Hsa-miR-6841-3p对宫颈癌细胞生物学功能的影响及机制的研究

发布时间：2018-03-04 00:12

本文选题：hsa-miR-6841-3p　切入点：三叶因子3　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本研究检测了MI RNA-6841-3P在HPV阳性宫颈癌组织、HPV阴性宫颈癌组织和正常宫颈组织中的表达,进一步探讨MIRNA-6841-3P对宫颈癌细胞生物学行为的影响,预测并验证MIRNA-6841-3P的靶基因,阐明MIRNA-6841-3P在宫颈癌发生发展中的作用,为深入理解宫颈癌发病的潜在分子机制和开发新的有效分子治疗靶点提供实验依据。方法:通过实时荧光定量PCR方法,检测MIRNA-6841-3P在38例HPV阳性宫颈癌组织和10例HPV阴性宫颈癌组织及8例正常宫颈组织中的表达;体外利用脂质体转染技术将MIRNA-6841-3P模拟物、MIRNA-6841-3P抑制物及其各自阴性对照转染至宫颈癌细胞系CASKI和SIHA,将转染后细胞系分为4组,实验分组分为MIR-6841-3P模拟物转染组(MIR-6841-3P),即含MIR-6841-3P模拟物(MIMICS);MIR-6841-3P模拟物对照组(MIR-6841-3P-NEGATIVE CONTROL-MIMICS,NC);MIR-6841-3P抑制物转染组(MIR-6841-3P-IN),即含MIR-6841-3P抑制物(INHIBITOR);MIR-6841-3P抑制物对照组(MIR-6841-3P-NEGATIVE CONTROL-INHIBITOR,NC-IN)。应用实时荧光定量PCR检测宫颈癌细胞系CASKI和SIHA转染后MIRNA-6841-3P M RNA以及其预测靶基因TFF3的转录表达,CCK-8法检测细胞增殖能力,划痕实验检测细胞迁移能力,TRANSWELL小室检测细胞侵袭能力。运用基因预测软件TARGET SCAN、MIRANDA以及PICTAR共同预测并分析MIRNA-6841-3P可能的潜在靶基因。WESTERN BLOT检验细胞转染后TFF3的表达水平的变化。采用的是SPSS 22.0统计学软件进行处理。结果:1.应用实时荧光定量PCR技术检测发现与正常组织相比MIRNA-6841-3P在宫颈癌组织表达明显降低,MIRNA-6841-3P在HPV阳性组织中表达量低于HPV阴性组织(P0.05),差异有统计学意义。2.实时荧光定量PCR显示,MIRNA-6841-3P模拟物转染组(MIR-6841-3P)与MIRNA-6841-3P模拟物对照组相比(NC),M RNA转录表达明显上调(P0.01),MIRNA-6841-3P抑制物转染组(MIR-6841-3P-IN)M RNA的转录表达低于MIRNA-6841-3P抑制物转染对照组(NC-IN)(P0.01),差异有统计学意义。3.与MI RNA-6841-3P抑制物转染对照组(NC-IN)比较,MIRNA-6841-3P抑制物转染组(MIR-6841-3P-IN)CASKI和SIHA细胞活性、迁移侵袭能力明显增强,与MIRNA-6841-3P模拟物转染对照组(NC)比较,转染MIRNA-6841-3P模拟物组(MI R-6841-3P),宫颈癌细胞系CASKI和SIHA细胞活性、迁移侵袭能力明显减弱(P0.05)。4.其预测靶基因TFF3 MRNA的转录表达在MIRNA-6841-3P模拟物转染组(MI R-6841-3P)明显低于MIRNA-6841-3P模拟物转染对照组(NC),MIRNA-6841-3P抑制物转染组(MI R-6841-3P-IN)明显高于MIRNA-6841-3P抑制物转染对照组(P0.01)。5.WESTERN BLOT实验表明,抑制MIRNA-6841-3P导致CASKI和SIHA细胞中TFF3的蛋白水平增加。另外,过表达MIRNA-6841-3P模拟物降低了这两种细胞中TFF3的表达。结论及意义:上调MIRNA-6841-3P表达可明显抑制宫颈癌细胞系CASKI和SIHA增殖,抑制其迁移、侵袭能力。TFF3可能是MIRNA-6841-3P的靶基因,并有可能为宫颈癌的治疗提供新的靶点。
[Abstract]:Objective: to detect the expression of MIRNA-6841-3P in HPV positive cervical carcinoma tissues and normal cervical tissues, to further investigate the effect of MIRNA-6841-3P on the biological behavior of cervical cancer cells, and to predict and verify the target gene of MIRNA-6841-3P. To elucidate the role of MIRNA-6841-3P in the carcinogenesis and development of cervical cancer, and to provide experimental basis for further understanding the underlying molecular mechanism of cervical cancer and to develop new effective molecular therapeutic targets. The expression of MIRNA-6841-3P was detected in 38 cases of HPV positive cervical carcinoma, 10 cases of HPV negative cervical carcinoma and 8 cases of normal cervix. MIRNA-6841-3P inhibitor (MIRNA-6841-3P) and its respective negative control were transfected into cervical cancer cell lines CASKI and SIHA by liposome transfection in vitro. The transfected cells were divided into 4 groups. The experiment was divided into two groups: MIR-6841-3PnP, MIR-6841-3P, MIR-6841-3P mimic, MIR-6841-3P-NEGATIVE ConNTROL-MISMICSMICMIR-6841-3P, MIR-6841-3P-INP, the MIR-6841-3P