大黄素逆转非小细胞肺癌EGFR-TKI耐药的机制研究

目的 探讨大黄素逆转非小细胞肺癌（NSCLC）表皮生长因子受体酪氨酸激酶抑制剂（EGFR TKI）耐药的作用机制。方法 应用持续诱导的方法构建NSCLC EGFR-TKI耐药细胞株HCC827／GR;应用MTS法检测大黄素（30μmol／L）、吉非替尼（1μmol／L）及两药联合处理HCC827和HCC827／GR细胞48h后细胞增殖能力的变化;应用Western blotting法检测HCC827和 HCC827／GR细胞中p EGFR、p-AKT、p-ERK1／2及p-MET蛋白表达水平的变化。结果 MTS法检测结果显示,经单药吉非替尼或大黄素处理后,HCC827／GR细胞增殖能力未减弱,而两药联合处理组的细胞增殖能力明显下降,差异有统计学意义（P＜0.05）。Western blotting检测结果显示,HCC827、HCC827／GR细胞中p-EGFR、p-ERK1/2明显表达,而p-AKT表达微弱;HCC827／GR 中p-MET表达水平较HCC827明显上调。经单药吉非替尼处理后,HCC827细胞株p-EGFR、p-ERK1/2表达水平下调,HCC827／GR细胞株p-EGFR表达明显下调;大黄素可显著下调HCC827／GR细胞株p-MET表达,但对p-EGFR、p-ERK1/2的表达无影响;而大黄素与吉非替尼两药联用可明显抑制HCC827／GR细胞株p-EGFR、p-ERK1/2以及p-MET的表达。结论 大黄素可以逆转NSCLC EGFR-TKI耐药,可能是通过抑制c-Met的活化来实现。     

BIM induction of apoptosis triggered by EGFR-sensitive and resistance cell lines of non-small-cell lung cancer

We sought to improve the understanding of oncogene-dependent and independent non-small-cell lung cancer (NSCLC), which could provide insight into mechanism of sensitivity and/or resistance to tyrosine kinase inhibitors or chemotherapeutics. NSCLC cell lines with different EGFR genotypes were used in this study; MTT assay and flow cytometry were applied to study the sensitivities of these cell lines to gefitinib and cisplatin. Western blot was performed to determine the expression levels of BIM and other Bcl-2 family proteins pre- and pro-treatment. Gefitinib provoked apoptosis of caspase activation via the intrinsic pathways and significantly up-regulated expression of BIM protein in drug-sensitive PC-9 cell line, but not resistant PC-9/BB4 cell line. The knockdown of BIM expression by RNA interference virtually eliminated gefitinib-induced cell killing in PC-9 cells in vitro. Cisplatin could induce apoptosis of the cell lines, including H1299, A549, PC-9, and PC-9/BB4 cells, but which was not associated with overexpression of BIM. BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cell line. Down-regulation of BIM and resistance to gefitinib were both seen in the acquired resistant PC-9/BB4 cell line. The induction of BIM may have a role in the treatment of TKI-resistant tumors.     

TIP30逆转人非小细胞肺癌吉非替尼耐药的实验研究

  目的  本实验旨在研究TIP30能否逆转非小细胞肺癌（non-small cell lung cancer,NSCLC）的吉非替尼耐药,并探讨其可能的机制。  方法  慢病毒LV-TIP30转染NSCLC吉非替尼耐药株PC9/GR上调TIP30,以PC9/GR、PC9/GR-LVTIP30和PC9/GR-LVNC为研究对象,分别加或不加5 μmol/L吉非替尼处理共6组细胞。CCK8检测细胞增殖抑制率,划痕修复实验、Transwell实验检测细胞迁移侵袭能力,免疫印迹实验检测p-AKT、p-ERK、p-MEK以及核内EGFR蛋白表达水平。  结果  非吉非替尼处理组中,PC9/GR-LVTIP30细胞的增殖、迁移和侵袭能力与PC9/GR细胞相比均受到明显的抑制（P < 0.05）,吉非替尼处理组中PC9/GR-LVTIP30细胞较PC9/GR细胞抑制作用更明显（P < 0.05）;过表达TIP30后,蛋白p-MEK、p-ERK、p-AKT表达水平降低,核内EGFR表达水平较PC9/GR降低,差异均具有统计学意义（P < 0.05）。  结论  上调TIP30能够逆转人非小细胞肺癌PC9/GR细胞对吉非替尼的耐药性,其发挥作用的机制可能是通过抑制EGFR核内化,进而抑制下游信号通路相关蛋白p-AKT、p-ERK、p-MEK激活发挥作用。      

