miRNA及其相关靶点介导的非小细胞肺癌不良转归及耐药的研究
摘要
背景：微小RNA（miRNAs）是一种19-25个核苷酸的非编码小RNA，是真核细胞重要的转录后调节机制，通过转录后水平调控靶基因的表达，参与细胞的凋亡、增殖、分化、发育和代谢等生理过程。研究提示miRNAs可通过调控其靶基因参与的信号通路，发挥类似癌基因（oncogene）和抑癌基因（tumor-suppressor）的作用参与肿瘤的发生发展。
目的：探讨原发性非小细胞肺癌（Non Small Cell Lung Cancer，NSCLC）组织中miRNA表达谱，进而分析其与NSCLC患者临床病理因素的关系，以评估其对预测患者预后的潜在临床价值。方法：采用铆钉核苷酸（Locked Nucleic Acid，LNA）高通量芯片技术筛选了8例NSCLC患者的癌和其配对的正常肺组织的miRNA差异表达谱。对芯片结果进行定量PCR验证，运用生物信息软件预测差异表达miRNA潜在的靶基因，并进行初步的临床分析。随后我们选取部分差异倍数较大的miRNA位点（miR-21、miR-143、miR-181a）在另外47例NSCLC患者的癌组织及其对应正常肺组织进行了TaqMan实时定量PCR检测，分析癌组织与正常肺组织miR-21、miR-143、miR-181a表达差异，并比较其与临床病理参数间的关联;运用Kaplan-Meier曲线和log-rank test评估miR-21、miR-143、miR-181a表达以及各项临床病理指标和患者术后生存期的关系；并进一步运用单因素Cox及多因素Cox回归模型评估miR-21、miR-143、miR-181a对患者预后的预测价值。
结果： miRNA芯片筛选出68个差异在1.5倍以上的miRNA（p<=0.05），基于差异表达的聚类分析可以较成功的区分肺癌和正常组织，其中差异在2倍以上的miRNA27个，包括9条在肺癌组织上调以及18条下调的miRNA。定量PCR验证部分芯片结果均与芯片结果一致。扩大样本对47对肺癌和其对应的正常肺组织的定量PCR分析得出肺癌组织中miR-21呈高表达水平(p=0.026)，而miR-143及miR-181a在肺癌组织里呈低表达(p=0.000，p=0.000)，再次验证了芯片的结果。我们进一步对上述三个miRNA位点进行了临床参数分析，发现miR-143与患者吸烟状态相关，miR-143在吸烟患者中的表达较非吸烟者低(p=0.026)，除此以外，上述miRNA位点的表达水平在各临床病理因素亚组间的差异无统计学意义；Kaplan–Meier生存分析显示miR-21高表达的患者相对于低表达的患者具有较短的术后生存期（p=0.002），miR-181a高表达的患者较低表达的患者术后生存期长（p=0.050）。而miR-143的表达与患者的术后生存无显著关联。临床分期和淋巴结转移情况亦和病人术后生存相关（p=0.000，p=0.000）。多因素Cox风险比例模型显示高表达的miR-21是一项独立于其他因素的的不良预后因子(Hazard Ratio HR,5.993;95%Confidence Interval CI,2.518–14.264; p=0.000)，低表达miR-181a亦是不良预后的独立预后因子(HR,0.328;95%CI,0.142–0.756;p=0.009)。除此以外，较晚的临床分期亦为独立的不良预后因素。
结论：本文研究识别了NSCLC相关的miRNA表达谱，并进一步得出NSCLC中失调的miR-21，miR-143以及miR-181a与NSCLC发生、发展及转归相关。提示这些miRNA可能可以作为NSCLC个体化诊治的新的分子标记物的候选位点，亦可能进一步成为新的治疗策略的靶点。
背景：铂类是NSCLC的一线药物，其中以顺铂最为常见，但肿瘤细胞对顺铂治疗不敏感和耐药是影响NSCLC患者化疗疗效和生存率的主要原因。近年来肿瘤耐药机制以及基于肿瘤分子分型的个体化治疗的研究已经取得一定进展，但至今临床上仍缺乏可以广泛推广应用的预测及监测疗效的分子标志物和基于耐药分子机制的有效干预策略。近年来研究表明， miRNAs是真核细胞重要的转录后调节机制，调节细胞凋亡、增殖、迁移等基本过程，进而参与肿瘤的发生发展，在肿瘤的个体化诊疗及耐药干预策略中具有重要的价值。因此我们推测miRNAs亦可能在NSCLC顺铂耐药中存在潜在作用。
目的：明确miRNA在NSCLC顺铂耐药中的作用，进而探讨miRNA及其关键靶点作为预测含铂方案疗效的生物标记物的可行性。
