miR-133a通过抑制ATP7B表达改变喉癌细胞的多药耐药性

发布时间：2018-06-18 12:25

本文选题：喉癌 + miR-133a　；参考：《吉林大学》2015年博士论文

【摘要】：研究背景及目的喉癌是我国北方地区发病率较高的肿瘤之一,发病率约占全身肿瘤的1~5%。尽管目前在喉癌的诊断和治疗方面取得了很大进展,但部分喉癌患者预后差,生存率低。对于晚期不能手术和术后复发的患者,常采用化学治疗方法,但化疗效果不佳,推测与喉癌细胞对化疗药物耐药有关。肿瘤细胞的多药耐药是化疗失败的主要原因之一,喉癌的化疗药物的耐药常表现为多药耐药。逆转肿瘤细胞的耐药一直是肿瘤研究的一个重点,近年来通过mi RNAs逆转肿瘤耐药的研究取得了进展,但应用mi RNAs针对喉癌多药耐药的干预研究较少。本论文采用微阵列芯片技术研究喉鳞癌及癌旁正常黏膜中mi RNAs表达谱差异,筛选参与喉癌发生的特异mi RNAs。通过比较特异mi RNA在亲本细胞Hep-2细胞与VCR耐药细胞株Hep-2/v之间的表达差异验证与喉癌多药耐药有关的mi RNAs。材料和方法分离8例喉癌组织及其对应癌旁正常黏膜中的mi RNA,采用荧光染料Cy3标记,并与制备好的哺乳动物mi RNA芯片V3.0杂交,用Lux Scan 10K/A扫描仪扫描荧光信号,采用Lux Scan3.0图像分析软件对芯片图像进行分析,用SAM和Cluster3.0软件进行差异基因筛选和聚类分析,对部分差异表达mi RNA采用RNA加尾和引物延伸实时定量RT-PCR法对芯片检测结果进行验证。通过给予50u M、100u M和200u M的顺铂作用Hep-2/v细胞。应用细胞增殖实验、Tunel细胞凋亡确定Hep-2和Hep-2/v对顺铂耐药的差别,确认Hep-2/v耐药株存在多药耐药性。采用Poly加尾PCR的方法检测正常咽上皮细胞NP69,Hep-2和Hep-2/v细胞mi R-133a的水平;采用免疫荧光染色和PCR、Western Blotting方法同时检测两种细胞在加入顺铂后ATP7B的变化。采用慢病毒重组载体p EZX-mi R-133a转染Hep-2细胞和Hep-2/v耐药细胞株,CCK8细胞活力实验观察不同浓度顺铂对细胞存活的影响;通过免疫荧光染色和实时定量PCR方法检测p EZX-mi R-133a转染细胞ATP7B的转录和蛋白表达变化。结果1.通过mi RNA表达谱分析共筛选出47个喉癌相关的差异表达mi RNAs基因,包括24个在喉癌组织中表达下调的mi RNAs和23个在喉癌组织中表达上调的mi RNAs。其中在喉癌组织中下调5倍以上的有mi R-1、mi R-486-5p、mi R-206、mi R-487a、mi R-375、mi R-422a、mi R-144、hsa-mi R-384、mi R-378和mi R-133a共10个。在喉癌组织中表达上调3倍以上的mi RNA有3个,分别是mi R-93、mi R-31和mi R-20b。在喉癌差异表达的mi RNA中有13个成5簇共表达,分别位于8、13、14、18和X染色体上。荧光定量RT PCR结果与mi RNAs芯片的结果较符合。2.50u M和100u M的顺铂作用24h后,Hep-2细胞存活为71%和73%,而对Hep-2/v的细胞的存活没有影响统计学分析有显著差异(p0.01);Hep-2/v的生存在100u M时为85%明显高于同剂量的Hep-2细胞(p0.05);50u M的顺铂作用Hep-2细胞24h,细胞凋亡指数为31.12±4.75,Hep-2/v细胞的凋亡指数仅为3.5±1.23,卡方检验p0.01;Hep-2/v细胞的ATP7B蛋白表达明显高于Hep-2细胞。3.顺铂作用后Hep-2和Hep-2/v表达ATP7B蛋白与对照组相比增强。实时定量PCR检测顺铂作用后不同时间ATP7A和ATP7B基因转录变化,结果发现50u M顺铂能够诱导Hep-2和Hep-2/v表达ATP7B,Hep-2/v细胞在作用后3h表达增强,6h达到峰值,持续表达24h无明显变化。Hep-2细胞和Hep-2/v表达ATP7B的荧光值明显高于NP-69细胞。4.Hep-2细胞mi R-133a的表达量与NP-69细胞相比下降4.76±0.54倍,Hep-2/v细胞的mi R-133a的表达量与NP-69相比下降5.55±0.14倍。重组慢病毒介导mi R-133a转染Hep-2细胞和Hep-2/v细胞48h后CCK8检测细胞存活。结果显示顺铂50u M和100u M作用组,转染p EZX-Control的Hep-2/v细胞存活明显高于转染p EZX-mi R-133a的Hep-2/v细胞;mi R-133a能够降低Hep-2/v细胞ATP7B的转录和表达。结论mi R-133a在喉癌组织中表达下调,喉癌组织中差异表达的mi R-133a可能是参与喉癌的发生的因素之一。喉癌耐药细胞Hep-2/v的ATP7B蛋白表达明显高于Hep-2细胞,ATP7B蛋白表达增高为喉癌细胞对顺铂耐药的研究提供了一个新思路。实验中发现了mi R-133a能重新提高喉癌耐药细胞对顺铂的化疗敏感性这一现象,推测其机制可能与mi R-133a下调ATP7B蛋白表达有关。
[Abstract]:Background and objective laryngeal cancer is one of the higher incidence of cancer in northern China. The incidence of the disease is about 1~5%. of the whole body tumor. Although great progress has been made in the diagnosis and treatment of larynx cancer, some patients with larynx cancer have poor prognosis and low survival rate. But the effect of chemotherapy is not good. It is presumed that the drug resistance of the laryngeal cancer cells is related to the drug resistance. The multidrug resistance of the tumor cells is one of the main reasons for the failure of chemotherapy. The drug resistance of the larynx cancer is often characterized by multidrug resistance. The reversal of the drug resistance of the tumor cells has been a key point in the cancer research. In recent years, the reversal of the tumor resistance through mi RNAs Progress has been made in the study, but the application of MI RNAs for multidrug resistance of larynx is less. This paper uses microarray technology to study the MI RNAs expression profiles in laryngeal squamous cell carcinoma and normal mucosa adjacent to cancer, and to screen specific mi RNAs. involved in the occurrence of larynx cancer by comparing the specific mi RNA in the parent cell Hep-2 cells and VCR resistant cell He. The expression difference between p-2/v and MI RNAs. related to multidrug resistance of larynx was used to separate 8 cases of larynx tissues and the MI RNA corresponding to the normal mucosa adjacent to the cancer, using a fluorescent dye Cy3 marker, and a V3.0 