非小细胞肺癌PC-9细胞厄洛替尼耐药潜在机制的RNA测序分析

目的:通过RNA测序分析比较亲本PC-9细胞和厄洛替尼获得性耐药PC-9细胞(PC-9/ER)表达谱的差异,揭示非小细胞肺癌(NSCLC)表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)耐药的潜在机制。方法:采用间歇诱导方法,建立PC-9/ER耐药细胞株,通过MTT实验绘制厄洛替尼药物浓度-细胞存活率曲线。通过RNA测序分析PC-9/ER细胞的差异表达基因并进行GO和KEGG功能富集分析;通过qRT-PCR进一步筛选可能参与EGFR-TKI耐药的潜在基因或可能的靶点。结果:厄洛替尼对PC-9/ER细胞增殖的抑制率显著低于PC-9细胞的抑制率,PC-9/ER细胞的耐药指数为41.92。与PC-9细胞相比,RNA测序PC-9/ER细胞筛选出1 028个差异表达基因,其中720个基因表达上调,308个基因表达下调,而且差异表达基因显著富集在PI3K-AKT通路和癌症通路。qRT-PCR验证差异表达基因的转录水平与测序结果基本一致。结论:ST6GALNAC3、CYP1A1、PAPPA2、INHBE和ACSS3等基因可能参与EGFR-TKI的耐药过程,针对PC-9/ER细胞差异表达基因的后续...     

MiR-130a Overcomes Gefitinib Resistance by Targeting Met in Non-Small Cell Lung Cancer Cell Lines

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most common cause oflung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is used for its treatment;however, drug resistance is a major obstacle. Expression of Met has been associated with both primary andacquired resistance to gefitinib, but the mechanisms regulating its expression are not fully understood. Recently,miRNAs such as miR-130a have been shown to play a role in gefitinib resistance, but importance in NSCLC andrelationships with Met have not been fully explored. Here we show that miR-130a is over-expressed in gefitinibsensitiveNSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Moreover, miR-130a expressionwas negatively correlated with that of Met. Further analysis revealed that over-expression of miR-130a increasedcell apoptosis and inhibited proliferation of NSCLC cells treated with gefitinib, whereas lowering the expressionof miR-130a decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in bothgefitinib-sensitive and -resistant NSCLC cell lines, suggesting that miR-130a overcomes gefitinib resistance.We also demonstrated that miR-130a binds to the 3’-UTR of Met and significantly suppresses its expression.Finally, our results showed that over-expressing Met could “rescue” the functions of miR-130a regarding cellapoptosis and proliferation after cells are treated with gefitinib. These findings indicate that the miR-130a/Metaxis plays an important role in gefitinib resistance in NSCLC. Thus, the miR-130a/Met axis may be an effectivetherapeutic target in gefitinib-resistant lung cancer patients.     

Implication of the insulin-like growth factor-IR pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been found to be effective against lung cancer in vitro, but clinical resistance to these agents has developed as their usage has increased. In this study, we determined whether the insulin-like growth factor I (IGF-I) signaling pathway induces resistance of non-small cell lung cancer (NSCLC) cells to the EGFR tyrosine kinase inhibitor gefitinib. EXPERIMENTAL DESIGN: The effects of gefitinib and cetuximab on NSCLC cells, alone or with an IGF-I receptor (IGF-IR) inhibitor, were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the flow cytometry-based terminal nucleotidyl transferase-mediated nick end labeling assay, coimmunoprecipitation, and Western blot analysis. EGFR and IGFR expression in NSCLC tissues were examined by Western blot analysis. RESULTS: Gefitinib inhibited NSCLC cell proliferation by inducing apoptosis when IGF-IR signaling was suppressed. Treatment with gefitinib, but not cetuximab, induced EGFR:IGF-IR heterodimerization and activation of IGF-IR and its downstream signaling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF-IR expression. Inhibition of IGF-IR activation and knockdown of survivin expression led to increased apoptosis. In contrast, overexpression of survivin protected cells with low IGF-IR expression from gefitinib-induced apoptosis. Most NSCLC tissues with EGFR overexpression had associated high levels of IGF-IR expression. CONCLUSIONS: IGF-IR expression may be useful as a predictive marker for gefitinib treatment of NSCLC. Suppression of IGF-IR signaling pathways may prevent or delay development of gefitinib resistance in patients with NSCLC.     

