本文選題：MALAT1 + Lin28A�。� 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:在對腫瘤的治療過程中,腫瘤細胞對藥物耐藥是一個函待解決的難題。課題組前期研究發(fā)現(xiàn),下調(diào)MALAT1的表達可以逆轉(zhuǎn)A549細胞株對吉非替尼的耐藥性。但是其中的具體機制我們并不清楚。Lin28是一個腫瘤干細胞相關(guān)基因,它在多種腫瘤中異常表達,并與腫瘤的低分化和不良預(yù)后有關(guān),Lin28(Lin28A)和它的同源基因Lin28B與惡性腫瘤的發(fā)生、進展、預(yù)后以及誘導(dǎo)多功能干細胞形成等密切相關(guān)。上皮間質(zhì)轉(zhuǎn)化(Epithelial-Mesenchymal Transition,EMT)是指在外界環(huán)境刺激下,上皮細胞轉(zhuǎn)化為間充質(zhì)細胞的現(xiàn)象。EMT的發(fā)生與腫瘤的形成和轉(zhuǎn)移密不可分。近年來,越來越多的研究發(fā)現(xiàn),腫瘤細胞的耐藥與腫瘤干細胞基因Lin28高表達和EMT的發(fā)生有密切的關(guān)系。本實驗主要探究抑制MALAT1表達是否可以通過影響Lin28表達和EMT的發(fā)生這兩條途徑來逆轉(zhuǎn)肺癌細胞株A549對吉非替尼的耐藥。方法:(1)用PCR和Western Blot方法分別檢測Lin28A和Lin28B在A549(耐藥)細胞株和HCC827(敏感)細胞株中表達水平。(2)用Western Blot方法分別檢測E鈣粘蛋白(E-Cadherin)和波形蛋白(Vimmentin)在A549(耐藥)細胞株和HCC827(敏感)細胞株中表達水平。(3)用siRNA轉(zhuǎn)染A549(耐藥)細胞,檢測轉(zhuǎn)染效率,抑制MALAT1表達。(4)CCK方法檢測轉(zhuǎn)染前后A549對吉非替尼的耐藥情況。(5)用PCR和Western Blot方法檢測Lin28在轉(zhuǎn)染組(MALAT1表達受到抑制)A549(耐藥)細胞株中表達水平。(6)用Western Blot方法分別檢測轉(zhuǎn)染組(MALAT1表達受到抑制)和未轉(zhuǎn)染組中E-Cadherin和Vimentin在A549(耐藥)細胞株表達水平。結(jié)果:(1)在A549細胞株與HCC827細胞株中,Lin28A表達量存在差異,但是差異不具有統(tǒng)計學(xué)意義(P0.05);Lin28B在A549中基因和蛋白水平均明顯高于HCC827,具有統(tǒng)計學(xué)意義(P0.05)。(2)與HCC827細胞株相比,A549細胞株中E-Cadherin蛋白的表達水平下調(diào),具有統(tǒng)計學(xué)意義(P0.05);Vimentin蛋白表達水平上調(diào),有統(tǒng)計學(xué)意義(P0.05)。(3)與未轉(zhuǎn)染組相比,轉(zhuǎn)染組對吉非替尼耐藥性下降,(P0.05),有統(tǒng)計學(xué)意義。(4)與未轉(zhuǎn)染組相比,細胞轉(zhuǎn)染組中MALAT1表達受到抑制,Lin28B基因和蛋白表達水平均下降且有統(tǒng)計學(xué)意義(P0.05);(5)與未轉(zhuǎn)染組相比,細胞轉(zhuǎn)染組E-Cadherin表達水平升高,具有統(tǒng)計學(xué)意義(P0.05);Vimentin表達水平降低,具有統(tǒng)計學(xué)意義(P0.05);結(jié)論:(1)Lin28B和EMT可能參與了 A549對吉非替尼耐藥的相關(guān)機制。(2)MALAT1可能通過影響Lin28B來逆轉(zhuǎn)A549對吉非替尼的耐藥。(3)MALAT1可能通過影響EMT來逆轉(zhuǎn)A549對吉非替尼的耐藥。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2
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本文編號：1811989