K562与K562/A02细胞株中白血病耐药相关microRNA的筛选研究
摘要
研究背景:化疗是白血病以及其它肿瘤的主要治疗手段,随着化疗药物的广泛应用,肿瘤的耐药性问题越来越突出,已成为肿瘤化疗失败的主要原因之一。肿瘤多药耐药(MDR)是指肿瘤细胞对一种抗肿瘤药物出现耐药性的同时,对其他多种化学结构不同、作用靶位不同的抗肿瘤药物也产生耐药。目前有关肿瘤耐药机制的研究主要是从多药耐药基因表达的蛋白入手,探讨这些蛋白在形成耐药机制中的作用。研究表明,P-gp糖蛋白、多药耐药相关蛋白、肺耐药蛋白、乳腺癌耐药相关蛋白、DNA拓扑异构酶Ⅱ和谷胱甘肽S转移酶等多药耐药相关蛋白质与肿瘤多药耐药性相关。虽然已有的研究揭示了一些肿瘤多药耐药机制,但目前仍然不能完全解释肿瘤多药耐药现象,临床上也缺乏逆转肿瘤细胞多药耐药的有效途径。
微小RNA(MicroRNA,简称miRNA)是一类长约19nt-23nt的单链非编码RNA,广泛存在于植物及动物中,具有物种及组织特异性,进化上高度保守。miRNA可与其靶mRNA的3′非翻译区(3′untranslated regions,3'-UTRs)特异性结合,导致靶mRNA的翻译抑制或降解,在细胞分化、增殖与凋亡、个体发育、机体代谢、病毒感染及肿瘤的发生发展等方面具有重要的生物学功能。新近,miRNA在肿瘤多药耐药中的作用也引起了关注,与乳腺癌、卵巢癌、胃癌等相关的多药耐药miRNA相继报道,但白血病耐药相关miRNA的研究在世界范围内尚未见报道。本研究拟通过白血病细胞株及其耐药株的对比研究,筛选出白血病的多药耐药相关miRNA,首次从miRNA调节环节探索白血病耐药机制,为进一步研究其在多药耐药机制中的作用奠定基础。
目的:采用microRNA芯片筛选出K562细胞及其耐药株K562/A02细胞差异表达的miRNA,并用荧光定量RT-PCR对筛选出来的miRNA进行验证,进一步确定白血病耐药相关miRNA。
方法:常规培养K562及K562/A02细胞株,取等量K562细胞和K562/A02细胞,Trizol法分别抽提两种细胞的总RNA;通过YM-100(Millipore)微离心过滤柱得到片段小于300nt的小RNA,对K562和K562/A02细胞的小RNA分别进行Cy3和Cy5荧光标记,然后进行microRNA芯片杂交反应;采集芯片原始信号值并将图像信号转化为数字信号,将两芯片的信号值进行比对筛选出差异表达miRNA;应用实时荧光定量RT-PCR方法对筛选出的miRNA进行可靠性验证,鉴定出白血病耐药相关miRNA。
结果:microRNA芯片结果显示,与K562细胞相比,K562/A02细胞中有22条miRNA存在表达差异(P＜0.01),表达差异在2倍以上的有9条,其中miR-221,miR-155,miR-451表达上调;miR-98,miR-181a,let-7f,miR-424,let-7g和miR-563表达下调。荧光定量RT-PCR对miR-221,miR-181a,miR-451和let-7f的检测结果与miRNA芯片的结果相符合,miR-451、let-7f在白血病细胞株表达差异显著。
结论:miR-221,miR-181a,miR-155,miR-451,miR-98,let-7f,miR-424,let-7g和miR-563在K562细胞和K562/A02细胞之间存在差异表达,提示这些miRNA尤其是miR-451与let-7f可能与白血病多药耐药相关。
Background:Chemotherapy is the main treatment for leukemia and other tumors. With the widely using of chemotherapy drugs, drug resistance is becoming increasingly prominent. Resistance to anticancer drugs has become one of the major reasons of the failure of tumor treatment. Multidrug resistance (MDR) describes a phenomenon of cross-resistance of tumor cells to several structurally unrelated chemotherapeutic agents after exposing to a single cytotoxic drug. The current mechanisms of tumor drug resistance focus on the proteins related to multidrug resistance. Several proteins have been found be related to MDR, such as P-glycoprotein (P-gp), multidrug resistance related protein (MRP), lung resistance related protein (LRP), breast cancer multidrug resistance related protein (BCRP), topoisomerase II (TOP II) and glutathione-S-transferase (GST). Although the genesis of MDR has been extendedly investigated, an efficient agent to overcome drug-resistance has not been available. The mechanisms of MDR remain unclear and require further study.
