小分子Ku70干扰RNA逆转肺癌耐药的实验研究
摘要
肺癌耐药是导致化疗失败的主要原因。研究发现肿瘤细胞耐药的分子机制很复杂,除了药物进入肿瘤细胞内浓度减少外,靶基因突变、扩增、DNA断裂修复加速、细胞抗/促凋亡基因表达差异等都会导致肿瘤细胞耐药,对肺癌耐药分子机制的进一步探讨和逆转肺癌耐药方法的研究已成为当今研究的热点。Ku70是Ku蛋白的组成成份之一,是DNA磷酸蛋白激酶的重要成分,参与DNA转录调节,可独立参与细胞抗凋亡及DNA修复。近年来的研究和我们的前期工作已证明,Ku70能够调控肿瘤细胞对放射线的敏感性;并与人肿瘤化学治疗耐受性密切相关。本研究尝试从封闭Ku70入手,通过RT-PCR方法、Western-blot方法、微阵列PCR基因芯片(PCR-Array)技术、MTT法、流式细胞术及分光光度法等,进一步探讨Ku70与细胞凋亡、肺癌耐药的关系;探讨封闭Ku70通过调控抗/促凋亡基因的表达,促进细胞凋亡进而改变肺癌的耐药性,以期为临床逆转肺癌耐药,提高化疗疗效寻找新靶点和途径。实验结果表明:Ku70不仅与部分抗/促凋亡相关基因共同参与了肺癌耐药的形成,并且证明了Ku70可作为上游调控点调控部分抗/促凋亡基因的表达,影响肿瘤细胞的耐药性;通过细胞膜电位的改变、Caspase-3活性变化、线粒体凋亡途径及细胞形态学的改变等进一步明确沉默Ku70后耐药的肺癌细胞A549DDP细胞对化疗药物的敏感性明显增加,细胞呈促凋亡状态,细胞的耐药性明显下降。Ku70可能成为临床监测肿瘤耐药性变化的一个指标。
首次将Ku70与细胞凋亡、肿瘤耐药联系起来,探讨Ku70通过细胞凋亡途径参与肿瘤耐药的分子机制及逆转耐药的可能途径。采用先进的、PCR-Array基因芯片技术,明确了Ku70与抗/促凋亡相关基因的关系。应用小分子RNA干扰技术封闭Ku70,证明了Ku70可作为上游调控点调控部分抗/促凋亡基因的表达,影响、甚至逆转肿瘤细胞的耐药性。通过多种临床简便、易行的手段从细胞形态学、药代动力学及电生理学等不同角度,证明沉默Ku70后耐药的肺癌细胞对化疗药物的敏感性明显增加,细胞凋亡增多,肺癌细胞的耐药性得以逆转。本研究提示Ku70可能成为临床监测肿瘤耐药性变化的一个指标,并提供了逆转肺癌耐药的一个新靶点。
Lung cancer is the leading cause of cancer death in the world and has been growing rapidly in recent years, especially Non-small cell lung cancer (NSCLC). According to the latest statistic, NSCLC accounts for 80-85% in all lung cancer cases. The patients with III stage account for more than 40% in NSCLCs, and survival rate of those patients with 2 years life span was less than 20%. Chemotherapy is a last choice for such patients. However, the occurrence of chemotherapy-resistance on lung cancer is the most important factor in failure of chemotherapy. Unfortunately, there is no effective way to the reversion of drug-resistant currently. Study found that the molecular mechanism of drug-resistant on tumor cells is very complex. In addition to the reduction of drug concentration in the tumor cells, the target gene mutation, amplification, acceleration of DNA break repair, alteration of apoptosis genes expression are all contribute to the occurrence of drug-resistance the cells Abnormalities of apoptosis signal conduction, abnormal expression of apoptosis inhibitor can lead to drug resistance of tumor cells. Studies have shown that a variety of apoptosis inhibitory factor, tumor inhibitory factor, and a variety of apoptotic signal was transmitted into cell by signaling pathways, thereby, apoptosis occurred ultimately. Apoptosis is an important mechanism that can regulate development of body, control cell senescence and to maintain a stable internal environment. Initiation and conduct of apoptosis are precisely regulated, which are thought to be a unique and complicated process. In addition, apoptosis is also related to cell cycle, extracellular matrix, epidermal growth factor receptor, transmembrane protein,tubulin and so on.
