miR-200c在膀胱癌上皮间质转化作用机制的研究
发布时间：2018-11-19 20:58
【摘要】：目的本研究应用实时荧光定量PCR法检测mi R-200c在膀胱癌细胞株中的表达情况,并且通过上调或下调mi R-200c在膀胱癌T24细胞中的表达来探索mi R-200c对膀胱癌细胞株侵袭迁移能力的影响及mi R-200c与Zebl构成的双向负反馈调节通路对膀胱癌上皮间质转化、侵袭迁移能力的调节作用。方法1.选取膀胱上皮永生化细胞SV-HUC-1、膀胱癌细胞T24、5637,通过RT-PCR检测各细胞系内mi R-200c的表达情况。2.对膀胱癌细胞系T24进行RT-PCR及Western blot检测ZEBl基因的表达,同时划痕试验、Transwell法检测T24细胞的侵袭迁移能力,验证ZEB1与肿瘤细胞侵袭及迁移能力的关系;3.利用脂质体Lipofectamin 2000将人工合成的mi R-200c过表达质粒及mi R-200c抑制剂转染至膀胱癌T24细胞中,观察ZEB1基因表达水平的变化及其对上皮间质转化和肿瘤细胞侵袭及迁移能力的调控。4.构建稳定过表达ZEB1的T24细胞株,观察ZEB1过表达后mi R-200c表达水平的变化及T24细胞侵袭迁移能力和上皮间质转化的变化。结果1.与膀胱上皮永生化细胞SV-HUC-1相比,膀胱癌细胞T24、5637中mi R-200c呈低表达;与分化较好、级别较低的膀胱癌细胞5637相比,T24细胞内mi R-200c的表达水平约为其1/4。2.mi R-200c过表达质粒转然组与对照组相比,前者细胞表达ZEB1的水平明显降低,且肿瘤细胞的侵袭迁移能力受到明显抑制,E-cadherin表达上调,Vimentin表达下调,细胞发生间质上皮转化;mi R-200c抑制剂转染组与对照组相比,前者ZEB1的表达水平明显增加,且肿瘤细胞的侵袭迁移能力增强,E-cadherin表达降低,Vimentin表达增加,细胞发生上皮间质转化。3.与对照组相比,过表达ZEB1的T24细胞mi R-200c的表达水平下降,细胞侵袭迁移能力增强,E-cadherin表达下调,Vimentin表达增加,细胞发生上皮间质转化。结论1.mi R-200c在高级别、低分化的膀胱癌细胞中呈低表达,提示mi R-200c可作为肿瘤抑制因子。2.mi R-200c可抑制膀胱癌细胞的侵袭转移能力,mi R-200c在膀胱癌细胞侵袭转移过程中发挥重要作用。3.mi R-200c与ZEB1形成了的双向抑制的负反馈环路,对膀胱癌的侵袭迁移能力和上皮间质转化起重要的调控作用。
[Abstract]:Objective to detect the expression of mi R-200c in bladder cancer cell line by real-time fluorescence quantitative PCR. By up-regulating or down-regulating the expression of mi R-200c in T24 cells, the effect of mi R-200c on the invasion and migration of bladder cancer cell line and the transformation of epithelial stroma between mi R-200c and Zebl were investigated. The regulation of invasion and migration ability. Method 1. The expression of mi R-200c in bladder epithelial immortalized cell line SV-HUC-1, was detected by RT-PCR. 2. RT-PCR and Western blot were used to detect the expression of ZEBl gene in bladder cancer cell line T24. At the same time, scratch test and Transwell assay were used to detect the invasion and migration ability of T24 cell line, and to verify the relationship between ZEB1 and tumor cell invasion and migration ability. 3. The synthetic mi R-200c overexpression plasmid and mi R-200c inhibitor were transfected into bladder cancer T24 cells by liposome Lipofectamin 2000. To observe the changes of ZEB1 gene expression and its regulation on epithelial mesenchymal transformation and tumor cell invasion and migration. 4. T24 cell line with stable overexpression of ZEB1 was constructed, and the expression level of mi R-200c and the changes of invasion and migration ability and epithelial interstitial transformation of T24 cells after overexpression of ZEB1 were observed. Result 1. Compared with the bladder epithelial immortalized cell SV-HUC-1, the expression of mi R-200c in bladder cancer cell line T24m5637 was low. The expression level of mi R-200c in T24 cells was significantly lower than that in the control group compared with that in the control group, and the expression level of mi R-200c in T24 cells was significantly lower than that in the control group. The invasion and migration ability of tumor cells was obviously inhibited, the expression of E-cadherin was up-regulated, the expression of Vimentin was down-regulated and the mesenchymal epithelium was transformed. Compared with the control group, the expression of ZEB1 in the mi R-200c inhibitor transfection group was significantly increased, and the invasion and migration ability of tumor cells was enhanced, the expression of E-cadherin was decreased, the expression of Vimentin was increased, and the epithelial interstitial transformation occurred. Compared with the control group, the expression of mi R-200c in T24 cells with overexpression of ZEB1 decreased, the ability of cell invasion and migration increased, the expression of E-cadherin decreased, the expression of Vimentin increased, and the epithelial interstitial transformation occurred. Conclusion the expression of 1.mi R-200c is low in high grade and poorly differentiated bladder cancer cells, suggesting that mi R-200c can be used as a tumor suppressor. 2.mi R-200c can inhibit the invasion and metastasis of bladder cancer cells. Mi R-200c plays an important role in the invasion and metastasis of bladder cancer cells. The negative feedback loop formed by 3.mi R-200c and ZEB1 plays an important role in regulating the invasion and migration ability and epithelial interstitial transformation of bladder cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14