MicroRNA(miRNA) is one kind of small noncoding RNA molecules (19nt-23nt) which regulates protein expression by binding 3'-UTRs(3' untranslated regions) of target mRNA of protein-coding genes, resulting in cleavage or translation repression of target mRNA. MiRNAs have been widely identified in plants and animals, and is conserved in sequence between distantly related organisms. MiRNA plays an important role in various cellular processes, such as cell differentiation, proliferation and apoptosis. It is one of regulators of individual development, metabolism, viral infection and the genesis, development of tumor. Recently,more and more attention has been paid on the role of miRNA in tumor multidrug resistance. Several multidrug resistance-related miRNAs of breast cancer, ovarian cancer, gastric cancer have been reported. In this study, for the first time,we will screen and search the multidrug resistance-associated miRNAs of leukemia by using microRNA microarry in combination with Real time PT-PCR.
Objective: Screen and search the miRNA, which differentially express in K562 cell line and it's adriaraycin -resistant cells --K562/A02 cell line by using microRNA Microarray in combination with Real time RT-PCR.
Methods: Total RNA of K562 cells and K562/A02 cells was isolated by Trizol. Small RNAs (<300nt) were isolated using a YM-100 Microcon centrifugal filter (from Millipore), which of K562 and K562/A02 cells were Labeled with Cy3 and Cy5 respectively. Then the miRNA was hybridized on microRNA Microarray. Original image signal were collected and digitized. The differentially expressed miRNA were screened and identified through comparison between the two chips. The results of microarray were confirmed by Real time RT-PCR.
Results: The results of microRNA microarray show that 22 miRNAs expressed differentially between K562 and K562/A02 cells (P<0.01) .As compared to K562 cells, expression of miR-221, miR-155 and miR-451 was up-regulated in K562/A02 cells by more than two folds, while expression of miR-98, miR-181a, let-7f, let-7g, miR-424 and miR-563 was down-regulated by more than two folds. MiR-221, miR-181a, miR-451 and let-7f were further confirmed by Real time RT-PCR. The results of Real time RT-PCR were consistent with that of microarray.
Conclusion: MiR-98, miR-221, miR-181a, miR-155, miR-424, miR-451, let-7f, let-7g and miR-563 were differentially expressed between K562 and K562/A02 cells. Our results suggest that these differentially expressed miRNAs particularly miR-451 and let-7f may play an important role in the mechanisms of leukemia MDR.
微小RNA(MicroRNA,简称miRNA)是一类长约19nt-23nt的单链非编码RNA,广泛存在于植物及动物中,具有物种及组织特异性,进化上高度保守。miRNA可与其靶mRNA的3′非翻译区(3′untranslated regions,3'-UTRs)特异性结合,导致靶mRNA的翻译抑制或降解,在细胞分化、增殖与凋亡、个体发育、机体代谢、病毒感染及肿瘤的发生发展等方面具有重要的生物学功能。新近,miRNA在肿瘤多药耐药中的作用也引起了关注,与乳腺癌、卵巢癌、胃癌等相关的多药耐药miRNA相继报道,但白血病耐药相关miRNA的研究在世界范围内尚未见报道。本研究拟通过白血病细胞株及其耐药株的对比研究,筛选出白血病的多药耐药相关miRNA,首次从miRNA调节环节探索白血病耐药机制,为进一步研究其在多药耐药机制中的作用奠定基础。
目的:采用microRNA芯片筛选出K562细胞及其耐药株K562/A02细胞差异表达的miRNA,并用荧光定量RT-PCR对筛选出来的miRNA进行验证,进一步确定白血病耐药相关miRNA。
方法:常规培养K562及K562/A02细胞株,取等量K562细胞和K562/A02细胞,Trizol法分别抽提两种细胞的总RNA;通过YM-100(Millipore)微离心过滤柱得到片段小于300nt的小RNA,对K562和K562/A02细胞的小RNA分别进行Cy3和Cy5荧光标记,然后进行microRNA芯片杂交反应;采集芯片原始信号值并将图像信号转化为数字信号,将两芯片的信号值进行比对筛选出差异表达miRNA;应用实时荧光定量RT-PCR方法对筛选出的miRNA进行可靠性验证,鉴定出白血病耐药相关miRNA。
结果:microRNA芯片结果显示,与K562细胞相比,K562/A02细胞中有22条miRNA存在表达差异(P＜0.01),表达差异在2倍以上的有9条,其中miR-221,miR-155,miR-451表达上调;miR-98,miR-181a,let-7f,miR-424,let-7g和miR-563表达下调。荧光定量RT-PCR对miR-221,miR-181a,miR-451和let-7f的检测结果与miRNA芯片的结果相符合,miR-451、let-7f在白血病细胞株表达差异显著。
结论:miR-221,miR-181a,miR-155,miR-451,miR-98,let-7f,miR-424,let-7g和miR-563在K562细胞和K562/A02细胞之间存在差异表达,提示这些miRNA尤其是miR-451与let-7f可能与白血病多药耐药相关。