Cisplatin is used to treat the lung cancer commonly as a chemotherapy drugs in clinic. Its toxicity and effectiveness of anti-tumor has been fully affirmed. However, drug-resistance can be easily formed by Cisplatin, including congenital drug resistance and acquired drug resistance, therefore, which greatly limits application of cisplatin in the clinic .Recently, study has also found that cisplatin-resistance was closely related to the differential expression of promoting apoptosis genes and anti-apoptosis genes. Further studies to explore the molecular mechanism on drug resistance and the methods of reversing drug resistance in lung cancer have become hot spots for the researcher and clinicians. Ku protein was first found in the blood serum of patients with connective tissue, and studies have shown that Ku protein plays an important role in cell apoptosis and DNA repair. Gama and other U.S. scientists have confirmed that expression of Ku protein was decreased in apoptotic cells. Recently, the study and our preliminary lab study found that Ku70 can sensitize tumor cells to radiation, and Ku70 is closely linked to the tolerance of chemotherapy on tumor in human. Studies have shown that Ku70-mRNA expression level in lung cancer was significantly higher than that in normal lung tissue (P <0.01); After several rounds of chemotherapy for lung cancer Ku70-mRNA expression was significantly higher than that without chemotherapy. (P <0.01).It has been proved that Ku70mRNA expression in drug-resistance of human non-small cell lung cancer A549DDP cells was significantly higher than that of primary human non-small cell lung cancer A549 cells by RT-PCR, which suggested that Ku70 may be involved in drug-resistance on human non-small cell lung cancer A549 cells. However, there are no studies showing that Ku70 was linked clearly between drug sensitivity with chemotherapy. Apoptosis resistance is one of the molecular mechanisms of drug resistance, therefore, To investigate the possibility of relationship among Ku70, apoptosis-related genes and non-small cell lung cancer resistant was the main topic between drug- resistance and reversion of drug-resistance through silence of Ku70 gene in non-small cell lung cancer. By using PCR-Array Assay gene chip technology, MTT assay, flow cytometry, spectrophotometry and immunofluorescence chemical technique, we have investigated the relationship between Ku70, apoptosis-related gene expression, apoptosis and the drug-resistant lung cancer cells. The goal of our study is to find a new way which might be helpful for the reversion of drug-resistance in lung cancer and could enhance the effects of chemotherapy for the lung cancer. At the same time, we anticipate that Ku70 might be a new target for the treatment of lung cancer and theoretical foundation on study of proteomics of apoptotic gene in drug-resistance. Methods and results:
Part one: The relationship between Ku70 and the drug-resistance in lung cell line
A549DDP cells were cultured with RPMI-1640 containing 10% fetal calf serum, gradually adding DDP to 1ug/ml. The small RNA fragment was transfected into cells by Lipofectamin 2000. Total RNA extraction and RT - PCR reactions were preformed according to the instructions from manufactory. Briefly, 1μg total RNA was used as template for reverse transcription, GAPDH was used as a positive control. The results showed that Ku70mRNA and protein in human drug-resistant non-small cell lung cacer cell lines were highly expressed. This suggested that drug-resistance of A549DDP cells is closely related to high expression of Ku70.
Part two: Detection of apoptosis-related genes expression in lung cancer cells by PCR Array gene chip technique.
The expression of cell apoptosis-related genes was detected in A549DDP with silence of Ku70 gene based on PCR-Array Assay Protocol. The results showed that promoting apoptosis genes including Bax, P53, P73 were significantly increased in A549DDP cells with silence of Ku70 gene compared to parental A549 cells and negative control group ( non-specific siRNA transfection group),anti-apoptosis gene TNFRSF1A, BFAR were significantly lower in A549DDP cells with silence of Ku70 gene compared to parental A549 cells and negative control group .
