NSCLC细胞株中人miR-93-5p表达及其意义

发布时间：2018-05-04 18:30

  本文选题：miR-93-5p + 肺腺癌A549细胞株　； 参考：《延边大学》2017年硕士论文

【摘要】：目的:探讨下调miR-93-5p表达对非小细胞肺癌(NSCLC)细胞株的抑制增殖和侵袭转移,分析miR-93-5p与下游靶基因的关系,为临床防治NSCLC提供实验依据。方法:1.采用慢病毒介导的miR-93-5p干扰法,制备表达miR-93-5p的肺腺癌A549细胞株和肺鳞癌SK-MES-1细胞株(实验组),另设未介导的两种细胞株作为对照组。MTT方法检测各组细胞株的增殖能力,采用免疫印迹法检测各组细胞株的miR-93-5p活性。2.利用平板克隆形成法检测介导后两种NSCLC细胞株单细胞克隆形成能力,流式细胞仪检测两种NSCLC细胞株周期阻滞和凋亡。3.Transwel1迁移和侵袭小室法检测两种NSCLC细胞的迁移和侵袭能力。4.Western blot和双荧光素酶基因方法分析miR-93-5p和靶基因PTEN和RB1调控关系。结果:与对照组比较 A549(1.82±0.099)和 SK-MES-1(1.90±0.099)实验组A549(0.53.±0.008)和SK-MES-1(0.65±0.08)细胞株的抑制率明显升高(79%,58%,均p0.001);与对照组比较(1.90±0.089),实验组A549 和SK-MES-1细胞的miR-93-5p细胞活性下降(71%,66%,均p0.001);两种细胞株抑制体外克隆形成能力下降(63%,75%,均p0.001);慢病毒介导miR-93-5p干扰后A549细胞周期阻滞于G1期(对照组细胞G1期48%,S期40%和G2/M期 12%;A549 细胞 G1 期 63%,S 期 26%和 G2/M 期 11%),SK-MES-1 细胞阻滞于G1期(对照组G1期49%,S期37%和G2/M'期14%;SK-MES-1细胞G1期57%,S期30%和G2/M期13%)。两种细胞株中G1期marker蛋白CDK2和cyclinD蛋白表达量显著下降。两种细胞株凋亡率分别(26.2%,18.9%,均p0.001)。两种细胞株中凋亡相关蛋白Bcl2的表达量下降,而Caspase 3的表达量上升。miR-93-5p干扰显著抑制A549和SK-MES-1细胞株体外迁移能力(抑制率 61.4%,78.5%,均p0.001)。miR-93-5p 干扰显著抑制 A549 和 SK-MES-1细胞的体外侵袭能力(抑制率62%和88.5%,均p0.001)。Western blot检测结果miR-93-5p干扰后两株细胞中的PTEN和RB1蛋白表达量显著上升,双荧光素酶基因实验发现miR-93-5p直接结合在PTEN和RB1的3'非编码区,从而抑制两个基因的翻译。结论:干扰miR-93-5p能够阻滞细胞周期和诱导细胞凋亡;干扰miR-93-5p能够抑制NSCLC细胞株体外增殖、克隆形成、迁移和侵袭能力;影响NSCLC的增殖、侵袭和迁移能力的机制是miR-93-5p直接结合在靶基因PTEN和RB1的3'非编码区域,抑制两个靶基因的蛋白表达而实现。
[Abstract]:Objective: to investigate the inhibitory effect of down-regulation of miR-93-5p expression on proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC) cell line, and to analyze the relationship between miR-93-5p and downstream target gene, and to provide experimental evidence for clinical prevention and treatment of NSCLC. Method 1: 1. Lung adenocarcinoma A549 cell line and lung squamous cell carcinoma SK-MES-1 cell line expressing miR-93-5p were prepared by lentivirus-mediated miR-93-5p interference method. The activity of miR-93-5p. 2. 2. The single cell clone forming ability of the two NSCLC cell lines was detected by plate clone formation method. Cell cycle arrest and apoptosis of two NSCLC cell lines were detected by flow cytometry. 3. Transwel1 migration and invasive chamber assay were used to detect the migration and invasion ability of two kinds of NSCLC cells. 4. Western blot and double luciferase gene method were used to analyze the regulatory relationship between miR-93-5p and target genes PTEN and RB1. Results: compared with the control group, the inhibition rate of A549A (1.82 卤0.099) and SK-MES-1(1.90 卤0.099 (A549A 0.53 卤0.008) and SK-MES-1(0.65 卤0.08) cell lines was significantly higher than that of the control group (P < 0.01). Compared with the control group, the activity of miR-93-5p cells in the A549 and SK-MES-1 cells in the experimental group was significantly decreased (P 0.001), and the inhibition effect of the two cell lines was significant (P 0.001). The cell cycle of A549 cells after lentivirus-mediated miR-93-5p interference was arrested in G1 phase (40% of the control cells in G1 phase and 40% of the control cells in G _ 2 / M phase 12C A549 cells, and 26% of the cells in the G _ 1 / M phase 63 ~ S phase and the G _ 2 / M phase 11Q SK-MES-1 cells were blocked in the G _ 1 phase. In the control group, 37% of S phase and 14% of G _ 2 / M 'phase in G _ 1 phase and G _ 2 / M ~ (-1) phase were 30% in S phase and 30% in G _ 2 / M phase, and 30% in S phase and 13% in G _ 2 / M phase. The expression of marker protein CDK2 and cyclinD protein in G 1 phase decreased significantly in the two cell lines. The apoptotic rates of the two cell lines were 26.2and 18.9g, respectively (P 0.001). The expression of apoptosis-related protein Bcl2 was decreased in two cell lines. However, the expression of Caspase 3 increased. MiR-93-5p interference significantly inhibited the migration ability of A549 and SK-MES-1 cell lines in vitro (inhibition rate 61.4% 78.5%, both p0.001).miR-93-5p interference significantly inhibited the invasion ability of A549 and SK-MES-1 cells in vitro (inhibition rate 62% and 88. 5%, respectively). The results of p0.001).Western blot detection showed that the inhibition rate of p0.001).miR-93-5p interference in A549 and SK-MES-1 cells was 88. 5% and 88. 5% respectively. After miR-93-5p interference, the expression of PTEN and RB1 protein increased significantly in the two cell lines. Double luciferase gene experiment found that miR-93-5p binds directly to the 3'noncoding region of PTEN and RB1, thus inhibiting the translation of the two genes. Conclusion: interfering with miR-93-5p can block cell cycle and induce apoptosis, interfere with miR-93-5p can inhibit the proliferation, clone formation, migration and invasion of NSCLC cell line in vitro, and affect the proliferation of NSCLC. The mechanism of invasion and migration is that miR-93-5p binds directly to the 3'non-coding region of target gene PTEN and RB1 and inhibits the protein expression of the two target genes.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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本文编号：1844120