趋化因子受体CCR7在乳腺癌细胞转移中的机制探讨

发布时间：2018-07-18 14:17

【摘要】：目的:本实验利用RNA干扰技术,以乳腺癌细胞为研究对象,下调乳腺癌细胞系中的基因CCR7的表达对乳腺癌细胞转移的影响,并行蛋白质谱检测,研究沉默CCR7后哪些蛋白和信号分子参与CCR7的调控作用,对探讨CCR7与乳腺癌转移的内在机制,发现新的诊断标记物及治疗靶点具有积极的意义。方法:1、常规培养乳腺癌细胞系MDA-MB-231、4T1-luc、MDA-MB-361、T47D、MDA-MB-468、MCF-7、MCF10A、184B5、184A1、HCC1806、HCC1937、HCC1580、MCF10A,用q-PCR和Westen blot实验筛选出高表达CCR7的乳腺癌细胞系。2、对筛选出来的乳腺癌细胞株进行转染,提取实验组(CCR7-siRNA)和对照组的总RNA和总蛋白,用q-PCR和Western blot检测干扰效果。3、利用划痕实验和transwell小室迁移实验检测了siCCR7转染对乳腺癌细胞迁移的影响。4、通过蛋白质谱结果筛选出差异表达蛋白,并用q-PCR验证参与CCR7的调控作用关键蛋白。结果:1、高表达CCR7的乳腺癌细胞系的筛选q-PCR结果显示:人乳腺癌细胞株T47D、HCC1500、SK-RB-3、BT474、MCF7的CCR7的mRNA表达水平较其他乳腺癌细胞株高。高表达CCR7的乳腺癌细胞系的筛选Western blot结果显示:CCR7在小鼠乳腺癌细胞株4T1-Luc、人乳腺癌细胞株T74D、MCF7中高表达,在部分乳腺癌细胞株中如MB231、MB468、HCC1937中有较高水平的表达。但是在正常乳腺细胞株如184B5、184A1和MCF10A中几乎无CCR7表达。最后我们选取MCF7及T47D乳腺癌细胞进行后续的实验。2、T47D、MCF7细胞在经过siRNA处理后,通过q-PCR检测对T47D、MCF7乳腺癌细胞的沉默效应,结果显示CCR7-siRNA 1#和CCR7-siRNA 2#能非常有效地抑制T47D、MCF7细胞中内源性CCR7的mRNA水平的表达。Western blot得到相似的结果。3、划痕实验和transwell小室迁移实验结果均表明,与shLacZ组比,shCCR7C组和shCCR7D组乳腺癌细胞迁移能力变弱。4、通过蛋白质谱结果筛选出差异表达蛋白如下:EPHA2、PVR、SQSTM1、ANXA6、PTRF、ALDH3A2、EPCAM。并用q-PCR方法及Western blot方法验证沉默CCR7后ALDH3A2和PTRF可能参与CCR7的调控作用。结论:(一)CCR7在不同的乳腺癌细胞株的表达量有所不同,在恶性程度较高的细胞中表达量更高,说明可能CCR7的表达水平的高低与乳腺癌转移的恶性程度有一定相关性。(二)敲低CCR7后抑制了乳腺癌细胞的迁移,引起了 ALDH3A2及PTRF的降低。(三)干扰CCR7抑制乳腺癌细胞的迁移可能通过下调ALDH3A2和PTRF的表达实现,这需要进一步研究。
[Abstract]:Objective: to down-regulate the expression of CCR7 gene in breast cancer cell line by RNA interference technique, and to detect the expression of CCR7 gene in breast cancer cell line, and to detect the expression of CCR7 in breast cancer cell line. The study of which proteins and signaling molecules involved in CCR7 regulation after CCR7 silencing has positive significance in exploring the intrinsic mechanism of CCR7 and breast cancer metastasis and finding new diagnostic markers and therapeutic targets. Methods the breast cancer cell line MDA-MB-231T 1-lucus MDA-MB-361MDA-MB-468 MDA-MB-468 MCF-7MCF10An1848B554A1806 HCC1937HCC1580MCF10A was screened by q-PCR and Westen blot assay. The breast cancer cell line with high expression of CCR7 was screened by q-PCR and Westen blot. The total RNA and total protein of the experimental group (CCR7-siRNA) and the control group were extracted from the breast cancer cell line MDA-MB-361MDA-MB-468, the total RNA and total protein of the experimental group (CCR7-siRNA) and the control group were extracted. The interference effect was detected by q-PCR and Western blot. The effect of siCCR7 transfection on the migration of breast cancer cells was detected by scratch test and transwell chamber migration assay. The differentially expressed proteins were screened by protein analysis. The key proteins involved in the regulation of CCR7 were verified by q-PCR. Results the screening of breast cancer cell lines with high expression of CCR7 by q-PCR showed that the expression level of CCR7 mRNA in human breast cancer cell line T47DHCC1500 SK-RB-3 BT474 MCF7 was higher than that in other breast cancer cell lines. The screening of breast cancer cell lines with high expression of CCR7 by Western blot showed that the high expression level was found in mouse breast cancer cell line 4T1-Lucand human breast cancer cell line T74DnMCF7, and in some breast cancer cell lines such as MB231M468HCC1937. But there was almost no CCR 7 expression in normal mammary gland cell lines such as 184 B 5, 184 A1 and MCF10 A. Finally, we selected MCF7 and T47D breast cancer cells for further experiment. After siRNA treatment, we detected the silencing effect of MCF7 cells on T47D- MCF7 cells by q-PCR. The results showed that CCR7-siRNA 1# and CCR7-siRNA 2# could effectively inhibit the expression of endogenous CCR7 mRNA in T47D- MCF7 cells. Western blot obtained similar results. Compared with shLacZ group, the migration ability of breast cancer cells in shCCR7C and shCCR7D groups was weaker than that in shLacZ group. The differentially expressed proteins were as follows: EPHA2, PVR, SQSTM1, ANXA6, PTRFV, ALDH3A2, EPCAM. Q-PCR and Western blot methods were used to verify that ALDH3A2 and PTRF might be involved in the regulation of CCR7 after CCR7 silencing. Conclusion: (1) the expression of CCR7 in different breast cancer cell lines is different, and the expression level is higher in the cells with higher malignant degree, which suggests that the level of CCR7 expression may be related to the malignant degree of breast cancer metastasis. (2) knocking down CCR7 inhibited the migration of breast cancer cells, and caused the decrease of ALDH3A2 and PTRF. (3) inhibiting the migration of breast cancer cells by CCR7 may be achieved by down-regulating the expression of ALDH3A2 and PTRF, which needs further study.
【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】：R737.9

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