Part three: Effect of cisplatin on proliferation of A549DDP cell To observe the role cisplatin on drug resistance in A549DDP cells with silence of Ku70 gene, MTT assay was used to detect cell proliferation. Briefly, the absorption density (OD) was measured by automatic microplate reader at 570nm wavelength. Cell growth inhibition rate, IC50 were calculated. The results showed that after Ku70 silence, cell proliferation of A549DDP decreased significantly compared to control group, IC50 of cisplatin was significantly dropped from 37.89 +4.17 to 8.75 +0.62 ( P <0.01 ), suggesting that siRNA-Ku70 transfected cells can significantly reverse drug-resistance in A549DDP cells.
Part four: Apoptosis-inducing ability of cisplatin on Ku70 silence A549DDP cells
Apoptosis-inducing ability of cisplatin on A549DDP cells increased after Ku70 silence. Detected by flow cytometry, the number of sub-diploid cells characterized by apoptotic cells increased after transfection with siRNA-Ku70 in A549DDP than transfection with siRNA-control in A549DDP. The same result also appeared in the mitochondrial membrane potential test and Caspase-3 activity assay.
Part five: The diversification of cell morphology
Using Hocheast33258 staining, the smaller nuclear part, membrane shrinkage, chromatin aggregation can be seen dramatically in A549DDP cells with Ku70 compared to other control groups. Especially, nuclear chromatin and apoptotic bodies, which is regarded as the feature of late apoptosis, can be seen in A549DDP cells with Ku70 silence. Conclusion:
First, Ku70 and some of apoptosis-related genes were participated in the formation of drug-resistance on non small cell lung cancer. Secondly, Ku70 may be involved in drug- resistance of tumor cells. Ku70 may regulate expression of promoting / anti-apoptosis gene groups as the upstream regulatory site. Thirdly, The drug-resistance of lung cancer cells was reversed after silence of Ku70 by inducing apoptosis, such that was confirmed through the cell membrane potential changes, Caspase-3 activity, mitochondrial pathway of apoptosis and the changes of cell morphological .Fourthly, sensitivity to chemotherapeutic drug significantly increased, apoptosis increased and resistance to chemotherapeutic drug obviously decreased in resistant lung cancer cells.Finally, Ku70 may be an indicator to monitor changes of tumor drug resistance in clinic, and may become a new target that may reverse drug resistance of lung cancer.
This is the first study linking the Ku70, cell apoptosis and drug-resistance together. The relationship among them was investigated by advanced PCR-array method. Sensitivity to chemotherapeutic drug significantly increased, apoptosis increased and resistance to chemotherapeutic drug obviously decreased in resistant lung cancer cells.The possible ways was explored on reversing resistance of non-small cell lung cancer with application of small molecule RNA interference to close Ku70 gene. Ku70 may be an indicator to monitor changes of tumor drug resistance in clinical, and may become a new target that may reverse drug resistance of lung cancer. This study will provide a new concept for further research on apoptosis resistance proteomics.
