本文選題：miR-93-5p + 肺腺癌A549細(xì)胞株　； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：目的:探討下調(diào)miR-93-5p表達(dá)對非小細(xì)胞肺癌(NSCLC)細(xì)胞株的抑制增殖和侵襲轉(zhuǎn)移,分析miR-93-5p與下游靶基因的關(guān)系,為臨床防治NSCLC提供實驗依據(jù)。方法:1.采用慢病毒介導(dǎo)的miR-93-5p干擾法,制備表達(dá)miR-93-5p的肺腺癌A549細(xì)胞株和肺鱗癌SK-MES-1細(xì)胞株(實驗組),另設(shè)未介導(dǎo)的兩種細(xì)胞株作為對照組。MTT方法檢測各組細(xì)胞株的增殖能力,采用免疫印跡法檢測各組細(xì)胞株的miR-93-5p活性。2.利用平板克隆形成法檢測介導(dǎo)后兩種NSCLC細(xì)胞株單細(xì)胞克隆形成能力,流式細(xì)胞儀檢測兩種NSCLC細(xì)胞株周期阻滯和凋亡。3.Transwel1遷移和侵襲小室法檢測兩種NSCLC細(xì)胞的遷移和侵襲能力。4.Western blot和雙熒光素酶基因方法分析miR-93-5p和靶基因PTEN和RB1調(diào)控關(guān)系。結(jié)果:與對照組比較 A549(1.82±0.099)和 SK-MES-1(1.90±0.099)實驗組A549(0.53.±0.008)和SK-MES-1(0.65±0.08)細(xì)胞株的抑制率明顯升高(79%,58%,均p0.001);與對照組比較(1.90±0.089),實驗組A549 和SK-MES-1細(xì)胞的miR-93-5p細(xì)胞活性下降(71%,66%,均p0.001);兩種細(xì)胞株抑制體外克隆形成能力下降(63%,75%,均p0.001);慢病毒介導(dǎo)miR-93-5p干擾后A549細(xì)胞周期阻滯于G1期(對照組細(xì)胞G1期48%,S期40%和G2/M期 12%;A549 細(xì)胞 G1 期 63%,S 期 26%和 G2/M 期 11%),SK-MES-1 細(xì)胞阻滯于G1期(對照組G1期49%,S期37%和G2/M'期14%;SK-MES-1細(xì)胞G1期57%,S期30%和G2/M期13%)。兩種細(xì)胞株中G1期marker蛋白CDK2和cyclinD蛋白表達(dá)量顯著下降。兩種細(xì)胞株凋亡率分別(26.2%,18.9%,均p0.001)。兩種細(xì)胞株中凋亡相關(guān)蛋白Bcl2的表達(dá)量下降,而Caspase 3的表達(dá)量上升。miR-93-5p干擾顯著抑制A549和SK-MES-1細(xì)胞株體外遷移能力(抑制率 61.4%,78.5%,均p0.001)。miR-93-5p 干擾顯著抑制 A549 和 SK-MES-1細(xì)胞的體外侵襲能力(抑制率62%和88.5%,均p0.001)。Western blot檢測結(jié)果miR-93-5p干擾后兩株細(xì)胞中的PTEN和RB1蛋白表達(dá)量顯著上升,雙熒光素酶基因?qū)嶒灠l(fā)現(xiàn)miR-93-5p直接結(jié)合在PTEN和RB1的3'非編碼區(qū),從而抑制兩個基因的翻譯。結(jié)論:干擾miR-93-5p能夠阻滯細(xì)胞周期和誘導(dǎo)細(xì)胞凋亡;干擾miR-93-5p能夠抑制NSCLC細(xì)胞株體外增殖、克隆形成、遷移和侵襲能力;影響NSCLC的增殖、侵襲和遷移能力的機(jī)制是miR-93-5p直接結(jié)合在靶基因PTEN和RB1的3'非編碼區(qū)域,抑制兩個靶基因的蛋白表達(dá)而實現(xiàn)。
[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 鹵0.099) and SK-MES-1(1.90 鹵0.099 (A549A 0.53 鹵0.008) and SK-MES-1(0.65 鹵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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2
，

本文編號：1844120