．01)；boyden chamber小室细胞侵袭实验结果siRNA转染组穿膜细胞数明显减少，与空白对照组和阴性对照组比较，差异有显著性(P＜0．05)。
4．干扰食管癌细胞KYSE150MMP-2基因表达后，基因芯片分析其表达谱的变化。结果发现330个探针表达明显改变，对应有明确名称的基因为235个，其中上调的127个，下调的108个；EST序列53个，其中上调17个，下调36个。共涉及87个信号通路，主要与细胞基质黏附、细胞迁移、细胞信号转导、细胞周期、细胞凋亡以及衰老等相关。
结论：1．食管癌细胞表达MMP-2与其恶性程度和临床病理改变相关，随恶性程度的升高其表达丰度升高；食管癌细胞KYSE150为MMP-2高表达细胞系，可作为进一步研究的实验材料。
2．RNA干扰技术可高效地下调食管癌细胞KYSE150MMP-2基因的表达，可作为一种基因治疗的策略深入研究。
3．食管癌细胞KYSE150MMP-2基因下调后，细胞迁移和侵袭能力显著被抑制，表明MMP-2可作为食管癌基因治疗的靶点。
4．食管癌细胞KYSE150MMP-2基因下调后，其细胞基因表达谱有明显变化，主要涉及细胞周期、凋亡、粘附、迁移、衰老等方面。表明MMP-2基因表达受很多相关基因及信号通路的调控，其分子机制有待进一步深入探讨。
Background and objective:Esophageal carcinoma is one of main malignancies inChina,and its invasion and metastasis are the leading cause of poor prognosis.Forthis reason,studying molecular mechanisms of invasion and metastasis in esophagealcarcinoma became one of the hotspots.Previous studies indicated that matrixmetalloproteinase(MMP)s,which can degrade and destruct the tissue around tumorsuch as basement membrane and extracellular matrix,play important roles in theprocess of tumor invasion and metastasis in several types of the solid tumors.Among all of the MMPs discovered recently,MMP-2 is one of MMPs closelyassociated with tumor invasion.The recent studies have demonstrated that MMP-2overexpersses in many kinds of malignant tumor,and the expression levels of MMP-2associated with the degree of tumor invasion and poor prognosis.RNAinterference(RNAi)is the process of sequence-specific post transcriptional genesilencing(PTGS)triggered by double stranded RNA(dsRNA).Now,RNAi is veryuseful technique and has been applied in gene function studies.In this study,wesuppressed MMP-2 expression in esophageal carcinoma cell line KYSE150 withRNA interference and then observed the behavior of KYSE150 in invasion andmigration after MMP-2 gene expression was silenced.We further analysized thealteration of downstream genes of MMP-2.
Methods:1.Four esophageal carcinoma cell lines(EC9706,KYSE510,KYSE150and TE13)were cultured using common methods.Western blot and real-timequantitative PCR were used to verify the expression level of MMP-2.Therelationship between MMP-2 expression level and the malignant degree ofesophageal carcinoma cell was analyzed.The MMP-2 over-expressed cell line wasselected.
2.Three MMP-2-targeting siRNAs were designed and synthesized.ThesesiRNAs were transiently transfected into KYSE150 via cationic liposomeLipofectamine 2000.Western Blot and real-time quantitative PCR were used to verifythe interference efficiency of MMP-2 protein and mRNA expression levels.ThesiRNA with high interference efficiency and the eligible siRNA density were selected.
3.After MMP-2 was down-regulated in esophageal carcinoma cell lineKYSE150 by siRNA,the ability of cell invasion was measured by Boyden Chamber,the ability of cell migration was evaluated by wound healing assay.
4.After silencing MMP-2 expression,the alteration of gene expression profiles was investigated through Affymetrix HU133 plus 2 gene chip,and the bioinformaticswas used to analysize data.
Results:1.MMP-2 expressed in all 4 esophageal carcinoma cell lines,the expressionlevel has a correlation with cell malignant degree.The MMP-2 was significantlyexpressed in KYSE150 as compared to the other cell lines(P＜0.01).
2.The levels MMP-2 mRNA and protein in KYSE150 were significantlydecreased by RNA interference(P＜0.01).The siRNA-1 is the most effective siRNA,and 50nM is the most eligible density.
3.After MMP-2 expression was down-regulated in KYSE150 by siRNA,bothcell invasion and migration ability were significantly inhibited(P＜0.01).
4.Microarray assay revealed that expression of 330 probes were altered inresponse to MMP-2 gene silencing,including 235 genes and 53 ESTs.Among the 235genes,127 genes were up-regulated and 108 were down-regulated.These genes areinvolved in cell cycle,apoptosis,cell signal transduction,cell adhesion,cell migrationand aging.
Conclusion:1.The expression level of MMP-2 in esophageal carcinoma cell lineshas a correlation with cell malignant degree.As the MMP-2 overexpression cell line,KYSE150 is an ideal material for follow-up research.
2.siRNA can efficiently down-regulate the MMP-2 gene in esophagealcarcinoma cell line KYSE150,siRNA should be a method for gene therapy.
3.The ability of migration and invasion of esophageal carcinoma cell lineKYSE150 can be inhibited by MMP-2 silencing.MMP-2 may be considered as atarget gene for therapy of esophageal carcinoma.
4.MMP-2 is an important gene to regulate adhesion and invasion in tumorcells.Silencing MMP-2 gene led to a significant regulation in the downstream genes.These genes are involved in cell cycle,apoptosis,cell signal transduction,celladhesion,cell migration and aging.However,the present study still can notcompletely reveal the MMP-2 signal pathways and the molecular mechanism.Furtherstudies are needed to be carried out.
引文
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