發(fā)布時(shí)間：2018-11-17 12:27

【摘要】：目的:在前期研究中已證實(shí)OCT4B1誘導(dǎo)結(jié)直腸癌細(xì)胞發(fā)生EMT而獲得干細(xì)胞特性,本研究探討其調(diào)控機(jī)制。方法:在前期研究中已證實(shí),通過(guò)懸浮培養(yǎng)的人結(jié)直腸癌腫瘤干細(xì)胞(SW480 CSCs)OCT4B1 m RNA水平較其親本細(xì)胞(SW480)明顯增高;采用慢病毒介導(dǎo)的RNA干擾技術(shù)沉默SW480 CSCs中OCT4B1基因(SW480 CSCs-RNAi)后,與陰性對(duì)照組(SW480 CSCs-NC)比較,OCT4B1 m RNA水平明顯降低,本研究對(duì)上述四組細(xì)胞作如下檢測(cè):(1)用RT-q PCR法及Western blot檢測(cè)調(diào)控腫瘤EMT過(guò)程的指標(biāo)PLK1m RNA及蛋白水平是否與OCT4B1呈相同趨勢(shì)變化;(2)行mi RNA芯片檢測(cè),篩選與OCT4B1及PLK1變化趨勢(shì)相反,且與PLK1存在結(jié)合位點(diǎn)的mi RNA;(3)用RT-q PCR方法驗(yàn)證該mi RNA在四組細(xì)胞中的表達(dá)是否與芯片檢測(cè)結(jié)果一致;(4)采用雙熒光素酶實(shí)驗(yàn)檢測(cè)篩選的mi RNA與PLK1是否存在直接調(diào)控作用。結(jié)果:(1)RT-q PCR檢測(cè)四組細(xì)胞PLK1 m RNA表達(dá),SW480與SW480 CSCs相對(duì)水平分別為(1.00±0.13)及(3.22±0.10),SW480 CSCs-NC與SW480 CSCs-RNAi相對(duì)水平分別為(1.99±0.17)及(0.92±0.09),差異均具有統(tǒng)計(jì)學(xué)意義(P0.01);Western blot檢測(cè)PLK1蛋白表達(dá),SW480與SW480 CSCs蛋白表達(dá)分別為(0.68±0.05)與(1.16±0.08),SW480 CSCs-NC與SW480 CSCs-RNAi蛋白表達(dá)分別為(1.50±0.11)與(0.27±0.06),差異均具有統(tǒng)計(jì)學(xué)意義(P0.01),提示PLK1與OCT4B1呈相同趨勢(shì)變化;(2)mi RNA芯片檢測(cè)發(fā)現(xiàn)mi R-8064與OCT4B1及PLK1變化趨勢(shì)相反,通過(guò)生物信息學(xué)軟件Target Scan human分析發(fā)現(xiàn)它與PLK1存在結(jié)合位點(diǎn);(3)進(jìn)一步通過(guò)RT-q PCR證實(shí)mi R-8064在四組細(xì)胞中表達(dá),SW480與SW480 CSCs相對(duì)水平分別為(1.00±0.12)及(0.40±0.06),SW480 CSCs-NC與SW480 CSCs-RNAi相對(duì)水平分別為(0.12±0.03)及(0.93±0.02),均具有統(tǒng)計(jì)學(xué)意義(P0.05),發(fā)現(xiàn)在四組細(xì)胞中mi R-8064表達(dá)與芯片檢測(cè)結(jié)果一致;(4)通過(guò)雙熒光素酶實(shí)驗(yàn)證實(shí)PLK1是mi R-8064直接靶基因。結(jié)論:OCT4B1誘導(dǎo)EMT使腫瘤細(xì)胞獲得干細(xì)胞特性,其機(jī)制部分與OCT4B1抑制mi RNA-8064表達(dá)進(jìn)而促進(jìn)靶基因PLK1表達(dá)有關(guān)。
[Abstract]:Aim: to investigate the regulatory mechanism of OCT4B1 induced EMT in colorectal cancer cells and obtain stem cell characteristics in previous studies. Methods: in previous studies, the OCT4B1 m RNA level of human colorectal cancer stem cells (SW480 CSCs) in suspension culture was significantly higher than that of their parent cells (SW480). After silencing OCT4B1 gene (SW480 CSCs-RNAi) in SW480 CSCs by lentivirus-mediated RNA interference technique, the level of OCT4B1 m RNA was significantly lower than that of negative control group (SW480 CSCs-NC). In this study, the above four groups of cells were tested as follows: (1) RT-q PCR and Western blot were used to detect the changes of PLK1m RNA and protein levels in the process of regulating tumor EMT. Whether the PLK1m RNA and protein levels showed the same trend as OCT4B1; (2) mi RNA microarray was used to screen mi RNA; (3, which was contrary to OCT4B1 and PLK1, and had binding site with PLK1. RT-q PCR method was used to verify whether the expression of mi RNA in the four groups of cells was consistent with the results of microarray detection. (4) double luciferase assay was used to detect the direct regulation of mi RNA and PLK1. Results: (1) the expression of PLK1 m RNA was detected by RT-q PCR. The relative levels of SW480 and SW480 CSCs were (1.00 鹵0.13) and (3.22 鹵0.10), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (1.99 鹵0.17) and (0.92 鹵0.09), respectively. The expression of SW480 and SW480 CSCs was (0.68 鹵0.05) and (1.16 鹵0.08) by Western blot, and the expression of SW480 CSCs-NC and SW480 CSCs-RNAi was (1.50 鹵0.11) and (0.27 鹵0.06), respectively. The differences were statistically significant (P0.01), indicating that PLK1 and OCT4B1 showed the same trend. (2) mi RNA chip detection showed that mi R-8064 had the opposite trend with OCT4B1 and PLK1, and Target Scan human analysis of bioinformatics software found that it had binding sites with PLK1. (3) the expression of mi R-8064 was further confirmed by RT-q PCR. The relative levels of SW480 and SW480 CSCs were (1.00 鹵0.12) and (0.40 鹵0.06), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (0.12 鹵0.03) and (0.93 鹵0.02), respectively, with statistical significance (P0.05). It was found that the expression of mi R-8064 in the four groups was consistent with the results of microarray detection. (4) double luciferase assay confirmed that PLK1 is a direct target gene of mi R-8064. Conclusion: OCT4B1 induces EMT to obtain stem cell characteristics, which is partly related to the inhibition of mi RNA-8064 expression by OCT4B1 and the promotion of target gene PLK1 expression.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.34

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