喉癌多药耐药相关基因的筛查及中药逆转机制的研究
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摘要
喉癌是头颈部较为常见的恶性肿瘤之一,但在其综合治疗中,化疗的疗效欠佳,其主要原因是由于多药耐药性的产生。因此研究喉癌多药耐药相关基因的表达和调控机制以及筛选具有高效、低毒及多靶点性的中药逆转剂,对于提高喉癌的疗效,尤其是远期疗效,有重要的意义。
本文以长春新碱为诱导剂,采用药物浓度梯度递增法体外诱导建立了喉癌多药耐药Hep-2/v细胞模型。在此基础上应用基因芯片技术研究喉癌耐药细胞株Hep-2/v与其亲本细胞Hep-2之间的多药耐药相关miRNA及mRNA表达谱,筛选出差异表达的miRNA及mRNA,并利用Target数据库初步预测了miRNA的靶基因。MTT、流式细胞术及RT-PCR、Western Blot等技术研究中药对喉癌细胞的逆转特性及多药耐药相关基因mRNA的表达变化。统计学方法对实验结果进行了分析。
实验结果如下:
(1)以药物浓度梯度递增法成功构建了喉癌多药耐药细胞模型。
(2)AFFX miRNA芯片筛查出差异表达的多药耐药相关miRNA共7个,其中表达上调2个,表达下调5个。
(3)全基因组寡核酸微阵列芯片筛查出差异表达的多药耐药相关基因共有605个,其中表达上调的基因270个,表达水平下调的基因335个。
(4)结合基因芯片筛查结果,初步预测miRNA-210的靶基因为HTRA1及NUPR1;miRNA-923的靶基因为NUPR1;miRNA-93的靶基因为RGS10。
(5)粉防己碱及川芎嗪对Hep-2/v细胞的逆转倍数分别为2.22倍和1.88倍。RT-PCR及Western Blot检测显示粉防己碱能够逆转Hep-2/v细胞内HTRA1、RGS10及MDR1基因的表达,但对NUPR1基因的表达无逆转作用。
本实验对喉癌多药耐药相关基因的表达及调控机制进行了研究,表明miRNA可通过调控其靶基因的表达参与喉癌的多药耐药性。同时对中药的逆转机制进行了研究,并筛选出对喉癌耐药细胞有明显逆转作用的中药逆转剂,为进一步研究奠定了基础。
Laryngeal cancer is one of the relative common malignant tumors of head and neck. However, operations are still the main clinical methods currently, with poor curative effects of chemotherapy; MDR in laryngeal cancer is the main reason for restricting the effects of chemotherapy. Studies show that MDR in laryngeal cancer is an intricate process with multi-factor participation. In recent years, with the development of molecular biology, new MDR-related genes have been discovered continuously, but the molecular mechanism of MDR has not been fully clarified. MiRNA is a class of non-coding small RNA widely existed in plants and animals bodies, found in recent years, can make post-transcriptional negative regulation of gene expression. MiRNA widely participates in the occurrence and development of tumor, each miRNA can regulate about 200 gene expressions. It is predicted that miRNA can regulate MDR related gene expression, thus affecting the drug resistant phenotype of the laryngeal cancer cell. Hence this study establishes MDR of laryngeal cancer cell model, using gene microarray to screen MDR related miRNA and mRNA expressions, screen out the differential expression of miRNA and mRNA, and discuss the expression and regulatory mechanism of MDR in laryngeal cancer.
Aiming at the MDR of laryngeal cancer, the effective therapy mean is to use drug resistant reversal agent, however, the toxicity of the present developed reversal agent restricted its clinical application, due to the complexity of the MDR mechanism, only repressing and blocking the single drug resistant mechanism is not enough for eliminating MDR. TCM is rich in resources with multi-target function, so seeking new high efficiency and low toxicity anti-tumor medicine and TCM reversal agent has become the key to the current drug resistant tumor research. This study is on the basis of the molecular mechanism of the MDR, with MTT、RT-PCR and Western blot methods, it detects the reversal function and reversal mechanism of TCM on laryngeal cancer Hep-2/v, in order to find a TCM reversal agent with clinical value.