hybridization with a prepared mammalian mi RNA chip. The fluorescent signal was scanned with Lux Scan 10K/A scanner, and the fluorescent signal was scanned by Lux Scan 10K/A scanner. Analysis software was used to analyze the chip image. The differential gene screening and cluster analysis were carried out with SAM and Cluster3.0 software. A partial differential expression of MI RNA was verified by RNA plus tail and primer extension real-time quantitative RT-PCR method. By giving 50U M, 100u M and 200u M with cisplatin, the cell proliferation was applied. Tunel cell apoptosis determined the difference between Hep-2 and Hep-2/v on cisplatin resistance, and confirmed the multidrug resistance of Hep-2/v resistant strains. Poly plus tail PCR was used to detect the level of MI R-133a in normal pharyngeal epithelial cells, NP69, Hep-2 and Hep-2/v cells, and two cells were simultaneously detected by immunofluorescence staining and PCR. Changes in ATP7B after cisplatin. Transfection of Hep-2 and Hep-2/v resistant cells with lentivirus recombinant vector p EZX-mi R-133a. The effect of cisplatin on cell viability was observed at different concentrations in CCK8 cell viability test. The transcriptional and protein expression changes of P EZX-mi R-133a transfected cell ATP7B were detected by immunofluorescence staining and real-time quantitative PCR method. Results 1. the differential expression of MI RNAs gene related to 47 laryngomas, including 24 down regulated mi RNAs in Laryngic carcinoma and 23 mi RNAs. in Laryngic cancer tissues, was screened by Mi RNA expression analysis. R-144, hsa-mi R-384, MI R-378 and MI R-133a are 10. There are 3 mi RNA expressed in the larynx cancer tissues, which are mi R-93. 13 of 5 clusters are co expressed in the differential expression of the larynx cancer. After 24h of.50u M and 100u M, Hep-2 cells survived 71% and 73%, while the survival of Hep-2/v cells had no significant difference (P0.01), and the survival of Hep-2/v was 85% higher than the same dose of Hep-2 cells at 100u M; the apoptosis index was 31.12 + 4.75. The apoptotic index of V cells was only 3.5 + 1.23, chi square test was P0.01, and the expression of ATP7B protein in Hep-2/v cells was significantly higher than that of.3. cisplatin in Hep-2 cells. Hep-2 and Hep-2/v expressed ATP7B protein compared with those of the control group. Hep-2 and Hep-2/v expressed ATP7B, and the expression of 3H was enhanced after the action of Hep-2/v cells, and the 6h reached its peak value. There was no obvious change in the expression of 24h. The fluorescent value of.Hep-2 cells and Hep-2/v expressed ATP7B was significantly higher than that of NP-69 cell.4.Hep-2 cells. P-69 decreased by 5.55 + 0.14 times. Recombinant lentivirus mediated mi R-133a transfection of Hep-2 cells and Hep-2/v cells for 48h to detect cell survival. The results showed that cisplatin 50U M and 100u M cells were significantly higher than those transfected with Hep-2 cells. Conclusion the expression of MI R-133a in larynx tissues is downregulated. The differential expression of MI R-133a in larynx cancer tissues may be one of the factors involved in the occurrence of larynx cancer. The expression of ATP7B protein in the Hep-2/v of larynx cancer cells is obviously higher than that of Hep-2 cells. The increase of ATP7B protein expression provides a new thought for the study of the larynx cell cell for the study of cisplatin resistance. In the experiment, it was found that MI R-133a could improve the chemosensitivity of cisplatin in larynx resistant cells. It is presumed that the mechanism may be related to the down regulation of ATP7B protein expression by Mi R-133a.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.65

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