Lin28B在MALAT1降低非小细胞肺癌对吉非替尼耐药机制的研究

发布时间：2018-04-27 19:21

  本文选题：MALAT1 + Lin28A　； 参考：《大连医科大学》2017年硕士论文

【摘要】：目的:在对肿瘤的治疗过程中,肿瘤细胞对药物耐药是一个函待解决的难题。课题组前期研究发现,下调MALAT1的表达可以逆转A549细胞株对吉非替尼的耐药性。但是其中的具体机制我们并不清楚。Lin28是一个肿瘤干细胞相关基因,它在多种肿瘤中异常表达,并与肿瘤的低分化和不良预后有关,Lin28(Lin28A)和它的同源基因Lin28B与恶性肿瘤的发生、进展、预后以及诱导多功能干细胞形成等密切相关。上皮间质转化(Epithelial-Mesenchymal Transition,EMT)是指在外界环境刺激下,上皮细胞转化为间充质细胞的现象。EMT的发生与肿瘤的形成和转移密不可分。近年来,越来越多的研究发现,肿瘤细胞的耐药与肿瘤干细胞基因Lin28高表达和EMT的发生有密切的关系。本实验主要探究抑制MALAT1表达是否可以通过影响Lin28表达和EMT的发生这两条途径来逆转肺癌细胞株A549对吉非替尼的耐药。方法:(1)用PCR和Western Blot方法分别检测Lin28A和Lin28B在A549(耐药)细胞株和HCC827(敏感)细胞株中表达水平。(2)用Western Blot方法分别检测E钙粘蛋白(E-Cadherin)和波形蛋白(Vimmentin)在A549(耐药)细胞株和HCC827(敏感)细胞株中表达水平。(3)用siRNA转染A549(耐药)细胞,检测转染效率,抑制MALAT1表达。(4)CCK方法检测转染前后A549对吉非替尼的耐药情况。(5)用PCR和Western Blot方法检测Lin28在转染组(MALAT1表达受到抑制)A549(耐药)细胞株中表达水平。(6)用Western Blot方法分别检测转染组(MALAT1表达受到抑制)和未转染组中E-Cadherin和Vimentin在A549(耐药)细胞株表达水平。结果:(1)在A549细胞株与HCC827细胞株中,Lin28A表达量存在差异,但是差异不具有统计学意义(P0.05);Lin28B在A549中基因和蛋白水平均明显高于HCC827,具有统计学意义(P0.05)。(2)与HCC827细胞株相比,A549细胞株中E-Cadherin蛋白的表达水平下调,具有统计学意义(P0.05);Vimentin蛋白表达水平上调,有统计学意义(P0.05)。(3)与未转染组相比,转染组对吉非替尼耐药性下降,(P0.05),有统计学意义。(4)与未转染组相比,细胞转染组中MALAT1表达受到抑制,Lin28B基因和蛋白表达水平均下降且有统计学意义(P0.05);(5)与未转染组相比,细胞转染组E-Cadherin表达水平升高,具有统计学意义(P0.05);Vimentin表达水平降低,具有统计学意义(P0.05);结论:(1)Lin28B和EMT可能参与了 A549对吉非替尼耐药的相关机制。(2)MALAT1可能通过影响Lin28B来逆转A549对吉非替尼的耐药。(3)MALAT1可能通过影响EMT来逆转A549对吉非替尼的耐药。
[Abstract]:Objective: in the course of tumor treatment, drug resistance of tumor cells is a difficult problem to be solved. Our previous study found that down-regulation of MALAT1 could reverse the drug resistance of A549 cell line to gefitinib. However, the specific mechanism of Lin28 is not clear. Lin28 is a tumor-stem cell related gene, which is abnormal expressed in many kinds of tumors, and is related to the low differentiation and poor prognosis of tumor. Lin28Lin28A) and its homologous gene Lin28B are related to the occurrence and progression of malignant tumor. Prognosis and induction of multifunctional stem cell formation are closely related. Epithelial-mesenchymal transition (EMTT) is a phenomenon in which epithelial cells are transformed into mesenchymal cells under the stimulation of external environment. The occurrence of EMT is closely related to tumor formation and metastasis. In recent years, more and more studies have found that drug resistance of tumor cells is closely related to the overexpression of tumor stem cell gene Lin28 and the occurrence of EMT. The aim of this study was to investigate whether inhibiting the expression of MALAT1 could reverse the drug resistance of lung cancer cell line A549 to gefitinib by affecting the expression of Lin28 and the development of EMT. Methods PCR and Western Blot were used to detect the expression of Lin28A and Lin28B in A549 cell line and HCC827 (sensitive) cell line, respectively. E-Cadherin and vimentin were detected by Western Blot and vimentin in A549 cell line and HCC827 cell line, respectively. SiRNA was used to transfect A549 (drug resistant) cells. Detection of transfection efficiency, Inhibition of MALAT1 expression. Detection of the drug resistance of A549 to gefitinib before and after transfection. Use of PCR and Western Blot methods to detect the expression level of Lin28 in the transfected cell line. 6) Western Blot method was used to detect the expression of Lin28 in the transfected cell line. The expression of E-Cadherin and Vimentin in A549 cell line was inhibited in the staining group and in the untransfected group. Results there was significant difference in the expression of HCC827 and A549 cell line in cell line 1 (1), and the expression of Lin28A in A549 cell line was significantly different from that in HCC827 cell line. However, there was no significant difference in the gene and protein levels in A549, which was significantly higher than that in HCC827. The expression of E-Cadherin protein was down-regulated in A549 cell line compared with that of HCC827 cell line, and the expression level of Vimentin protein was up-regulated in A549 cell line, and the expression of Vimentin protein in A549 cell line was significantly higher than that in A549 cell line. Compared with the untransfected group, the drug resistance of the transfection group to gefitinib decreased, and the drug resistance of the transfection group was significantly lower than that of the untransfected group, and the drug resistance of the transfected group was significantly lower than that of the untransfected group, and the drug resistance of the transfected group was significantly lower than that of the untransfected group. The expression level of MALAT1 in the transfected group was significantly lower than that in the non-transfected group. The expression level of E-Cadherin in the transfected group was significantly higher than that in the non-transfected group, and the expression level of Vimentin in the transfected group was significantly lower than that in the non-transfected group. Conclusion: 1: 1) Lin28B and EMT may be involved in the mechanism of A549's resistance to gefitinib. [WT5BZ] MALAT1 may reverse A549's resistance to gifetini by affecting Lin28B, which may reverse A549's resistance to gefitinib by affecting EMT.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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本文编号：1811989