煤煙誘導(dǎo)支氣管上皮細(xì)胞轉(zhuǎn)化和MUC1基因表達(dá)對(duì)肺癌細(xì)胞生物學(xué)特性的影響
發(fā)布時(shí)間：2018-12-11 11:45
【摘要】：[背景]基因的表觀遺傳學(xué)被認(rèn)為在腫瘤的發(fā)生和發(fā)展中起到重要作用。而DNA甲基化模式改變引起的MUC1基因表達(dá)的改變,對(duì)腫瘤細(xì)胞的生物學(xué)行為的影響起著重要的作用。本研究致力于對(duì)MUC1基因的表達(dá)情況,以及其在肺癌細(xì)胞發(fā)生發(fā)展中生物學(xué)功能進(jìn)行探究。[目的]揭示煤煙誘導(dǎo)對(duì)支氣管上皮細(xì)胞轉(zhuǎn)化的影響和MUC1基因表達(dá)改變與支氣管上皮細(xì)胞生物學(xué)特性改變的相關(guān)性及在肺癌增殖、侵襲中的作用。為深入理解MUC1基因在肺癌中的作用進(jìn)行初步探索,以期為肺癌新的診斷和治療方法提供研究基礎(chǔ)。[方法]①用1.0umol/L,C1煤煙試劑誘導(dǎo)支氣管上皮細(xì)胞(Beas-2bCoal細(xì)胞),通過RT-PCR檢測(cè)MUC1的表達(dá)情況,采用CCK-8試劑檢測(cè)誘導(dǎo)細(xì)胞的增殖生長(zhǎng)情況,用平板細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞的增殖能力和群體依賴性,劃痕實(shí)驗(yàn)和Transwell檢測(cè)細(xì)胞遷移愈合能力。②設(shè)計(jì)shRNA,篩選有效shRNA,通過脂質(zhì)體介導(dǎo)的轉(zhuǎn)染試驗(yàn),同時(shí)設(shè)立Blank組(空白對(duì)照組)、shNC陰性對(duì)照組(無關(guān)干擾序列陰性對(duì)照組),檢測(cè)MUC1基因的表達(dá)對(duì)宣威肺癌細(xì)胞的生物學(xué)特性的影響:(1)通過觀察熒光及RT-PCR篩選轉(zhuǎn)染細(xì)胞株。(2)用CCK-8試劑檢測(cè)MUC1基因沉默后細(xì)胞的增殖生長(zhǎng)情況。(3)用平板細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)MUC1基因沉默后細(xì)胞的增殖能力和群體依賴性。(4)劃痕實(shí)驗(yàn)和Transwell小室實(shí)驗(yàn)檢測(cè)MUC1基因沉默后細(xì)胞的細(xì)胞劃痕愈合以及遷移侵襲能力。③用5 umol/L的5--Aza-CdR甲基化酶抑制,處理Beas-2b,煤煙誘導(dǎo)Beas-2b和XWLC-05細(xì)胞,檢測(cè)MUCl基因表達(dá)對(duì)宣威肺癌細(xì)胞的生物學(xué)特性的影響:(1)通過RT-PCR檢測(cè)MUC1的表達(dá)情況。(2)用CCK-8試劑檢測(cè)甲基化酶抑制劑處理后細(xì)胞的增殖生長(zhǎng)情況。(3)用平板細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)MUC1基因去甲基化后細(xì)胞的增殖能力和群體依賴性。(4) Transwell小室實(shí)驗(yàn)檢測(cè)MUC1基因去甲基化后細(xì)胞的細(xì)胞劃痕愈合以及遷移侵襲能力。[結(jié)果]①CCK-8檢測(cè)顯示Beas-2b Coal細(xì)胞的增殖生長(zhǎng)能力較正常Beas-2b細(xì)胞增強(qiáng)。劃痕試驗(yàn)顯示Beas-2b Coal細(xì)胞劃痕愈合能力較正常Beas-2b細(xì)胞明顯增強(qiáng)。提示煤煙引起支氣管上皮細(xì)胞發(fā)生惡性傾向。②shRNA干擾MUC1基因表達(dá)沉默,CCK-8檢測(cè)顯示MUC1基因沉默后,宣威肺癌細(xì)胞的增殖能力下降,平板克隆和Transwell小室實(shí)驗(yàn)顯示細(xì)胞克隆形成數(shù)目明顯減少以及侵襲遷移能力受抑。③5-Aza-CdR處理后,CCK-8檢測(cè)顯示MUC1基因去甲基化表達(dá)上調(diào)后,宣威肺癌細(xì)胞的增殖能力增強(qiáng),劃痕試驗(yàn)和Transwell小室實(shí)驗(yàn)顯示細(xì)胞的遷移愈合能力以及侵襲遷移能力增強(qiáng)。[結(jié)論]①煤煙誘導(dǎo)支氣管上皮細(xì)胞的增殖生長(zhǎng)能力和劃痕愈合能力明顯增強(qiáng)。提示煤煙引起支氣管上皮細(xì)胞發(fā)生惡性傾向。②通過shRNA有效抑制XWLC-05細(xì)胞MUC1基因的表達(dá),可明顯降低細(xì)胞增殖、克隆形成以及劃痕愈合和侵襲遷移能力;提示MUC1基因可能在宣威肺癌的侵襲轉(zhuǎn)移中起著重要作用。③細(xì)胞株體外實(shí)驗(yàn)結(jié)果顯示,低劑量5-Aza-CdR甲基化酶抑制劑可促進(jìn)MUC1基因去甲基化表達(dá)增高;并對(duì)宣威肺癌細(xì)胞的增殖、克隆形成以及劃痕愈合和侵襲遷移能力有促進(jìn)作用。
[Abstract]:[Background] The epigenetics of the gene is thought to play an important role in the occurrence and development of the tumor. The change of MUC1 gene expression caused by the change of DNA methylation pattern plays an important role in the biological behavior of tumor cells. This study is devoted to the expression of MUC1 gene and its biological function in the development of lung cancer cells.[Objective] To reveal the effect of soot induction on the transformation of bronchial epithelial cells and the relationship between the change of the expression of MUC1 gene and the biological characteristics of bronchial epithelial cells and the role of the change of the expression of MUC1 gene in the proliferation and invasion of lung cancer. In order to further understand the role of MUC1 gene in lung cancer, it is necessary to provide a basis for the new diagnosis and treatment of lung cancer.[Methods] The bronchial epithelial cells (Beas-2bCal cells) were induced by using a 1. 