方法：miRNA芯片检测NSCLC耐顺铂CDDP细胞株A549/CDDP及对应的亲体细胞A549细胞中miRNA表达差异；采用定量PCR验证结果，经文献检索、结合芯片结果和我们第一部分实验结论，我们挑选miR-21进行进一步的研究。我们运用细胞转染，细胞增殖和凋亡检测，验证其在肺癌细胞耐药中的作用。进一步运用文本挖掘和生物信息学方法分析miR-21在肺癌耐药中的潜在靶点和信号通路，之后在体外功能实验中我们验证了部分可能的作用靶点，探究其在顺铂耐药机制中的潜在作用。最后我们还运用Real-time PCR检测了58例接受了顺铂为基础的术后辅助化疗的NSCLC患者癌组织中miR-21的表达，分析miR-21的表达和患者术后含铂辅助化疗的疗效相关性；在入组患者中，32例患者有对应的血浆标本，我们亦对其血浆标本进行了了miR-21表达的检测，比较血浆miR-21与癌组织miR-21表达的相关性，并分析其与患者治疗疗效之间的关联。
结果：芯片结果显示，A549/CDDP与亲本细胞系A459比较有21条差异表达的miRNA（p <0.05,差异比值>1.5倍）。体外药物敏感试验及凋亡检测实验发现，抑制miR-21表达可以增加顺铂诱导的肺癌细胞凋亡，阻滞细胞增殖，从而使肺癌细胞对顺铂的敏感性明显增加，减轻了肿瘤耐药；而过表达miR-21抑制了顺铂诱导的肺癌细胞凋亡，促进细胞增殖，从而可导致细胞对顺铂的耐药性增加，提示miR-21参与了NSCLC顺铂耐药。我们进而运用生物信息学系统的分析了与miR-21潜在靶点和肺癌耐药相关的信号通路及其网络结构，初步筛选出肺癌细胞耐药相关的miR-21可能的24个枢纽靶点，并推测miR-21是通过调控这些靶点相关的信号通路：JAK/STAT信号通路；MAPK信号通路；Wnt信号通路；细胞周期相关通路；PPAR信号通路；凋亡相关通路及其他一些通路参与了肺癌细胞耐药。之后我们运用体外细胞转染，蛋白检测等方法证实了部分靶点：在A549/CDDP中转染Anti-miR-21可以增加抑癌基因PTEN和RECK蛋白的表达；可以增加促凋亡因子FASLG的表达而降低凋亡抵抗因子BCL-2的表达；相反地，转染pre-miR-21可以降低A549细胞PTEN、RECK、FASLG蛋白的表达而增加BCL-2蛋白的表达。进一步的临床分析研究表明：在接受术后以铂类为基础的化疗的NSCLC患者中，与铂类治疗敏感组患者相比，肺癌组织miR-21在耐药组患者中显著升高(n=58, p=0.000)。并且增高的miR-21与较短的无疾病生存期DFS相关(p=0.008)，是独立的预测因子(HR,2.820;95%CI,1.091-7.285; p=0.032)。58例接受了顺铂为基础的术后辅助化疗患者中，32例患者存有配对的血浆标本，大部分（68.75%）患者血浆标本miR-21的表达与癌组织miR-21的表达趋势一致，相关系数为0.375（p=0.034）。结论：本文识别了NSCLC细胞对铂类耐药相关的miRNA表达谱，并初步证实miR-21可以通过调控一系列重要的靶点介导了NSCLC的顺铂耐药，miR-21不仅可以作为候选的生物标记物预测NSCLC患者辅助含铂化疗的疗效和治疗转归，并且很可能可以作为逆转化疗耐药新策略中的一个潜在靶标。
Context: MicroRNAs (miRNAs) represent a class of small non-coding RNAs thatregulate the gene expressions at the posttranscriptional level, subsequently controlcrucial physiological processes. Recent evidence demonstrates that some miRNAshave the functions similar to oncogene or tumor suppressors, it may play importantroles in tumorigenesis. MiRNA expression profiles may become useful biomarkersfor cancer diagnostics, prognosis and prediction of response to treatment, and it couldbe a powerful tool for cancer prevention and therapeutics.