ID1对非小细胞肺癌EGFR-TKI耐药的影响

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是当今世界上发病率和死亡率最高的恶性肿瘤之一,而表皮生长因子受体-酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitors, EG-FR-TKI）整体有效率为30%-40%,无进展生存期（progression-free survival, PFS）为12个月。但EGFR-TKI在临床中的耐药现象也很普遍,严重影响了其抑瘤作用。因此,克服耐药、寻找一种新的与肺癌耐药相关的预后因子势在必行。本研究旨在通过体内外实验探讨DNA结合抑制因子1（differentiation inhibitory factor 1, ID1）与NSCLCEGFR-TKI耐药之间的关系,看其是否有统计学意义,并初步探讨其机制。方法免疫组化（immunohistochemistry, IHC）检测手术标本（肺癌组织和癌旁组织）1D1的表达;qRT-PCR、Western-blot检测并比较肺癌细胞敏感株与耐药株中ID1的表达变化;MTT检测吉非替尼对ID1慢病毒载体处理肺癌细胞的细胞增殖情况,将肺癌细胞接种至裸鼠腋下,待肿瘤生长至一定体积使用吉非替尼治疗,估算肿瘤体积。结果 ID1在肺癌组织中的表达明显高于正常组织（P<0.05）;ID1的表达与NSCLC EGFR-TKI耐药呈正相关（P<0.05）。结论 ID1在NSCLC中高表达,并且参与了NSCLC EGFR-TKI的耐药,其机制可能与STAT3磷酸化程度增加有关。     

非小细胞肺癌吉非替尼耐药相关miRNAs的筛选鉴定

  目的  miRNA是一类通过结合mRNA调节基因表达的非编码单链小分子RNA,本研究目的是探讨非小细胞肺癌（NSCLC）中miRNA与吉非替尼耐药的关系。  方法  CCK8法检测NSCLC吉非替尼耐药细胞PC9/GR相对于亲本细胞PC9的耐药倍数;miRNA芯片检测PC9/GR与PC9中miRNA的表达差异;RT-PCR验证miRNA芯片结果。将差异表达的miRNA模拟物/抑制剂转染至PC9/GR中,观察其对吉非替尼敏感性的影响。  结果  吉非替尼对PC9和PC9/GR的IC₅₀值分别为42.89 nmoL/L和3.87 μ moL/L,耐药倍数为90.23倍。miRNA芯片结果显示,PC9/GR与PC9比较55条有差异表达miRNAs（P < 0.01）,其中在PC9/GR上调的miRNAs有21条,包括miRNA-1 246、miRNA-125b等;下调的miRNAs有34条,包括miRNA-224、miRNA-125a~5p等。RT-PCR进一步验证其中9条miRNAs,有8条与芯片结果趋势一致。将上述8条miRNAs的模拟物/抑制剂转染至PC9/GR中,发现miRNA-125a~5p模拟物可降低吉非替尼敏感性。  结论  PC9/GR与PC9的miRNA表达存在差异,miRNA可能与NSCLC吉非替尼耐药相关,miRNA-125a~5p可促进PC9/GR对吉非替尼产生耐药。      

MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET

In non-small-cell lung cancer (NSCLC) that harbours an activating epidermal growth factor receptor (EGFR) mutation, over-expression of hepatocyte growth factor (HGF) is an important mechanism involved in the acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) by restoring activity of the PI3K/Akt pathway via phosphorylation of MET. In our study, we found that the forced expression of miR-34a inhibited cell growth and induced apoptosis partly by targeting MET in HGF-induced gefitinib-resistant HCC827 and PC-9 cells. Furthermore, dramatic tumour regression was observed in the miR-34a plus gefitinib group in HGF-induced gefitinib resistant mouse xenograft models. This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells.     

The P21-activated kinase expression pattern is different in non-small cell lung cancer and affects lung cancer cell sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors

Exploring methods for increasing epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) sensitivity has become a major focus in non-small cell lung cancer (NSCLC). Major downstream effectors of the Rho family small guanosine triphosphatases, P21-activated kinases (PAKs) activate the main signaling pathways downstream of EGFR and thus promote tumor cell proliferation. In this study, we explored the expression pattern of phosphorylated PAKs in NSCLC and their potential value as drug targets for treating cancer. The expression and prognostic significance of phosphorylated group I and II PAKs were evaluated in 182 patients with NSCLC. Immunohistochemical analysis revealed low group I PAK expression in normal lung tissues and increased expressed in the cytoplasm, particularly in lung squamous cell carcinoma. Abnormal group I PAK expression was associated with lymph node metastases and high tumor-node-metastases (TNM) stage in NSCLC patients and correlated with poor prognosis. We used group I PAK inhibitor (IPA3) to specifically decrease group I PAK activity in human lung cancer cell lines. Decreased group I PAK activity inhibited cell proliferation and combined IPA3 and EGFR-TKI (gefitinib) treatment inhibited cell proliferation in an obvious manner. Together, our results revealed the PAK expression pattern in NSCLC, and a role for group I PAK in cell proliferation, which provides evidence that decreased PAK activity may have a potential application as a molecular targeted therapy in advanced NSCLC.     