0umol/ L and a C1 soot reagent, and the expression of MUC1 was detected by RT-PCR. The proliferation and growth of the induced cells were detected by using the CCK-8 reagent. The scratch test and the Transwell test cell migration healing ability. The shRNA was designed and the effective shRNA was selected, and the expression of MUC1 gene was detected by liposome-mediated transfection, and the expression of MUC1 gene was affected by the biological characteristics of Xuanwei lung cancer cells. and (1) screening the transfected cell strain by observing the fluorescence and the RT-PCR. (2) The proliferation and growth of MUC1 gene after silencing by using CCK-8 reagent. (3) The proliferation ability and population-dependence of the cell after the MUC1 gene silencing were detected by the plate cell clone formation. (4) The scratch test and the Transwell chamber experiment were used to detect the cell scratch and the invasion ability of the cells after the MUC1 gene was silent. The effects of MUCl gene expression on the biological characteristics of Xuanwei lung cancer cells were detected by 5--Aza-CdR methylase inhibition with 5 umol/ L, and the expression of MUC1 was detected by RT-PCR. (2) The proliferation and growth of the cells treated with the methylase inhibitor were detected by the CCK-8 reagent. (3) The proliferation ability and population-dependence of MUC1 gene after demethylation of MUC1 gene were detected by plate cell clone formation. (4) Transwell chamber experiment was used to detect the cell scratch and invasion ability of MUC1 gene after demethylation of MUC1 gene.[Results] The proliferation and growth ability of Beas-2b Coal cells was enhanced by CCK-8 detection. The scratch test showed that the scratch-healing ability of the Beas-2b Coal cells was stronger than that of the normal Beas-2b cells. It is suggested that the soot induced the malignant tendency of the bronchial epithelial cells. The expression of MUC1 gene was blocked by shRNA. After the expression of MUC1 gene was detected by CCK-8, the proliferation ability of the cell of Xuanwei lung cancer was decreased, and the number of cell clone formation and the ability of invasion and migration were significantly reduced. After the 5-Aza-CdR treatment, the expression of the demethylation of MUC1 gene was detected by CCK-8, and the proliferation ability of the lung cancer cells of the Xuanwei lung cancer was enhanced, the scratch test and the Transwell cell experiment showed the migration and healing ability of the cells and the enhancement of the invasion and migration ability.[Conclusion] The proliferation and growth ability of the bronchial epithelial cells and the ability of the healing of the scratch-healing are obviously enhanced. It is suggested that the soot induced the malignant tendency of the bronchial epithelial cells. The expression of the MUC1 gene of the XWLC-05 cell can be effectively inhibited by shRNA, and the cell proliferation, the clone formation and the scratch healing and the invasion and migration ability can be obviously reduced; and the MUC1 gene can play an important role in the invasion and metastasis of the Xuanwei lung cancer. The results of the in vitro experiments show that the low dose of 5-Aza-CdR methylase inhibitor can promote the increase of the demethylation of MUC1 gene, and can promote the proliferation, cloning and formation of the lung cancer cells of the Xuanwei lung cancer and the ability of the scratch healing and the invasion and migration.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
本文編號(hào)：2372490
[Abstract]:[Background] The epigenetics of the gene is thought to play an important role in the occurrence and development of the tumor. The change of MUC1 gene expression caused by the change of DNA methylation pattern plays an important role in the biological behavior of tumor cells. This study is devoted to the expression of MUC1 gene and its biological function in the development of lung cancer cells.[Objective] To reveal the effect of soot induction on the transformation of bronchial epithelial cells and the relationship between the change of the expression of MUC1 gene and the biological characteristics of bronchial epithelial cells and the role of the change of the expression of MUC1 gene in the proliferation and invasion of lung cancer. In order to further understand the role of MUC1 gene in lung cancer, it is necessary to provide a basis for the new diagnosis and treatment of lung cancer.