miR-495表达对非小细胞肺癌细胞生物学行为的影响

发布时间：2018-04-22 11:30

本文选题：非小细胞肺癌 + mi　；参考：《郑州大学》2015年博士论文

【摘要】：背景和目的肺癌是死亡率较高的肿瘤之一,其中非小细胞肺癌占几乎80%。尽管诊治水平不断进步,仍有30%-40%的非小细胞肺癌患者因肿瘤的转移扩散预后极差。而对肿瘤发生发展的潜在机制研究有助于我们进一步认识了解肿瘤的发病机制,并开发新的治疗手段。微小RNA,又名micro RNA(mi RNA),是非编码、单链、小分子RNA(22个左右的核糖核苷酸),在物种之间及进化上都具有高度的保守性,所有的真核细胞生物几乎都可以有表达。mi RNA与其靶基因m RNA 3’端非编码区(3’UTR)结合,主要结合的方式为完全或者不完全的互补结合,调控着靶基因的转录或者翻译水平,进而影响细胞的生物学行为的变化,包括增殖、分化、细胞周期和凋亡。从而也使得mi RNA在肿瘤发生发展的过程中扮演着癌基因或者抑癌基因的重要角色。随着研究进展,在多种恶性肿瘤中越来越多的mi RNA被发现存在异常表达,包括大肠癌、白血病、乳腺癌、肺癌、前列腺癌及脑癌等。在非小细胞肺癌组织中mi R-495的表达水平及具体的生物学作用和调控机制需进一步探讨。转移相关蛋白3(metastasisassociated protein 3,MTA3)是转移相关蛋白家族中一种关键的共调节蛋白,该家族还包括MTA1和MTA2。证据显示在包括食管、肺、乳腺、子宫和鼻咽等在内的多种癌症中MTA3表达上调,并能够通过作用于多种细胞信号通路,影响肿瘤的发展与转移。在Agilent mi RNA芯片检测发现mi R-495低表达的基础上,通过生物信息学分析,我们预测MTA3基因为mi R-495的靶基因,根据靶基因在肺癌的研究,本课题拟首先验证mi R-495在非小细胞肺癌中的表达,同时研究MTA3在非小细胞肺癌中的表达情况,然后观察调控mi R-495表达对非小细胞肺癌细胞Calu-3的生长、侵袭和细胞周期的影响,并同时初步探讨mi R-495调控细胞生物学行为改变的分子机制。本课题包括以下两部分:第一部分:非小细胞肺癌组织中mi RNA和MTA3异常表达及其与临床病理特征的相关性分析;第二部分:体内、体外调控mi R-495和表达对非小细胞肺癌细胞系Calu-3的生长、侵袭和细胞周期的影响,并对其肿瘤抑制作用机制进行初步研究。第一部分非小细胞肺癌组织中mi R-495和MAT3表达及与临床病理学特征的相关性分析方法 1.收集标本,72例非小细胞肺癌组织与其向对应的72例癌旁正常组织标本。2.运用Agilent mi RNA芯片的方法检测3例非小细胞肺癌组织与与其向对应的72例癌旁正常组织标本中mi RNA表达情况。3.运用q RT-PCR检测72例标本中mi R-495的表达水平,同时运用q RT-PCR和Western blot方法检测72例标本中MTA3的表达水平,并应用Spearman相关分析二者的相关性。4.依据mi R-495和MTA3的表达水平,运用双变量相关性分别分析二者与非小细胞肺癌患者性别、年龄、病理类型、肿瘤分化程度、TNM分期及其有无淋巴结转移之间的相关性。5.统计学分析:采用SPSS 21.0统计软件进行数据分析,单因素方差用于分析多项指标间的比较,LSD-t检验用于比较两项指标间的比较;t检验用于计量资料;Spearman相关分析应用于相关性检验,以α=0.05为显著性水准。结果 1.Agilent mi RNA芯片检测分析得到56个差异表达3倍以上的mi RNAs,其中有26个mi RNAs表达为上调,30个mi RNAs表达下调。2.mi R-495在非小细胞肺癌组织中低表达,MTA3呈高表达水平,且二者呈负相关(P0.05)。3.通过双变量相关性的分析结果发现非小细胞肺癌组织中mi R-495和MTA3的表达水平均与有无淋巴结的转移、分化程度及TNM分期相关(P0.05),与患者的年龄、性别和病理类型无相关性(P0.05)。第二部分调控mi R-495的表达对非小细胞肺癌细胞系Calu-3生物学行为的影响及其相关机制的初步分析方法 1.运用荧光定量PCR和Western-blot检测非小细胞肺癌细胞系A549,Calu-3,H460,H1299,H1650和SPC-A-1中mi R-495和MTA3的表达水平。2.分别合成mi R-495agomir和mi R-495scramble,选择对数生长期的Calu-3细胞株,根据实验分组分别使用LipofectamineTM2000进行脂质体的转染。3.荧光定量PCR检测各实验组细胞中mi R-495的表达变化。4.运用CCK8方法和克隆形成实验检测各实验组Calu-3细胞增殖和生长情况变化。5.裸鼠移植瘤实验检测各实验组移植瘤生长及MTA3蛋白表达的情况。6.流式细胞仪检测各实验组Calu-3细胞周期的变化及Western blot检测相关周期蛋白表达的情况。7.应用Transwell侵袭实验和划痕实验检测各实验组的Calu-3细胞侵袭转移能力的变化。8.采用生物信息学分析软件预测mi R-495的作用靶标。构建MTA3 3’UTR区的野生型及突变型重组双荧光报告基因载体,分别将其与mi R-495 agomir或者mi R-495scramble共转染入Calu-3细胞中,通过双荧光素酶报告实验证实mi R-495的作用靶标。9.构建无3’UTR区MTA3的表达载体,单独转入或与mi R-495 agomir共转染入Calu-3细胞中,通过restore方法分析mi R-495靶向调控MTA3表达的作用机制。10.比较Calu-3细胞中转染si RNA-MTA3与过表达mi R-495对Calu-3细胞增殖、侵袭和细胞周期的影响。11.统计学分析:采用SPSS 21.0软件进行数据分析。结果 1.非小细胞肺癌细胞系中,mi R-495低表达水平见于A549,Calu-3,H460,H1650以及SPC-A-1细胞,但在H1299细胞中相对高表达(P0.05)。MTA3在A549,Calu-3,H460,H1650以及SPC-A-1细胞呈现高表达水平,但在H1299细胞中相对低表达(P0.05)。2.mi R-495组非小细胞肺癌细胞Calu-3的mi R-495表达水平明显高于NC组和Mock组(P0.05)。提示转染mi R-495后,Calu-3细胞的mi R-495表达水平上调。3.CCK-8实验和克隆形成实验结果表明,与NC组和Mock组相比,mi R-495组Calu-3细胞的增殖能力在转染后2天开始出现显著抑制(P0.05),并且随时间延长日益显著。4.mi R-495组裸鼠的生长在转染后2周后出现抑制,并且随时间的延长而日益显著(P0.05)。4周后处死小鼠,剥离肿瘤组织,计算裸鼠移植瘤体积,结果显示mi R-495组肿瘤体积显著小于NC组和Mock组肿瘤体积(P0.05)。各组裸鼠移植瘤中MTA3的表达在mi R-495组的表达水平与相对于NC组和Mock组中的表达水平明显升高(P0.05)。5.流式细胞仪分析结果显示,相对于NC组和Mock组mi R-495组的细胞处于G0/G1期比例显著升高,而处于S期比例降低(P0.05)。Western blot的方法分析细胞周期相关蛋白的表达变化,相比NC组和Mock组,mi R-495组周期蛋白A,周期蛋白D1,磷酸化Rb的表达水平均显著降低(P0.05)。6.Transwell和划痕实验检测结果显示mi R-495组的细胞侵袭能力显著低于NC组和Mock组(P0.05)。说明mi R-495过表达可抑制Calu-3细胞的侵袭能力。7.生物信息学分析和双荧光素酶报告实验结果提示,MTA3是mi R-495的作用靶标。8.Restore实验结果提示,无3'UTR区的MTA3的表达不受mi R-495的调控,且其作用可覆盖mi R-495上调对非小细胞肺癌细胞生物学行为的作用。9.Calu-3细胞中si RNA-MTA3转染与过表达mi R-495的作用比较结果进一步提示了mi R-495通过作用于MTA3的3'UTR区,负向调控MTA3蛋白的表达,导致非小细胞肺癌细胞的增殖、侵袭能力受到抑制,细胞周期受到阻滞。结论 1.非小细胞肺癌组织中检测到56个差异表达的mi RNAs,其中有26个mi RNAs表达为上调,30个mi RNAs表达下调。非小细胞肺癌组织中,mi R-495呈低表达,其表达的水平均与有无淋巴结的转移、分化程度以及TNM分期相关,且与MTA3的表达水平具有负相关性。2.在非小细胞肺癌细胞Calu-3中上调mi R-495表达通过作用于MTA3的3'UTR区,负向调控MTA3的表达,导致Calu-3细胞生长和侵袭抑制,细胞周期阻滞。mi R-495有望成为非小细胞肺癌治疗的新靶点。