Objective: To explore the global expression profile of miRNAs in Non Small CellLung Cancer (NSCLC) and its potential relevance to clinicopathologicalcharacteristics and prognosis.
Methods: LNA microRNA microarrays were utilized to detect miRNA expressionlevels in eight surgically removed lung carcinoma tissues (LCT) and theircorresponding normal lung tissues (NT). After initial microarray validation byquantitative real-time reverse transcription polymerase chain reaction assays(qRT-PCR), miR-21, miR-143and miR-181a were selected for further study inanother47paired LCTs and matched NTs by qRT-PCR using Taqman microRNAassay.
Result: Twenty-seven microRNAs were observed to be deregulated greater than two-fold in LCT compared with NT by microarray. Consistenting with the microarray,the expression level of miR-21by qRT-PCR was significantly higher in tumor tissuesthan in adjacent normal tissues (p=0.026); while miR-143(p=0.000) and miR-181a(p=0.000) were downregulated in LCT. Interestingly, among the47NSCLC cases,low level expression of miR-143was significantly correlated with smoking status(p=0.026), high miR-21expression (hazard ratio,5.993;95%confidence interval,2.518–14.264; p=0.000) and low miR-181a expression (hazard ratio,0.328;95%confidence interval0.142–0.756; p=0.009) were associated with poor survival,independent of clinical covariates, including TNM staging, lymph note status.
Conclusion： Our data thus indicating the potential of miR-21, miR-143andmiR-181a as a novel diagnostic or prognostic biomarker for NSCLC. Besides, thesedata will guide further studies of specific microRNAs might become potential targetsfor therapeutic intervention.
Context: Recent developments in clinical trials for adjuvant chemotherapy usingplatinum based regimens have proven to prolong survival after surgery of NSCLC.However, the ability of cancer cells to become resistant to platinum remains asignificant impediment to successful chemotherapy, which usually leads to a relapseand worsening of prognosis, even though such treatment is associated with seriousadverse effects. The mechanisms regulating lung cancer resistance to chemotherapyagents are poorly understood and biomarkers which can predict a good outcome fromadjuvant platinum based chemotherapy are required. A better understanding of theprocesses and mechanisms leading to platinum resistance of NSCLC is necessary todevelop effective therapies that can improve the prognosis of patients with this deadlydisease. Recent evidence has indicated that miRNAs are involved in tumor formationand progression by serving as either oncogenes or tumor suppressor genes, as well asby offering resistance to cytotoxic anticancer therapy. Considering the critical role ofmiRNA in cancer, it was hypothesized that miRNAs could also affect the response toplatinum by regulating the biological processes which are relevant to lung cancerchemoresistance.
Purpose: To investigate the possible role of microRNAs in the resistance toplatinum based chemotherapy in Non Small Cell Lung Cancer (NSCLC), explore their potential role and find potential biomarkers for prediction of the response toplatinum.
Methods: Microarray was employed to compare the expression of miRNAs betweenA549and A549/CDDP cells. The effect of a differently expressed miRNA (miR-21)was examined on the sensitivity of cells to platinumAfter a systematic review ofEnglish language studies of lung cancer-related molecules were pooled; genes wereclassified in three functional groups by gene ontology (GO) analysis. The keymolecules were indentified by establishing lung cancer related networks andpathways. MiR-21targets were predicted by computational method, followed byscreening for matched gene symbols in NCBI human sequences and GO、pathwayand network analysis. MiR-21targets and their network, which are involved in theresistant mechanisms of lung cancer, were obtained by the final integrative analysis.In vitro study such as trasfection and western blot were used to validate some ofpotential targets. Moreover, miR-21expression in NSCLC tumor tissues and matchedplasma sample was also analyzed by Real-time PCR.
Results:21miRNAs were deregulated in A549/CDDP. Increased miR-21expression significantly increased the resistance of A549cell to platinum, whereasreduced miR-21decreased the resistance of A549/CDDP cell. In thesystematic-analysis of lung cancer related miR-21-targets analysis,24Hub geneswere identified by overlap calculation, suggesting that miR-21may play an importantrole in the development and progression of lung cancer through JAK/STAT signalpathway, MAPK signaling pathway, Wnt signaling pathway, cell cycle, PPARsignaling pathway, apoptosis pathway and other pathways. Finally, transfection ofA549/CDDP with anti-miR-21increased the expression of PTEN, RECK, FASLGand decreased BCL-2expression. In contrast, pre-miR-21decreased the expression ofPTEN, RECK, FASLG and increased BCL-2in A549. This finding was further validated in the tissue samples of58patients and it was found that miR-21expressionwas significantly increased in platinum based chemotherapy-resistant patients (n=58,p=0.000). And increased miR-21expression was associated with the shorter DFS (p=0.008). Among these58patients,32had the corresponding plasma samples andsimilar tendencies were detected in68.75%patients.
