EGFR-TKIs通過下調(diào)黏附分子CD44表達(dá)抑制非小細(xì)胞肺癌的轉(zhuǎn)移
[Abstract]:Objective: Lung cancer is one of the most common malignant tumors in the world, in which non-small cell lung cancer accounts for 80-85% of all lung cancers. In recent years, low-toxicity molecular targeted drugs, represented by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), have made breakthroughs in the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), and have been used by the National Compreh Cancer Network (NCN). The main mechanism of EGFR-TKI is to block the abnormal activation of tyrosine kinase domains in the intracellular domain of EGFR, thereby blocking the downstream signal. However, some patients taking EGFR-TKI not only inhibited tumor growth, but also affected the overall metastasis trend. This suggests that EGFR-TKI may not only affect tumor cells themselves, but also possibly regulate them through some potential pathways. Adhesion molecule CD44 is one of the important members of the adhesion molecule family in tumor microenvironment, which mediates cell-to-cell and cell-to-matrix adhesion, and provides sufficient information for tumor metastasis by participating in a series of signal transduction pathways. Dynamics. In the study of head and neck tumors, breast cancer and so on, we found that there may be some cross-synergistic relationship between EGFR signaling pathway and CD44, which plays an important role in tumor metastasis, but whether EGFR signaling pathway and CD44 signaling pathway also have this interaction in non-small cell lung cancer (NSCLC) and affect metastasis. The purpose of this study is to investigate whether the mechanism of EGFR-TKI inhibiting tumor metastasis is related to the down-regulation of CD44 expression in NSCLC cell lines with EGFR mutation and to explore the key signaling molecules linking the two signaling pathways. Methods: 1. HCC 827 cells (EGFR19 exon mutant lung adenocarcinoma cells) and A549 cells (EGFR wild type lung adenocarcinoma cells) were cultured in RPMI-1640 medium containing 15% fetal bovine serum, 100U/ml penicillin and 100ug/ml streptomycin. HCC 827 cells (EGFR mutant) and A549 cells (EGFR wild type) were tested for their sensitivity to erlotinib, a representative of EGFR-TKI, by real-time labeled cell proliferation assay. Cell proliferation inhibitory effect of erlotinib on HCC 827 was detected by MTT assay. The proliferation inhibitory rate and half inhibitory concentration (IC50) were calculated 48 hours after the drug was added to the corresponding concentration of erlotinib and the appropriate concentration of EGF stimulator (50ng/ml) was added to simulate the human body environment. The invasion and migration of tumor cells were observed by Transwell chamber invasion test and scratch test respectively in four groups (control, EGF (50ng/ml), erlotinib (0.3ugM) + EGF (50ng/ml) and CD44 neutralizing antibody (20ug/ml) + EGF (50ng/ml). Flow cytometry was used to detect the invasion and migration of tumor cells in three groups (control, EGF (50n/ml). The expression of CD44 on the surface of tumor cells was detected by Western blot. The expression of CD44 on tumor cells was detected by Q RT-PCR in three groups (control, EGF (50ng/ml) and erlotinib (0.3 mu M) + EGF (50ng/ml) respectively. The expression of CD44m RNA in tumor cells of EGF (50ng/ml) stimulation group, Erlotinib (0.3ugM) + EGF (50ng/ml) treatment group and Western blot assay were used to detect CD44, STAT3, phosphoric acid in four groups (control, EGF (50ng/ml) stimulation group, Erlotinib (0.3ugM) + EGF (50ng/ml) treatment group, STAT3 blocker (S3I-20150ugM) + EGF (50ng/ml) treatment group. Statistical methods: SPSS 21.0 statistical software was used to process the data. The measurement data were expressed as (?) + s or M + QR. Single factor analysis of variance or rank sum test were used to analyze the statistical data. LSD method was used to compare the expression of STAT3 protein between groups. The test level was alpha = 0.05, and the difference was statistically significant with P 0.05. Real-time label-free cell proliferation experiment showed that the EGFR-TKI representative drug Erlotinib (0.3 mu M) inhibited the proliferation of non-small cell lung cancer cell line HCC827 with EGFR mutation in a time-dependent manner, but had little effect on wild-type lung adenocarcinoma cell line A549 with EGFR. HCC827 was selected for follow-up study. 