【摘要】：目的本研究應(yīng)用實時熒光定量PCR法檢測mi R-200c在膀胱癌細(xì)胞株中的表達(dá)情況,并且通過上調(diào)或下調(diào)mi R-200c在膀胱癌T24細(xì)胞中的表達(dá)來探索mi R-200c對膀胱癌細(xì)胞株侵襲遷移能力的影響及mi R-200c與Zebl構(gòu)成的雙向負(fù)反饋調(diào)節(jié)通路對膀胱癌上皮間質(zhì)轉(zhuǎn)化、侵襲遷移能力的調(diào)節(jié)作用。方法1.選取膀胱上皮永生化細(xì)胞SV-HUC-1、膀胱癌細(xì)胞T24、5637,通過RT-PCR檢測各細(xì)胞系內(nèi)mi R-200c的表達(dá)情況。2.對膀胱癌細(xì)胞系T24進(jìn)行RT-PCR及Western blot檢測ZEBl基因的表達(dá),同時劃痕試驗、Transwell法檢測T24細(xì)胞的侵襲遷移能力,驗證ZEB1與腫瘤細(xì)胞侵襲及遷移能力的關(guān)系;3.利用脂質(zhì)體Lipofectamin 2000將人工合成的mi R-200c過表達(dá)質(zhì)粒及mi R-200c抑制劑轉(zhuǎn)染至膀胱癌T24細(xì)胞中,觀察ZEB1基因表達(dá)水平的變化及其對上皮間質(zhì)轉(zhuǎn)化和腫瘤細(xì)胞侵襲及遷移能力的調(diào)控。4.構(gòu)建穩(wěn)定過表達(dá)ZEB1的T24細(xì)胞株,觀察ZEB1過表達(dá)后mi R-200c表達(dá)水平的變化及T24細(xì)胞侵襲遷移能力和上皮間質(zhì)轉(zhuǎn)化的變化。結(jié)果1.與膀胱上皮永生化細(xì)胞SV-HUC-1相比,膀胱癌細(xì)胞T24、5637中mi R-200c呈低表達(dá);與分化較好、級別較低的膀胱癌細(xì)胞5637相比,T24細(xì)胞內(nèi)mi R-200c的表達(dá)水平約為其1/4。2.mi R-200c過表達(dá)質(zhì)粒轉(zhuǎn)然組與對照組相比,前者細(xì)胞表達(dá)ZEB1的水平明顯降低,且腫瘤細(xì)胞的侵襲遷移能力受到明顯抑制,E-cadherin表達(dá)上調(diào),Vimentin表達(dá)下調(diào),細(xì)胞發(fā)生間質(zhì)上皮轉(zhuǎn)化;mi R-200c抑制劑轉(zhuǎn)染組與對照組相比,前者ZEB1的表達(dá)水平明顯增加,且腫瘤細(xì)胞的侵襲遷移能力增強,E-cadherin表達(dá)降低,Vimentin表達(dá)增加,細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化。3.與對照組相比,過表達(dá)ZEB1的T24細(xì)胞mi R-200c的表達(dá)水平下降,細(xì)胞侵襲遷移能力增強,E-cadherin表達(dá)下調(diào),Vimentin表達(dá)增加,細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化。結(jié)論1.mi R-200c在高級別、低分化的膀胱癌細(xì)胞中呈低表達(dá),提示mi R-200c可作為腫瘤抑制因子。2.mi R-200c可抑制膀胱癌細(xì)胞的侵襲轉(zhuǎn)移能力,mi R-200c在膀胱癌細(xì)胞侵襲轉(zhuǎn)移過程中發(fā)揮重要作用。3.mi R-200c與ZEB1形成了的雙向抑制的負(fù)反饋環(huán)路,對膀胱癌的侵襲遷移能力和上皮間質(zhì)轉(zhuǎn)化起重要的調(diào)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R737.14

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