OCT4B1诱导结直肠癌细胞发生上皮间质转化而获得干细胞特性的机制研究
发布时间：2018-11-17 12:27
【摘要】：目的:在前期研究中已证实OCT4B1诱导结直肠癌细胞发生EMT而获得干细胞特性,本研究探讨其调控机制。方法:在前期研究中已证实,通过悬浮培养的人结直肠癌肿瘤干细胞(SW480 CSCs)OCT4B1 m RNA水平较其亲本细胞(SW480)明显增高;采用慢病毒介导的RNA干扰技术沉默SW480 CSCs中OCT4B1基因(SW480 CSCs-RNAi)后,与阴性对照组(SW480 CSCs-NC)比较,OCT4B1 m RNA水平明显降低,本研究对上述四组细胞作如下检测:(1)用RT-q PCR法及Western blot检测调控肿瘤EMT过程的指标PLK1m RNA及蛋白水平是否与OCT4B1呈相同趋势变化;(2)行mi RNA芯片检测,筛选与OCT4B1及PLK1变化趋势相反,且与PLK1存在结合位点的mi RNA;(3)用RT-q PCR方法验证该mi RNA在四组细胞中的表达是否与芯片检测结果一致;(4)采用双荧光素酶实验检测筛选的mi RNA与PLK1是否存在直接调控作用。结果:(1)RT-q PCR检测四组细胞PLK1 m RNA表达,SW480与SW480 CSCs相对水平分别为(1.00±0.13)及(3.22±0.10),SW480 CSCs-NC与SW480 CSCs-RNAi相对水平分别为(1.99±0.17)及(0.92±0.09),差异均具有统计学意义(P0.01);Western blot检测PLK1蛋白表达,SW480与SW480 CSCs蛋白表达分别为(0.68±0.05)与(1.16±0.08),SW480 CSCs-NC与SW480 CSCs-RNAi蛋白表达分别为(1.50±0.11)与(0.27±0.06),差异均具有统计学意义(P0.01),提示PLK1与OCT4B1呈相同趋势变化;(2)mi RNA芯片检测发现mi R-8064与OCT4B1及PLK1变化趋势相反,通过生物信息学软件Target Scan human分析发现它与PLK1存在结合位点;(3)进一步通过RT-q PCR证实mi R-8064在四组细胞中表达,SW480与SW480 CSCs相对水平分别为(1.00±0.12)及(0.40±0.06),SW480 CSCs-NC与SW480 CSCs-RNAi相对水平分别为(0.12±0.03)及(0.93±0.02),均具有统计学意义(P0.05),发现在四组细胞中mi R-8064表达与芯片检测结果一致;(4)通过双荧光素酶实验证实PLK1是mi R-8064直接靶基因。结论:OCT4B1诱导EMT使肿瘤细胞获得干细胞特性,其机制部分与OCT4B1抑制mi RNA-8064表达进而促进靶基因PLK1表达有关。
[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 卤0.13) and (3.22 卤0.10), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (1.99 卤0.17) and (0.92 卤0.09), respectively. The expression of SW480 and SW480 CSCs was (0.68 卤0.05) and (1.16 卤0.08) by Western blot, and the expression of SW480 CSCs-NC and SW480 CSCs-RNAi was (1.50 卤0.11) and (0.27 卤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 卤0.12) and (0.40 卤0.06), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (0.12 卤0.03) and (0.93 卤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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
[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 卤0.13) and (3.22 卤0.10), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (1.99 卤0.17) and (0.92 卤0.09), respectively. The expression of SW480 and SW480 CSCs was (0.68 卤0.05) and (1.16 卤0.08) by Western blot, and the expression of SW480 CSCs-NC and SW480 CSCs-RNAi was (1.50 卤0.11) and (0.27 卤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 卤0.12) and (0.40 卤0.06), respectively. The relative levels of SW480 CSCs-NC and SW480 CSCs-RNAi were (0.12 卤0.03) and (0.93 卤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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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