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

VEGF-D基因在肺腺癌、吉非替尼继发耐药肺腺癌及正常肺组织中的表达

背景与目的 表皮生长因子酪氨酸激酶受体抑制剂(epidermal growth factor receptor tyrosine inhibitor,EGFR-TKI)在治疗非小细胞肺癌中逐渐开始发挥重要作用,对EGFR-TKI继发耐药的机制亦日益受到关注.本研究旨在了解血管内皮生长因子D(vascular endothelial growth factor D,VEGF-D)基因在肺腺癌、吉非替尼继发耐药肺腺癌以及正常肺组织中的表达情况,以了解其在吉非替尼继发耐药中的作用.方法 取外科切除的肺腺癌组织及癌旁正常肺组织、吉非替尼继发耐药肺腺癌组织(包括转移的淋巴结).以β-actin为内参照,SYBR Green法实时定量PCR检测VEGF-D基因相对表达水平.确定阳性表达结果后,对表达率和表达水平进行定性和定量分析.结果 对吉非替尼继发耐药肺腺癌(1/6,16.7%)、肺腺癌(7/14,50.0%)和正常肺组织(16/16,100.0%)中VEGF-D基因表达率用计算确切概率值法比较后,三组样本VEGF-D的表达率有显著差别(P=0.000 091 7).7例VEGF-D基因表达阳性的肺癌组织VEGF-D表达水平经非参数检验显著低于对应的正常肺组织(P=0.000).结论 VEGF-D基因在正常肺组织中处于高表达状态,而在肺腺癌组织和吉非替尼继发耐药肺腺癌表达降低,说明其在肺癌及吉非替尼耐药肺癌组织所起的作用不同于正常肺组织.     

Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-β signaling pathway

Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.     

N-cadherin expression is a potential survival mechanism of gefitinib-resistant lung cancer cells

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and fatal cancer worldwide. Specific tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), such as gefitinib, have been effective in some NSCLC patients and are being used in the clinical setting as pioneer molecularly targeted cancer drugs. However, many patients have not responded to these drugs, and have acquired resistance after long-term treatment. To identify other potential NSCLC molecular targets, we used DNA microarrays to examine gene expression profiles of gefitinib-resistant PC9/ZD cells that are derived from gefitinib-sensitive PC9 cells and harbor a threonine to methionine mutation at codon 790 (T790M) in EGFR, a known mechanism of acquired resistance to gefitinib. We found that N-cadherin expression was significantly upregulated in PC9/ZD cells compared with PC9 cells. Inhibition of N-cadherin expression by siRNA or treatment with antibodies against N-cadherin induced apoptosis of PC9/ZD cells in association with reduced phosphorylation of Akt and Bad, a proapoptotic protein. Moreover, inhibition of Akt expression by siRNA or treatment with an inhibitor for phosphatidylinositol (PI)-3 kinase reduced survival of PC9/ZD cells. In addition, we found several N-cadherin-expressing lung cancer cells that showed inherent resistance to gefitinib treatment and reduced survival owing to siRNA-induced inhibition of N-cadherin expression. Thus, it appears that N-cadherin maintains the survival of the gefitinib-resistant lung cancer cells via the PI-3 kinase/Akt survival pathway. From these results, we propose that N-cadherin signaling contributes, at least in part, to the survival mechanisms of gefitinib-resistant NSCLC cells and that N-cadherin is a potential molecular target in the treatment of NSCLC.     

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

The efficacy of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification. ( Cancer Sci 2009)     

Notch-1 contributes to epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance in non-small cell lung cancer in vitro and in vivo

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs in non-small cell lung cancer (NSCLC) patients who initially respond to TKI treatment but whose cancer then progresses. Recent studies have shown that Notch signal is associated with drug resistance. However, the exact mechanism of Notch during acquisition of resistance to EGFR-TKI in human lung cancer remains unclear. In the present study, we showed that the expression of Notch-1 was highly upregulated in EGFR-TKI acquired resistant lung cancer cells. More importantly, Notch-1 contributed to the acquisition of the epithelial–mesenchymal transition (EMT) phenotype, which was critically associated with acquired resistance to EGFR-TKI. Silencing of Notch-1 using siRNA resulted in mesenchymal–epithelial transition (MET), which was associated with impaired invasion and anchorage-independent growth of lung cancer and resensitisation to gefitinib in acquired resistant NSCLC cells. Finally, gefitinib treatment of Balb/c nu/nu with acquired resistant lung cancer xenografts in combination with Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) resulted in effective tumour growth retardation, with decreased proliferative activity and increased apoptotic activity. Collectively, these data suggest that Notch-1 might play a novel role in acquired resistance to gefitinib, which could be reversed by inhibiting Notch-1.     