2 MTT assay was used to detect the inhibitory effect of different concentrations of erlotinib on the proliferation of HCC 827 cells. The results showed that with the increase of the concentration of erlotinib (0.001, 0.01, 0.1, 0.5, 1, 10 mu M), the inhibitory rate of proliferation of HCC 827 cells increased gradually after 48 hours and was dose-dependent (Fig.2, Table1, P 0.05). Combined with the inhibitory rate of cell proliferation, the inhibitory rate of erlotinib was linear. Regression analysis showed that IC50 was 0.323 mu M; 3 Transwell invasion test results were shown as Fig.3, Fig.4 and Table2: control, EGF stimulation group, erlotinib + EGF treatment group, CD44 neutralizing antibody + EGF treatment group, the number of penetrating membrane cells were (64.07 [1.51], (129.53 [4.20], (21.0 [1.06], (23.87 [1.70], respectively; the experimental group compared with the control group. Scratch test showed that the migration distances of HCC 827 cells in each group were (78.65 (+ 3.19) micron, (119.98 (+ 1.62) micron, (51.73 (+ 4.23) micron, (53.18 (+ 6.71) micron), respectively. There was significant difference between the experimental group and the control group (P 0.05); the results showed that erlotinib could inhibit the invasion and migration of HCC 827 cells. The expression of CD44 on the surface of HCC 827 cells in three groups (control, EGF (50ng/ml) and erlotinib (0.3 mu M) + EGF (50ng/ml) was detected by flow cytometry. The results showed that the expression of CD44 on the surface of HCC 827 cells in the control, EGF (50 ng/ml) and erlotinib (0.3 mu) + EGF (50 ng/ml) groups was significantly lower than that in the former two groups (Fig) respectively. The semi-quantitative results of CD44 protein in tumor cells of the three groups (the same 4) by Western-blot were (0.72.03), (0.83.04), (0.21.03), respectively. The expression of CD44 protein in erlotinib group was significantly lower than that in control group and EGF stimulation group (Fig.8, Table5, P 0.05). Lotinib could down-regulate the expression of CD44. 6 qRT-PCR assay was used to detect the expression of CD44 m RNA in tumor cells of three groups (the same 4). The expression of CD44 m RNA in EGF-stimulated group and Erlotinib+EGF-treated group was (2.22+0.17) and (0.50+0.04) times higher than that in control group (Fig.9, Table6, P 0.05), respectively. Western-blot method was used to detect the expression of CD44, STAT3 and phosphorylated STAT3 protein in tumor cells of four groups (control, EGF (50ng/ml) stimulation group, erlotinib (0.3ugm) + EGF (50ng/ml) treatment group, STAT3 blocker (S3I-201 50ugM) + EGF (50ng/ml) treatment group). Compared with the control group, the expression of CD44 and p-STAT3 protein in the erlotinib group were significantly down-regulated (P 0.05), while the expression of CD44 protein was still down-regulated after STAT3 specific blocker was used to block the STAT3 signaling pathway (P 0.05); based on the above results, it was preliminarily concluded that erlotinib blocked the EGFR signaling pathway and indirectly down-regulated the adhesion molecule C. The expression of D44 may be mediated by EGFR/STAT3 signaling pathway, but its mechanism still needs further exploration. Conclusion: 1. HCC827 cells are highly sensitive to erlotinib, which is the representative drug of EGFR-TKI. 2 EGFR-TKI drugs can not only inhibit the proliferation of EGFR mutant NSCLC cell line HCC827, but also inhibit its invasion. The ability of metastasis may be related to the down-regulation of CD44. 3 EGFR-TKI can down-regulate the expression of CD44 and may be related to the EGFR/STAT3 signaling pathway.
【學(xué)位授予單位】:河北醫(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
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 張映城;顏兵;施俊;張璇;唐繼貴;孫昱瑋;秦志豐;;胃癌細(xì)胞中CD44陽性細(xì)胞具有腫瘤干細(xì)胞特征[J];中國腫瘤生物治療雜志;2016年04期
2 蔡迎彬;王巖;郭文佳;李可;;CD44基因在腸息肉和結(jié)直腸癌中的表達(dá)及其臨床意義[J];腫瘤學(xué)雜志;2016年02期
3 李小平;張曉偉;郭偉劍;鄭磊貞;;CD44在胰腺癌中的表達(dá)及其臨床意義[J];上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2015年09期
4 楊家佳;張琳;鄭茂金;王慶苓;徐玉婷;柳紅;;胃癌中VEGF、NRP1和干細(xì)胞標(biāo)志物CD44的表達(dá)及相關(guān)性[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2015年02期
5 李潔瑤;Guru Sah Sharan;張毅;陳新峰;王麗萍;;CD24和CD44在非小細(xì)胞肺癌中的表達(dá)和意義[J];醫(yī)藥論壇雜志;2014年03期
6 尹志永;王興波;陳麗娜;任士俊;趙金;楊大勇;;非小細(xì)胞肺癌中CD44、HER-2、P53、VEGF的表達(dá)及與臨床病理特征的關(guān)系[J];中華臨床醫(yī)師雜志(電子版);2013年18期
7 張秀亮;朱志圖;哈敏文;;CD44V6,HIF-1α,,VEGF與非小細(xì)胞肺癌侵襲和轉(zhuǎn)移的關(guān)系[J];遼寧醫(yī)學(xué)院學(xué)報(bào);2008年01期
本文編號(hào):2196676
本文鏈接:http://sikaile.net/shoufeilunwen/mpalunwen/2196676.html