The experiments as follow:
(1) Establishment of MDR cell lines Hep-2/v in laryngeal cancer
With vincristinc (VCR) as the inducer, induce laryngeal cancer Hep-2 cell by increasing the concentration of drug. First using MTT method, detect that the half repressed concentration(IC50) of VCR on the parent cell in laryngeal cancer is 0.04umol/L, the first adding drug concentration is 0.02umol/L VCR, gradually increase the concentration of drug till the cells grow stably and go down to posterity. After 12 months, the cells can grow stably in the concentration of 0.96 umol/L, and this cell lines are named Hep-2/v. Through inverted microscope and electron microscope, observe the morphology change of tumor cells. MTT method is used to detect the MDR of tumor cells, the cell periodical distribution and the accumulation of Rh123 in cells are detected by flow cytometry, and the RT-PCR and Westren blot technology are used to detect the mdr1/P-gp expression on gene and protein levels.
Results: IC50 of Hep-2/v cells is detected as 1.8umol/L with MTT method, the drug resistant to VCR is 45 times of Hep-2 cells, while this cell line has drug resistance on different degree on the anti-cancer drugs with different structures and action mechanisms, such as 5-Fu and DDP. It is seen that the cells of Hep-2/v are relatively big, and the surface microvillus disappears, the intracellular granules increases, the adherence ability is reduced than that of the parent cells. The detection by flow cytometry shows that Hep-2/v cells are higher than cells in G0/G1 period, while cells in S period are reduced obviously; Rhodamine positive rate of Hep-2/v cells is much higher than Hep-2/v cells. RT-PCR and Western blot detected at the gene and protein levels shows expression difference of MDR1/P-gp between Hep-2 and Hep-2/v has a significant meaning.
(2) Screening and identification of MDR related miRNA in laryngeal cancer
Screen the expression profiling of MDR related miRNA in laryngeal cancer using AFFX miRNA microarray, SAM software screen the differential expression miRNA, the screening standard is: q-value≤5%, meanwhile, Fold Change≥2 or Fold Change≤0.5, including miRNA of expression upregulation and downregulation miRNAs. RT-PCR verifies the differential expression miRNA for the screening parts.
Results: AFFX miRNA micorarray screens out 7 MDR related miRNAs of differential expressions, among them, 2 are upregulated, and 5 are downregulated. RT-PCR detection shows that the expression changes of miRNA-210,miRNA-923 and miRNA-93 are in conformity with the microarray results.
(3) Screening and identification of MDR related mRNA in laryngeal cancer
Screen the expression profiling of MDR related mRNA in laryngeal cancer using whole-genome oligonucleotide microarray and SAM software to make analysis, FDR is controlled within 5%, in two times standard to screen differential expression genes. Target database predicts the target genes of miRNA-210, miRNA-923 and miRNA-93, and verify their expressions using RT-PCR method.
Results: The whole-genome oligonucleotide microarray screen out total 605 MDR related genes of differential expression, among them 270 are expression upregulated genes, and 335 are downregulated genes. Through primary prediction of Target database, the target genes of miRNA-210 are HTRA1 and NUPR1; the target gene of miRNA-923 is NUPR1; miRNA-93’s gene is RGS10. RT-PCR detection shows that the expression changes of HTRA1, NUPR1and RGS10 are in conformity with the microarray results.
(4) Studies on reversal MDR of TCM in laryngeal cancer
Detect the non-toxicity doses of TCM Tetrandrine and Tetramethylp yrazine on Hep-2/v cells, and the reversal function on Hep-2/v cells under the doses using MTT method. Flow cytometry detects the Rhodamine accumulation in Hep-2/v cells before and after TCM reversal, RT-PCR and Western Blot detect the expression changes of HTRA1, NUPR1, RGS10 and MDR1 genes.
Results: The reversal folds of Tetrandrine and tetramethylp yrazine to Hep-2/v cells are 2.22 and 1.88 respectively. Flow cytometry detection shows that the Rhodamine positive rate increased obviously after adding Tetrandrine and tetramethylp yrazine in Hep-2/v cells respectively. RT-PCR and Western Blot detection shows that Tetrandrine can reverse the expression of HTRA1, RGS10 and MDR1 genes in Hep-2/v cell, but the expression of NUPR1 no reversal.
Conclusions:
(1) Hep-2/v cells are MDR cells with stable drug resistance; Hep-2/v cells have basic biological characteristics of drug resistant cells, which are the ideal cell models for study the drug resistant mechanism and screening reversal agent.
(2) The differential expression miRNA screened out from Hep-2 and Hep-2/v cells in laryngeal cancer may take part in the occurrence of MDR of laryngeal cancer.
(3) The differential expression miRNA screened out from Hep-2 and Hep-2/v cells in laryngeal cancer might take part in the occurrence of MDR of laryngeal cancer. Gene microarray technology is an effective method to make parallel analysis of multi-gene, and discuss the MDR mechanism.