miR-379-5p靶向CTSL调控肺癌细胞增殖、迁移、侵袭及凋亡的分子机制

目的:探讨微小RNA-379-5p（miR-379-5p）通过靶向组织蛋白酶L(CTSL)调控肺癌细胞增殖、迁移、侵袭及凋亡的作用机制。方法:选择本院收治的肺癌患者57例,取患者肺癌组织与癌旁组织,应用qRT-PCR法检测miR-379-5p与CTSL mRNA的表达水平;免疫组化法检测CTSL蛋白的表达情况。miR-con（miR-con组）、miR-379-5p mimics（miR-379-5p组）分别转染肺癌NC-H446细胞,miR-379-5p mimics与pcDNA（miR-379-5p+pcDNA组）、miR-379-5p mimics与pcDNA-CTSL（miR-379-5p+pcDNA-CTSL组）分别共转染肺癌NC-H446细胞,MTT检测细胞增殖情况;流式细胞术检测细胞凋亡率;Transwell迁移与侵袭实验检测细胞迁移与侵袭能力。双荧光素酶报告实验验证miR-379-5p与CTSL之间的靶向关系。Western blot检测CTSL、Cyclin D1、MMP-2、MMP-9、Cleaved caspase-3蛋白的表达。结果:肺癌组织中miR-379-5p的表达水平显著低于癌旁组织（P＜0.05）,而CTSL mRNA及蛋白阳性率均显著高于癌旁组织（P＜0.05）;与miR-con组比较,miR-379-5p组细胞增殖、迁移、侵袭能力显著降低（P＜0.05）,细胞凋亡率显著增加（P＜0.05）,明显抑制Cyclin D1、MMP-2、MMP-9蛋白表达（P＜0.05）,而促进Cleaved caspase-3蛋白表达（P＜0.05）;双荧光素酶报告实验证明miR-379-5p可负向调控CTSL表达与活性;CTSL过表达可逆转miR-379-5p过表达对肺癌细胞增殖、迁移、侵袭及凋亡的调控作用。结论:miR-379-5p过表达通过靶向CTSL而减弱肺癌细胞增殖、迁移及侵袭能力,诱导细胞凋亡。     

miR-125a-5p通过靶向APAF1 增强非小细胞肺癌细胞吉非替尼耐药性

目的：探讨miR-125a-5p 在诱导非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞吉非替尼（gefitinib，Gef）耐药中的作用及其机制。方法：选用人NSCLC 耐药细胞株A549/GR 和NSCLC细胞株A549，将miR-125a-5p mimic、miR-125a-5p inhibitor、pcDNA3.1-APAF1、空载体pcDNA3.1 转染至A549/GR细胞。用qPCR检测细胞中miR-125a-5p 的表达水平，用MTT法、Transwell 小室法和流式细胞术检测Gef 对细胞增殖、迁移和凋亡的影响。用双荧光素酶报告基因实验验证miR-125a-5p 与细胞凋亡蛋白酶活化因子1（apoptotic peptidase activating factor 1，APAF1）的靶向关系，用Western blotting检测A549/GR细胞中APAF1蛋白水平，用比色法测定细胞中caspase-3 及caspase-9 表达水平。结果：A549/GR 细胞中miR-125a-5p 表达水平显著高于A549 细胞（P<0.01）。敲降miR-125a-5p 显著增强Gef 对A549/GR细胞增殖、迁移的抑制作用（均P<0.05），并促进细胞凋亡（P<0.01）。双荧光素酶报告基因实验证实miR-125a-5p 靶向APAF1，并负调控其表达。进一步实验显示，miR-125a-5p 通过靶向下调APAF1 缓解Gef 对A549/G 细胞增殖、迁移的抑制作用及凋亡的促进作用（均P<0.05），减弱Gef引起的凋亡相关蛋白caspase-3及caspase-9表达的上调（均P<0.05）。结论：miR-125a-5p 促进NSCLC细胞Gef 耐药，其机制是通过靶向APAF1 而促进细胞的增殖、迁移并抑制凋亡。