[Abstract]:Background and objective lung cancer is one of the higher mortality rates, in which non small cell lung cancer accounts for almost 80%., although the level of diagnosis and treatment is progressing, the prognosis of non small cell lung cancer patients with 30%-40% is still very poor because of the metastasis and diffusion of tumor. The small RNA, also known as micro RNA (MI RNA), is a non coding, single chain, small molecule RNA (about 22 ribonucleotides), and is highly conserved between species and evolution, and all eukaryotic cells can almost have a combination of.Mi RNA with its target gene m RNA 3 'end non coding region (3' UTR). The main combination is a complete or incomplete complementary combination that regulates the transcriptional or translation level of the target gene, thereby affecting the changes in biological behavior of the cells, including proliferation, differentiation, cell cycle and apoptosis, thus making mi RNA an important corner of the oncogene or tumor suppressor gene in the progression of tumor development. As the research progresses, more and more mi RNA in a variety of malignant tumors have been found to have abnormal expression, including colorectal cancer, leukemia, breast cancer, lung cancer, prostate cancer and brain cancer. The expression level of MI R-495 in non-small cell lung cancer and the specific biological use and regulation mechanism need to be further discussed. Transfer related protein 3 (M Etastasisassociated protein 3, MTA3) is a key co regulated protein in the metastasis related protein family. The family also includes MTA1 and MTA2. evidence that the expression of MTA3 is up-regulated in a variety of cancers, including the esophagus, lungs, breast, uterus and nasopharynx, and can affect the development of cancer by acting on a variety of cell signaling pathways. On the basis of the detection of the low expression of MI R-495 by Agilent mi RNA chip, we predict that the MTA3 gene is the target gene of MI R-495 through bioinformatics analysis. According to the target gene in the study of lung cancer, this topic is to first verify the expression of MI R-495 in non-small cell lung cancer and to study the expression of MTA3 in non-small cell lung cancer. And then observe the effects of MI R-495 expression on the growth, invasion and cell cycle of non small cell lung cancer cell Calu-3, and discuss the molecular mechanism of MI R-495 regulating cell biological behavior changes. This topic includes the following two parts: the first part: the abnormal expression of MI RNA and MTA3 in non-small cell lung cancer and its clinical significance. The correlation analysis of pathological features; the second part: in vitro, the effect of MI R-495 and expression on the growth, invasion and cell cycle of non small cell lung cancer cell line Calu-3 in vitro, and the mechanism of its tumor inhibition are preliminarily studied. The expression of MI R-495 and MAT3 in the first part of non small cell lung cancer and its clinicopathological features Correlation analysis method 1. specimens were collected, 72 non-small cell