inhibitor, MIR-6841-3P inhibitor, MIR-6841-3P-NEGATIVE NTROL-INHIBITORNC-INNIN. The real-time quantitative PCR was used to detect the cervical cancer cell line CASKI and MIR-6841-3P, the MIR-6841-3P-NEGATE-CONTROL-INHIBITORNC-INP. The transcriptional expression of MIRNA-6841-3P M RNA and its predicted target gene TFF3 were detected by CCK-8 method after SIHA transfection. Scratch assay was used to detect cell migration ability and TRANSWELL chamber was used to detect cell invasion. Gene prediction software TARGET SCANN MIRANDA and PICTAR were used to predict and analyze MIRNA-6841-3P potential target gene. Western BLOT to detect the change of TFF3 expression level after transfection. SPSS 22.0 statistical software was used to process it. Results 1. The expression of MIRNA-6841-3P in cervical cancer tissues was significantly lower than that in normal tissues by real-time fluorescence quantitative PCR. The expression of MIRNA-6841-3P in HPV positive tissues was lower than that in HPV positive tissues. Real time fluorescence quantitative PCR showed that MIRNA-6841-3P mimic transfection group had significantly up-regulated MIR-6841-3P RNA expression compared with MIRNA-6841-3P mimic control group, and the expression of MIR-6841-3P RNA was significantly higher than that of MIRNA-6841-3P inhibition group compared with that of MIRNA-6841-3P mimetic control group (P < 0.05). The expression of MIR-6841-3P-INM RNA in MIR-6841-3P transfection group was significantly higher than that in MIRNA-6841-3P mimetic control group (P < 0.05). The expression of MIR-6841-3P-INM RNA was significantly higher in the transfected group than that in the MIRNA-6841-3P control group. The activity of MIRNA-6841-3P inhibitor was compared with that of MIRNA-6841-3P inhibitor transfection group (MIR-6841-3P-CASKI and SIHA cells). The ability of migration and invasion was significantly enhanced. Compared with the control group transfected with MIRNA-6841-3P mimics, the activity of CASKI and SIHA cells was increased in the MIRNA-6841-3P mimetic group. The transcriptional expression of the target gene TFF3 MRNA in the MIRNA-6841-3P mimic transfection group was significantly lower than that in the MIRNA-6841-3P mimic transfection group compared with the control group. The expression of MIR-6841-3P-INwas significantly higher than that of the MIRNA-6841-3P inhibitor transfection control group (P0.01N. 5. WESTERN BLOT). Inhibition of MIRNA-6841-3P induced an increase in TFF3 protein levels in CASKI and SIHA cells. In addition, overexpression of MIRNA-6841-3P mimics decreased the expression of TFF3 in these two cell lines. Conclusion: up-regulation of MIRNA-6841-3P expression can significantly inhibit the proliferation of CASKI and SIHA cells. Inhibiting its migration, invasiveness. TFF3 may be the target gene of MIRNA-6841-3P, and may provide a new target for the treatment of cervical cancer.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

【相似文献】