(4) MiRNA could regulate the expression of its target genes thus to affect the sensitivity of laryngeal cancer for chemotherapy.
(5) Tetrandrine could reverse the MDR of laryngeal cancer by a variety of mechanisms, which is able to become the MDR reversal agent with clinical value.
本文以长春新碱为诱导剂,采用药物浓度梯度递增法体外诱导建立了喉癌多药耐药Hep-2/v细胞模型。在此基础上应用基因芯片技术研究喉癌耐药细胞株Hep-2/v与其亲本细胞Hep-2之间的多药耐药相关miRNA及mRNA表达谱,筛选出差异表达的miRNA及mRNA,并利用Target数据库初步预测了miRNA的靶基因。MTT、流式细胞术及RT-PCR、Western Blot等技术研究中药对喉癌细胞的逆转特性及多药耐药相关基因mRNA的表达变化。统计学方法对实验结果进行了分析。
实验结果如下:
(1)以药物浓度梯度递增法成功构建了喉癌多药耐药细胞模型。
(2)AFFX miRNA芯片筛查出差异表达的多药耐药相关miRNA共7个,其中表达上调2个,表达下调5个。
(3)全基因组寡核酸微阵列芯片筛查出差异表达的多药耐药相关基因共有605个,其中表达上调的基因270个,表达水平下调的基因335个。
(4)结合基因芯片筛查结果,初步预测miRNA-210的靶基因为HTRA1及NUPR1;miRNA-923的靶基因为NUPR1;miRNA-93的靶基因为RGS10。
(5)粉防己碱及川芎嗪对Hep-2/v细胞的逆转倍数分别为2.22倍和1.88倍。RT-PCR及Western Blot检测显示粉防己碱能够逆转Hep-2/v细胞内HTRA1、RGS10及MDR1基因的表达,但对NUPR1基因的表达无逆转作用。
本实验对喉癌多药耐药相关基因的表达及调控机制进行了研究,表明miRNA可通过调控其靶基因的表达参与喉癌的多药耐药性。同时对中药的逆转机制进行了研究,并筛选出对喉癌耐药细胞有明显逆转作用的中药逆转剂,为进一步研究奠定了基础。
Laryngeal cancer is one of the relative common malignant tumors of head and neck. However, operations are still the main clinical methods currently, with poor curative effects of chemotherapy; MDR in laryngeal cancer is the main reason for restricting the effects of chemotherapy. Studies show that MDR in laryngeal cancer is an intricate process with multi-factor participation. In recent years, with the development of molecular biology, new MDR-related genes have been discovered continuously, but the molecular mechanism of MDR has not been fully clarified. MiRNA is a class of non-coding small RNA widely existed in plants and animals bodies, found in recent years, can make post-transcriptional negative regulation of gene expression. MiRNA widely participates in the occurrence and development of tumor, each miRNA can regulate about 200 gene expressions. It is predicted that miRNA can regulate MDR related gene expression, thus affecting the drug resistant phenotype of the laryngeal cancer cell. Hence this study establishes MDR of laryngeal cancer cell model, using gene microarray to screen MDR related miRNA and mRNA expressions, screen out the differential expression of miRNA and mRNA, and discuss the expression and regulatory mechanism of MDR in laryngeal cancer.
Aiming at the MDR of laryngeal cancer, the effective therapy mean is to use drug resistant reversal agent, however, the toxicity of the present developed reversal agent restricted its clinical application, due to the complexity of the MDR mechanism, only repressing and blocking the single drug resistant mechanism is not enough for eliminating MDR. TCM is rich in resources with multi-target function, so seeking new high efficiency and low toxicity anti-tumor medicine and TCM reversal agent has become the key to the current drug resistant tumor research. This study is on the basis of the molecular mechanism of the MDR, with MTT、RT-PCR and Western blot methods, it detects the reversal function and reversal mechanism of TCM on laryngeal cancer Hep-2/v, in order to find a TCM reversal agent with clinical value.
The experiments as follow:
(1) Establishment of MDR cell lines Hep-2/v in laryngeal cancer
With vincristinc (VCR) as the inducer, induce laryngeal cancer Hep-2 cell by increasing the concentration of drug. First using MTT method, detect that the half repressed concentration(IC50) of VCR on the parent cell in laryngeal cancer is 0.04umol/L, the first adding drug concentration is 0.02umol/L VCR, gradually increase the concentration of drug till the cells grow stably and go down to posterity. After 12 months, the cells can grow stably in the concentration of 0.96 umol/L, and this cell lines are named Hep-2/v. Through inverted microscope and electron microscope, observe the morphology change of tumor cells. MTT method is used to detect the MDR of tumor cells, the cell periodical distribution and the accumulation of Rh123 in cells are detected by flow cytometry, and the RT-PCR and Westren blot technology are used to detect the mdr1/P-gp expression on gene and protein levels.