lung cancer tissues and 72 normal paracancerous tissue specimens of corresponding.2. were used to detect the expression of MI RNA in 3 non small cell lung cancer tissues and 72 cases of normal tissues adjacent to cancer by Agilent mi RNA chip. The MI R-4 in 72 cases of Q RT-PCR detected by Q RT-PCR was used for MI R-4. The expression level of 95 was 95, and the expression level of MTA3 in 72 specimens was detected by Q RT-PCR and Western blot, and the correlation.4. of the Spearman correlation analysis was based on the expression level of MI R-495 and MTA3. The sex, age, pathological type and tumor differentiation of the two patients with non-small cell lung cancer were analyzed by bivariate correlation. The correlation of degree, TNM staging and the correlation between lymph node metastasis and.5. statistical analysis: using SPSS 21 statistical software for data analysis, single factor variance used to compare the comparison among multiple indexes, LSD-t test was used to compare the comparison among the two indexes; t test was used to measure the material; Spearman correlation analysis was applied to correlation test, to alpha Results =0.05 was a significant level. Results 1.Agilent mi RNA chip detection and analysis obtained 56 differential expressions of more than 3 times of MI RNAs, of which 26 mi RNAs were expressed as up, 30 mi RNAs expressed downregulation of.2.mi R-495 in non small cell lung cancer tissues and high expression, and the two were negatively correlated by bivariate correlation. The results of the analysis showed that the expression level of MI R-495 and MTA3 in non small cell lung cancer tissues was related to the metastasis of lymph node, the degree of differentiation and the TNM staging (P0.05), and no correlation with the age, sex and pathological type of the patients (P0.05). The second part regulated the expression of MI R-495 in the expression of Calu-3 biological behavior of non-small cell lung cancer cell lines. Preliminary analysis of its related mechanisms and its related mechanisms 1. using fluorescent quantitative PCR and Western-blot to detect non small cell lung cancer cell lines A549, Calu-3, H460, H1299, MI R-495 and MTA3 expression level.2. in H1650 and SPC-A-1, respectively. The transfection of liposomes using LipofectamineTM2000 to detect the expression of MI R-495 in the cells of each experimental group by transfection of.3. PCR and.4. using CCK8 method and clone formation test to detect the proliferation and growth of Calu-3 cells in each experimental group. The growth of the transplanted tumor and the expression of MTA3 protein in the experimental group were detected by the experimental group of.5. nude mice. Detection of the changes of Calu-3 cell cycle in each experimental group by.6. flow cytometry and the expression of periodic protein by Western blot detection.7. application of Transwell invasion experiment and scratch test to detect the change of Calu-3 cell invasion and metastasis ability of each experimental group.8. using bioinformatics analysis soft ware to predict the function target of MI R-495. The wild type and mutant recombinant double fluorescent reporter gene vector of 3 'UTR region was transfected into Calu-3 cells with MI R-495 agomir or MI R-495scramble respectively. The target.9. of MI R-495 was confirmed by the double Luciferase Report experiment, and the target.9. of MI R-495 was constructed without 3' UTR