Background:Chemotherapy is the main treatment for leukemia and other tumors. With the widely using of chemotherapy drugs, drug resistance is becoming increasingly prominent. Resistance to anticancer drugs has become one of the major reasons of the failure of tumor treatment. Multidrug resistance (MDR) describes a phenomenon of cross-resistance of tumor cells to several structurally unrelated chemotherapeutic agents after exposing to a single cytotoxic drug. The current mechanisms of tumor drug resistance focus on the proteins related to multidrug resistance. Several proteins have been found be related to MDR, such as P-glycoprotein (P-gp), multidrug resistance related protein (MRP), lung resistance related protein (LRP), breast cancer multidrug resistance related protein (BCRP), topoisomerase II (TOP II) and glutathione-S-transferase (GST). Although the genesis of MDR has been extendedly investigated, an efficient agent to overcome drug-resistance has not been available. The mechanisms of MDR remain unclear and require further study.
MicroRNA(miRNA) is one kind of small noncoding RNA molecules (19nt-23nt) which regulates protein expression by binding 3'-UTRs(3' untranslated regions) of target mRNA of protein-coding genes, resulting in cleavage or translation repression of target mRNA. MiRNAs have been widely identified in plants and animals, and is conserved in sequence between distantly related organisms. MiRNA plays an important role in various cellular processes, such as cell differentiation, proliferation and apoptosis. It is one of regulators of individual development, metabolism, viral infection and the genesis, development of tumor. Recently,more and more attention has been paid on the role of miRNA in tumor multidrug resistance. Several multidrug resistance-related miRNAs of breast cancer, ovarian cancer, gastric cancer have been reported. In this study, for the first time,we will screen and search the multidrug resistance-associated miRNAs of leukemia by using microRNA microarry in combination with Real time PT-PCR.
Objective: Screen and search the miRNA, which differentially express in K562 cell line and it's adriaraycin -resistant cells --K562/A02 cell line by using microRNA Microarray in combination with Real time RT-PCR.
Methods: Total RNA of K562 cells and K562/A02 cells was isolated by Trizol. Small RNAs (<300nt) were isolated using a YM-100 Microcon centrifugal filter (from Millipore), which of K562 and K562/A02 cells were Labeled with Cy3 and Cy5 respectively. Then the miRNA was hybridized on microRNA Microarray. Original image signal were collected and digitized. The differentially expressed miRNA were screened and identified through comparison between the two chips. The results of microarray were confirmed by Real time RT-PCR.
Results: The results of microRNA microarray show that 22 miRNAs expressed differentially between K562 and K562/A02 cells (P<0.01) .As compared to K562 cells, expression of miR-221, miR-155 and miR-451 was up-regulated in K562/A02 cells by more than two folds, while expression of miR-98, miR-181a, let-7f, let-7g, miR-424 and miR-563 was down-regulated by more than two folds. MiR-221, miR-181a, miR-451 and let-7f were further confirmed by Real time RT-PCR. The results of Real time RT-PCR were consistent with that of microarray.
Conclusion: MiR-98, miR-221, miR-181a, miR-155, miR-424, miR-451, let-7f, let-7g and miR-563 were differentially expressed between K562 and K562/A02 cells. Our results suggest that these differentially expressed miRNAs particularly miR-451 and let-7f may play an important role in the mechanisms of leukemia MDR.
引文
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