首次将Ku70与细胞凋亡、肿瘤耐药联系起来,探讨Ku70通过细胞凋亡途径参与肿瘤耐药的分子机制及逆转耐药的可能途径。采用先进的、PCR-Array基因芯片技术,明确了Ku70与抗/促凋亡相关基因的关系。应用小分子RNA干扰技术封闭Ku70,证明了Ku70可作为上游调控点调控部分抗/促凋亡基因的表达,影响、甚至逆转肿瘤细胞的耐药性。通过多种临床简便、易行的手段从细胞形态学、药代动力学及电生理学等不同角度,证明沉默Ku70后耐药的肺癌细胞对化疗药物的敏感性明显增加,细胞凋亡增多,肺癌细胞的耐药性得以逆转。本研究提示Ku70可能成为临床监测肿瘤耐药性变化的一个指标,并提供了逆转肺癌耐药的一个新靶点。
Lung cancer is the leading cause of cancer death in the world and has been growing rapidly in recent years, especially Non-small cell lung cancer (NSCLC). According to the latest statistic, NSCLC accounts for 80-85% in all lung cancer cases. The patients with III stage account for more than 40% in NSCLCs, and survival rate of those patients with 2 years life span was less than 20%. Chemotherapy is a last choice for such patients. However, the occurrence of chemotherapy-resistance on lung cancer is the most important factor in failure of chemotherapy. Unfortunately, there is no effective way to the reversion of drug-resistant currently. Study found that the molecular mechanism of drug-resistant on tumor cells is very complex. In addition to the reduction of drug concentration in the tumor cells, the target gene mutation, amplification, acceleration of DNA break repair, alteration of apoptosis genes expression are all contribute to the occurrence of drug-resistance the cells Abnormalities of apoptosis signal conduction, abnormal expression of apoptosis inhibitor can lead to drug resistance of tumor cells. Studies have shown that a variety of apoptosis inhibitory factor, tumor inhibitory factor, and a variety of apoptotic signal was transmitted into cell by signaling pathways, thereby, apoptosis occurred ultimately. Apoptosis is an important mechanism that can regulate development of body, control cell senescence and to maintain a stable internal environment. Initiation and conduct of apoptosis are precisely regulated, which are thought to be a unique and complicated process. In addition, apoptosis is also related to cell cycle, extracellular matrix, epidermal growth factor receptor, transmembrane protein,tubulin and so on.
Cisplatin is used to treat the lung cancer commonly as a chemotherapy drugs in clinic. Its toxicity and effectiveness of anti-tumor has been fully affirmed. However, drug-resistance can be easily formed by Cisplatin, including congenital drug resistance and acquired drug resistance, therefore, which greatly limits application of cisplatin in the clinic .Recently, study has also found that cisplatin-resistance was closely related to the differential expression of promoting apoptosis genes and anti-apoptosis genes. Further studies to explore the molecular mechanism on drug resistance and the methods of reversing drug resistance in lung cancer have become hot spots for the researcher and clinicians. Ku protein was first found in the blood serum of patients with connective tissue, and studies have shown that Ku protein plays an important role in cell apoptosis and DNA repair. Gama and other U.S. scientists have confirmed that expression of Ku protein was decreased in apoptotic cells. Recently, the study and our preliminary lab study found that Ku70 can sensitize tumor cells to radiation, and Ku70 is closely linked to the tolerance of chemotherapy on tumor in human. Studies have shown that Ku70-mRNA expression level in lung cancer was significantly higher than that in normal lung tissue (P <0.01); After several rounds of chemotherapy for lung cancer Ku70-mRNA expression was significantly higher than that without chemotherapy. (P <0.01).It has been proved that Ku70mRNA expression in drug-resistance of human non-small cell lung cancer A549DDP cells was significantly higher than that of primary human non-small cell lung cancer A549 cells by RT-PCR, which suggested that Ku70 may be involved in drug-resistance on human non-small cell lung cancer A549 cells. However, there are no studies showing that Ku70 was linked clearly between drug sensitivity with chemotherapy. Apoptosis resistance is one of the molecular mechanisms of drug resistance, therefore, To investigate the possibility of relationship among Ku70, apoptosis-related genes and non-small cell lung cancer resistant was the main topic between drug- resistance and reversion of drug-resistance through silence of Ku70 gene in non-small cell lung cancer. By using PCR-Array Assay gene chip technology, MTT assay, flow cytometry, spectrophotometry and immunofluorescence chemical technique, we have investigated the relationship between Ku70, apoptosis-related gene expression, apoptosis and the drug-resistant lung cancer cells. The goal of our study is to find a new way which might be helpful for the reversion of drug-resistance in lung cancer and could enhance the effects of chemotherapy for the lung cancer. At the same time, we anticipate that Ku70 might be a new target for the treatment of lung cancer and theoretical foundation on study of proteomics of apoptotic gene in drug-resistance. Methods and results:
Part one: The relationship between Ku70 and the drug-resistance in lung cell line
A549DDP cells were cultured with RPMI-1640 containing 10% fetal calf serum, gradually adding DDP to 1ug/ml. The small RNA fragment was transfected into cells by Lipofectamin 2000. Total RNA extraction and RT - PCR reactions were preformed according to the instructions from manufactory. Briefly, 1μg total RNA was used as template for reverse transcription, GAPDH was used as a positive control. The results showed that Ku70mRNA and protein in human drug-resistant non-small cell lung cacer cell lines were highly expressed. This suggested that drug-resistance of A549DDP cells is closely related to high expression of Ku70.
