煤烟诱导支气管上皮细胞转化和MUC1基因表达对肺癌细胞生物学特性的影响
发布时间：2018-12-11 11:45
【摘要】：[背景]基因的表观遗传学被认为在肿瘤的发生和发展中起到重要作用。而DNA甲基化模式改变引起的MUC1基因表达的改变,对肿瘤细胞的生物学行为的影响起着重要的作用。本研究致力于对MUC1基因的表达情况,以及其在肺癌细胞发生发展中生物学功能进行探究。[目的]揭示煤烟诱导对支气管上皮细胞转化的影响和MUC1基因表达改变与支气管上皮细胞生物学特性改变的相关性及在肺癌增殖、侵袭中的作用。为深入理解MUC1基因在肺癌中的作用进行初步探索,以期为肺癌新的诊断和治疗方法提供研究基础。[方法]①用1.0umol/L,C1煤烟试剂诱导支气管上皮细胞(Beas-2bCoal细胞),通过RT-PCR检测MUC1的表达情况,采用CCK-8试剂检测诱导细胞的增殖生长情况,用平板细胞克隆形成实验检测细胞的增殖能力和群体依赖性,划痕实验和Transwell检测细胞迁移愈合能力。②设计shRNA,筛选有效shRNA,通过脂质体介导的转染试验,同时设立Blank组(空白对照组)、shNC阴性对照组(无关干扰序列阴性对照组),检测MUC1基因的表达对宣威肺癌细胞的生物学特性的影响:(1)通过观察荧光及RT-PCR筛选转染细胞株。(2)用CCK-8试剂检测MUC1基因沉默后细胞的增殖生长情况。(3)用平板细胞克隆形成实验检测MUC1基因沉默后细胞的增殖能力和群体依赖性。(4)划痕实验和Transwell小室实验检测MUC1基因沉默后细胞的细胞划痕愈合以及迁移侵袭能力。③用5 umol/L的5--Aza-CdR甲基化酶抑制,处理Beas-2b,煤烟诱导Beas-2b和XWLC-05细胞,检测MUCl基因表达对宣威肺癌细胞的生物学特性的影响:(1)通过RT-PCR检测MUC1的表达情况。(2)用CCK-8试剂检测甲基化酶抑制剂处理后细胞的增殖生长情况。(3)用平板细胞克隆形成实验检测MUC1基因去甲基化后细胞的增殖能力和群体依赖性。(4) Transwell小室实验检测MUC1基因去甲基化后细胞的细胞划痕愈合以及迁移侵袭能力。[结果]①CCK-8检测显示Beas-2b Coal细胞的增殖生长能力较正常Beas-2b细胞增强。划痕试验显示Beas-2b Coal细胞划痕愈合能力较正常Beas-2b细胞明显增强。提示煤烟引起支气管上皮细胞发生恶性倾向。②shRNA干扰MUC1基因表达沉默,CCK-8检测显示MUC1基因沉默后,宣威肺癌细胞的增殖能力下降,平板克隆和Transwell小室实验显示细胞克隆形成数目明显减少以及侵袭迁移能力受抑。③5-Aza-CdR处理后,CCK-8检测显示MUC1基因去甲基化表达上调后,宣威肺癌细胞的增殖能力增强,划痕试验和Transwell小室实验显示细胞的迁移愈合能力以及侵袭迁移能力增强。[结论]①煤烟诱导支气管上皮细胞的增殖生长能力和划痕愈合能力明显增强。提示煤烟引起支气管上皮细胞发生恶性倾向。②通过shRNA有效抑制XWLC-05细胞MUC1基因的表达,可明显降低细胞增殖、克隆形成以及划痕愈合和侵袭迁移能力;提示MUC1基因可能在宣威肺癌的侵袭转移中起着重要作用。③细胞株体外实验结果显示,低剂量5-Aza-CdR甲基化酶抑制剂可促进MUC1基因去甲基化表达增高;并对宣威肺癌细胞的增殖、克隆形成以及划痕愈合和侵袭迁移能力有促进作用。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
本文编号：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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
【参考文献】
相关期刊论文 前5条
1 王静;杨永秀;毕学汉;;MUC1与E-cadherin在子宫内膜癌中的表达及意义[J];卫生职业教育;2016年08期
2 李引钰;;MYC和MUC1 mRNA联合检测在乳腺癌诊断中的应用[J];现代肿瘤医学;2015年22期
3 庄婷;薛梅;房爱菊;管冰心;梁艳;王志蕙;王妍;孟斌;;黏蛋白(MUC1、MUC2、MUC5AC和MUC6)在乳腺浸润性导管癌中的表达及意义[J];山东大学学报(医学版);2012年05期
4 蔡祖勋,梁庆正,张学宪,王旭;非小细胞肺癌组织中p53和MUC1的表达及其意义[J];肿瘤防治杂志;2004年07期
5 李芸,沈文会,赵维敏,邱琦,吴元德,仲伟鉴,张荣泉,郑方园,张国雄;煤烟提取物致癌作用机制的研究[J];卫生毒理学杂志;2000年02期
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