[Methods] The bronchial epithelial cells (Beas-2bCal cells) were induced by using a 1. 0umol/ L and a C1 soot reagent, and the expression of MUC1 was detected by RT-PCR. The proliferation and growth of the induced cells were detected by using the CCK-8 reagent. The scratch test and the Transwell test cell migration healing ability. The shRNA was designed and the effective shRNA was selected, and the expression of MUC1 gene was detected by liposome-mediated transfection, and the expression of MUC1 gene was affected by the biological characteristics of Xuanwei lung cancer cells. and (1) screening the transfected cell strain by observing the fluorescence and the RT-PCR. (2) The proliferation and growth of MUC1 gene after silencing by using CCK-8 reagent. (3) The proliferation ability and population-dependence of the cell after the MUC1 gene silencing were detected by the plate cell clone formation. (4) The scratch test and the Transwell chamber experiment were used to detect the cell scratch and the invasion ability of the cells after the MUC1 gene was silent. The effects of MUCl gene expression on the biological characteristics of Xuanwei lung cancer cells were detected by 5--Aza-CdR methylase inhibition with 5 umol/ L, and the expression of MUC1 was detected by RT-PCR. (2) The proliferation and growth of the cells treated with the methylase inhibitor were detected by the CCK-8 reagent. (3) The proliferation ability and population-dependence of MUC1 gene after demethylation of MUC1 gene were detected by plate cell clone formation. (4) Transwell chamber experiment was used to detect the cell scratch and invasion ability of MUC1 gene after demethylation of MUC1 gene.[Results] The proliferation and growth ability of Beas-2b Coal cells was enhanced by CCK-8 detection. The scratch test showed that the scratch-healing ability of the Beas-2b Coal cells was stronger than that of the normal Beas-2b cells. It is suggested that the soot induced the malignant tendency of the bronchial epithelial cells. The expression of MUC1 gene was blocked by shRNA. After the expression of MUC1 gene was detected by CCK-8, the proliferation ability of the cell of Xuanwei lung cancer was decreased, and the number of cell clone formation and the ability of invasion and migration were significantly reduced. After the 5-Aza-CdR treatment, the expression of the demethylation of MUC1 gene was detected by CCK-8, and the proliferation ability of the lung cancer cells of the Xuanwei lung cancer was enhanced, the scratch test and the Transwell cell experiment showed the migration and healing ability of the cells and the enhancement of the invasion and migration ability.[Conclusion] The proliferation and growth ability of the bronchial epithelial cells and the ability of the healing of the scratch-healing are obviously enhanced. It is suggested that the soot induced the malignant tendency of the bronchial epithelial cells. The expression of the MUC1 gene of the XWLC-05 cell can be effectively inhibited by shRNA, and the cell proliferation, the clone formation and the scratch healing and the invasion and migration ability can be obviously reduced; and the MUC1 gene can play an important role in the invasion and metastasis of the Xuanwei lung cancer. The results of the in vitro experiments show that the low dose of 5-Aza-CdR methylase inhibitor can promote the increase of the demethylation of MUC1 gene, and can promote the proliferation, cloning and formation of the lung cancer cells of the Xuanwei lung cancer and the ability of the scratch healing and the invasion and migration.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
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