Part two: Detection of apoptosis-related genes expression in lung cancer cells by PCR Array gene chip technique.
The expression of cell apoptosis-related genes was detected in A549DDP with silence of Ku70 gene based on PCR-Array Assay Protocol. The results showed that promoting apoptosis genes including Bax, P53, P73 were significantly increased in A549DDP cells with silence of Ku70 gene compared to parental A549 cells and negative control group ( non-specific siRNA transfection group),anti-apoptosis gene TNFRSF1A, BFAR were significantly lower in A549DDP cells with silence of Ku70 gene compared to parental A549 cells and negative control group .
Part three: Effect of cisplatin on proliferation of A549DDP cell To observe the role cisplatin on drug resistance in A549DDP cells with silence of Ku70 gene, MTT assay was used to detect cell proliferation. Briefly, the absorption density (OD) was measured by automatic microplate reader at 570nm wavelength. Cell growth inhibition rate, IC50 were calculated. The results showed that after Ku70 silence, cell proliferation of A549DDP decreased significantly compared to control group, IC50 of cisplatin was significantly dropped from 37.89 +4.17 to 8.75 +0.62 ( P <0.01 ), suggesting that siRNA-Ku70 transfected cells can significantly reverse drug-resistance in A549DDP cells.
Part four: Apoptosis-inducing ability of cisplatin on Ku70 silence A549DDP cells
Apoptosis-inducing ability of cisplatin on A549DDP cells increased after Ku70 silence. Detected by flow cytometry, the number of sub-diploid cells characterized by apoptotic cells increased after transfection with siRNA-Ku70 in A549DDP than transfection with siRNA-control in A549DDP. The same result also appeared in the mitochondrial membrane potential test and Caspase-3 activity assay.
Part five: The diversification of cell morphology
Using Hocheast33258 staining, the smaller nuclear part, membrane shrinkage, chromatin aggregation can be seen dramatically in A549DDP cells with Ku70 compared to other control groups. Especially, nuclear chromatin and apoptotic bodies, which is regarded as the feature of late apoptosis, can be seen in A549DDP cells with Ku70 silence. Conclusion:
First, Ku70 and some of apoptosis-related genes were participated in the formation of drug-resistance on non small cell lung cancer. Secondly, Ku70 may be involved in drug- resistance of tumor cells. Ku70 may regulate expression of promoting / anti-apoptosis gene groups as the upstream regulatory site. Thirdly, The drug-resistance of lung cancer cells was reversed after silence of Ku70 by inducing apoptosis, such that was confirmed through the cell membrane potential changes, Caspase-3 activity, mitochondrial pathway of apoptosis and the changes of cell morphological .Fourthly, sensitivity to chemotherapeutic drug significantly increased, apoptosis increased and resistance to chemotherapeutic drug obviously decreased in resistant lung cancer cells.Finally, Ku70 may be an indicator to monitor changes of tumor drug resistance in clinic, and may become a new target that may reverse drug resistance of lung cancer.
This is the first study linking the Ku70, cell apoptosis and drug-resistance together. The relationship among them was investigated by advanced PCR-array method. Sensitivity to chemotherapeutic drug significantly increased, apoptosis increased and resistance to chemotherapeutic drug obviously decreased in resistant lung cancer cells.The possible ways was explored on reversing resistance of non-small cell lung cancer with application of small molecule RNA interference to close Ku70 gene. Ku70 may be an indicator to monitor changes of tumor drug resistance in clinical, and may become a new target that may reverse drug resistance of lung cancer. This study will provide a new concept for further research on apoptosis resistance proteomics.
引文
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