MTA3. In lu-3 cells, the restore method was used to analyze the mechanism of MI R-495 targeting the expression of MTA3,.10. compared the effect of Si RNA-MTA3 and overexpression of MI R-495 on the proliferation, invasion and cell cycle of Calu-3 cells in Calu-3 cells. The low expression level of I R-495 is found in A549, Calu-3, H460, H1650 and SPC-A-1 cells, but the relatively high expression of (P0.05).MTA3 in H1299 cells is in A549. It was higher than group NC and group Mock (P0.05). After transfection of MI R-495, the expression level of MI R-495 in Calu-3 cells was up-regulated and the experimental results of.3.CCK-8 experiment and clone formation showed that the proliferation ability of the MI cells began to appear significantly at the 2 day after the transfection. The growth of the nude mice was inhibited 2 weeks after the transfection, and the mice were sacrificed more and more significantly (P0.05).4 weeks after the transfection. The tumor tissue was stripped and the tumor volume of nude mice was calculated. The results showed that the tumor volume of the MI R-495 group was significantly smaller than that of the NC group and the Mock group (P0.05). The expression of MTA3 in the xenografts in nude mice was in the MI R-495 group. The expression level and the expression level relative to the group NC and the Mock group increased significantly (P0.05).5. flow cytometry analysis showed that the proportion of the cells in the MI R-495 group in the NC group and the Mock group was significantly higher than that in the MI R-495 group, while the reduction (P0.05) in the S phase (P0.05) was compared to the changes in the expression of the cell cycle related proteins. The expression level of A, cyclin D1, and phosphorylated Rb in group NC and Mock group decreased significantly (P0.05).6.Transwell and scratch test results showed that the invasive ability of MI R-495 group was significantly lower than that of NC and other groups. And the results of the double Luciferase Report showed that MTA3 was the target of the action target of MI R-495. The expression of MTA3 without 3'UTR region was not regulated by Mi R-495, and the effect could cover the up regulation of MI R-495 on the biological behavior of non-small cell lung cancer cells. The results further suggest that MI R-495 can regulate the expression of MTA3 in the 3'UTR region of MTA3, negative regulation of the expression of MTA3 protein, resulting in the proliferation of non small cell lung cancer cells, the inhibition of invasion, and the cell cycle arrest. Conclusion 1. non small cell lung cancer tissues have detected 56 differentially expressed mi RNAs, of which 26 mi RNAs expressions are expressed. For up regulation, the expression of 30 mi RNAs was down. The expression level of MI R-495 in non-small cell lung cancer was low, and the expression level was related to the metastasis of lymph node, the degree of differentiation and the TNM staging, and the expression level of MTA3 was negatively correlated with.2. in non small cell lung cancer cell Calu-3 up regulation of MI R-495 expression passed the MTA3 3'UTR region. Negative regulation of MTA3 expression leads to Calu-3 cell growth and invasion inhibition. Cell cycle arrest of.Mi R-495 is expected to become a new target for non-small cell lung cancer treatment.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

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