[Abstract]:Objective to detect the expression of mi R-200c in bladder cancer cell line by real-time fluorescence quantitative PCR. By up-regulating or down-regulating the expression of mi R-200c in T24 cells, the effect of mi R-200c on the invasion and migration of bladder cancer cell line and the transformation of epithelial stroma between mi R-200c and Zebl were investigated. The regulation of invasion and migration ability. Method 1. The expression of mi R-200c in bladder epithelial immortalized cell line SV-HUC-1, was detected by RT-PCR. 2. RT-PCR and Western blot were used to detect the expression of ZEBl gene in bladder cancer cell line T24. At the same time, scratch test and Transwell assay were used to detect the invasion and migration ability of T24 cell line, and to verify the relationship between ZEB1 and tumor cell invasion and migration ability. 3. The synthetic mi R-200c overexpression plasmid and mi R-200c inhibitor were transfected into bladder cancer T24 cells by liposome Lipofectamin 2000. To observe the changes of ZEB1 gene expression and its regulation on epithelial mesenchymal transformation and tumor cell invasion and migration. 4. T24 cell line with stable overexpression of ZEB1 was constructed, and the expression level of mi R-200c and the changes of invasion and migration ability and epithelial interstitial transformation of T24 cells after overexpression of ZEB1 were observed. Result 1. Compared with the bladder epithelial immortalized cell SV-HUC-1, the expression of mi R-200c in bladder cancer cell line T24m5637 was low. The expression level of mi R-200c in T24 cells was significantly lower than that in the control group compared with that in the control group, and the expression level of mi R-200c in T24 cells was significantly lower than that in the control group. The invasion and migration ability of tumor cells was obviously inhibited, the expression of E-cadherin was up-regulated, the expression of Vimentin was down-regulated and the mesenchymal epithelium was transformed. Compared with the control group, the expression of ZEB1 in the mi R-200c inhibitor transfection group was significantly increased, and the invasion and migration ability of tumor cells was enhanced, the expression of E-cadherin was decreased, the expression of Vimentin was increased, and the epithelial interstitial transformation occurred. Compared with the control group, the expression of mi R-200c in T24 cells with overexpression of ZEB1 decreased, the ability of cell invasion and migration increased, the expression of E-cadherin decreased, the expression of Vimentin increased, and the epithelial interstitial transformation occurred. Conclusion the expression of 1.mi R-200c is low in high grade and poorly differentiated bladder cancer cells, suggesting that mi R-200c can be used as a tumor suppressor. 2.mi R-200c can inhibit the invasion and metastasis of bladder cancer cells. Mi R-200c plays an important role in the invasion and metastasis of bladder cancer cells. The negative feedback loop formed by 3.mi R-200c and ZEB1 plays an important role in regulating the invasion and migration ability and epithelial interstitial transformation of bladder cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14
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