Results: IC50 of Hep-2/v cells is detected as 1.8umol/L with MTT method, the drug resistant to VCR is 45 times of Hep-2 cells, while this cell line has drug resistance on different degree on the anti-cancer drugs with different structures and action mechanisms, such as 5-Fu and DDP. It is seen that the cells of Hep-2/v are relatively big, and the surface microvillus disappears, the intracellular granules increases, the adherence ability is reduced than that of the parent cells. The detection by flow cytometry shows that Hep-2/v cells are higher than cells in G0/G1 period, while cells in S period are reduced obviously; Rhodamine positive rate of Hep-2/v cells is much higher than Hep-2/v cells. RT-PCR and Western blot detected at the gene and protein levels shows expression difference of MDR1/P-gp between Hep-2 and Hep-2/v has a significant meaning.
(2) Screening and identification of MDR related miRNA in laryngeal cancer
Screen the expression profiling of MDR related miRNA in laryngeal cancer using AFFX miRNA microarray, SAM software screen the differential expression miRNA, the screening standard is: q-value≤5%, meanwhile, Fold Change≥2 or Fold Change≤0.5, including miRNA of expression upregulation and downregulation miRNAs. RT-PCR verifies the differential expression miRNA for the screening parts.
Results: AFFX miRNA micorarray screens out 7 MDR related miRNAs of differential expressions, among them, 2 are upregulated, and 5 are downregulated. RT-PCR detection shows that the expression changes of miRNA-210,miRNA-923 and miRNA-93 are in conformity with the microarray results.
(3) Screening and identification of MDR related mRNA in laryngeal cancer
Screen the expression profiling of MDR related mRNA in laryngeal cancer using whole-genome oligonucleotide microarray and SAM software to make analysis, FDR is controlled within 5%, in two times standard to screen differential expression genes. Target database predicts the target genes of miRNA-210, miRNA-923 and miRNA-93, and verify their expressions using RT-PCR method.
Results: The whole-genome oligonucleotide microarray screen out total 605 MDR related genes of differential expression, among them 270 are expression upregulated genes, and 335 are downregulated genes. Through primary prediction of Target database, the target genes of miRNA-210 are HTRA1 and NUPR1; the target gene of miRNA-923 is NUPR1; miRNA-93’s gene is RGS10. RT-PCR detection shows that the expression changes of HTRA1, NUPR1and RGS10 are in conformity with the microarray results.
(4) Studies on reversal MDR of TCM in laryngeal cancer
Detect the non-toxicity doses of TCM Tetrandrine and Tetramethylp yrazine on Hep-2/v cells, and the reversal function on Hep-2/v cells under the doses using MTT method. Flow cytometry detects the Rhodamine accumulation in Hep-2/v cells before and after TCM reversal, RT-PCR and Western Blot detect the expression changes of HTRA1, NUPR1, RGS10 and MDR1 genes.
Results: The reversal folds of Tetrandrine and tetramethylp yrazine to Hep-2/v cells are 2.22 and 1.88 respectively. Flow cytometry detection shows that the Rhodamine positive rate increased obviously after adding Tetrandrine and tetramethylp yrazine in Hep-2/v cells respectively. RT-PCR and Western Blot detection shows that Tetrandrine can reverse the expression of HTRA1, RGS10 and MDR1 genes in Hep-2/v cell, but the expression of NUPR1 no reversal.
Conclusions:
(1) Hep-2/v cells are MDR cells with stable drug resistance; Hep-2/v cells have basic biological characteristics of drug resistant cells, which are the ideal cell models for study the drug resistant mechanism and screening reversal agent.
(2) The differential expression miRNA screened out from Hep-2 and Hep-2/v cells in laryngeal cancer may take part in the occurrence of MDR of laryngeal cancer.
(3) The differential expression miRNA screened out from Hep-2 and Hep-2/v cells in laryngeal cancer might take part in the occurrence of MDR of laryngeal cancer. Gene microarray technology is an effective method to make parallel analysis of multi-gene, and discuss the MDR mechanism.
(4) MiRNA could regulate the expression of its target genes thus to affect the sensitivity of laryngeal cancer for chemotherapy.
(5) Tetrandrine could reverse the MDR of laryngeal cancer by a variety of mechanisms, which is able to become the MDR reversal agent with clinical value.
引文
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