Conclusion: Our data suggests that the expression level of miR-21in tumor tissueand plasma might be used as a biomarker to predict adjuvant platinum basedchemotherapy response and disease free survival in patients with NSCLC. Thus, itmay serve as a novel therapeutic target to modulate platinum-based chemotherapy.
目的：探讨原发性非小细胞肺癌（Non Small Cell Lung Cancer，NSCLC）组织中miRNA表达谱，进而分析其与NSCLC患者临床病理因素的关系，以评估其对预测患者预后的潜在临床价值。方法：采用铆钉核苷酸（Locked Nucleic Acid，LNA）高通量芯片技术筛选了8例NSCLC患者的癌和其配对的正常肺组织的miRNA差异表达谱。对芯片结果进行定量PCR验证，运用生物信息软件预测差异表达miRNA潜在的靶基因，并进行初步的临床分析。随后我们选取部分差异倍数较大的miRNA位点（miR-21、miR-143、miR-181a）在另外47例NSCLC患者的癌组织及其对应正常肺组织进行了TaqMan实时定量PCR检测，分析癌组织与正常肺组织miR-21、miR-143、miR-181a表达差异，并比较其与临床病理参数间的关联;运用Kaplan-Meier曲线和log-rank test评估miR-21、miR-143、miR-181a表达以及各项临床病理指标和患者术后生存期的关系；并进一步运用单因素Cox及多因素Cox回归模型评估miR-21、miR-143、miR-181a对患者预后的预测价值。
结果： miRNA芯片筛选出68个差异在1.5倍以上的miRNA（p<=0.05），基于差异表达的聚类分析可以较成功的区分肺癌和正常组织，其中差异在2倍以上的miRNA27个，包括9条在肺癌组织上调以及18条下调的miRNA。定量PCR验证部分芯片结果均与芯片结果一致。扩大样本对47对肺癌和其对应的正常肺组织的定量PCR分析得出肺癌组织中miR-21呈高表达水平(p=0.026)，而miR-143及miR-181a在肺癌组织里呈低表达(p=0.000，p=0.000)，再次验证了芯片的结果。我们进一步对上述三个miRNA位点进行了临床参数分析，发现miR-143与患者吸烟状态相关，miR-143在吸烟患者中的表达较非吸烟者低(p=0.026)，除此以外，上述miRNA位点的表达水平在各临床病理因素亚组间的差异无统计学意义；Kaplan–Meier生存分析显示miR-21高表达的患者相对于低表达的患者具有较短的术后生存期（p=0.002），miR-181a高表达的患者较低表达的患者术后生存期长（p=0.050）。而miR-143的表达与患者的术后生存无显著关联。临床分期和淋巴结转移情况亦和病人术后生存相关（p=0.000，p=0.000）。多因素Cox风险比例模型显示高表达的miR-21是一项独立于其他因素的的不良预后因子(Hazard Ratio HR,5.993;95%Confidence Interval CI,2.518–14.264; p=0.000)，低表达miR-181a亦是不良预后的独立预后因子(HR,0.328;95%CI,0.142–0.756;p=0.009)。除此以外，较晚的临床分期亦为独立的不良预后因素。
结论：本文研究识别了NSCLC相关的miRNA表达谱，并进一步得出NSCLC中失调的miR-21，miR-143以及miR-181a与NSCLC发生、发展及转归相关。提示这些miRNA可能可以作为NSCLC个体化诊治的新的分子标记物的候选位点，亦可能进一步成为新的治疗策略的靶点。
背景：铂类是NSCLC的一线药物，其中以顺铂最为常见，但肿瘤细胞对顺铂治疗不敏感和耐药是影响NSCLC患者化疗疗效和生存率的主要原因。近年来肿瘤耐药机制以及基于肿瘤分子分型的个体化治疗的研究已经取得一定进展，但至今临床上仍缺乏可以广泛推广应用的预测及监测疗效的分子标志物和基于耐药分子机制的有效干预策略。近年来研究表明， miRNAs是真核细胞重要的转录后调节机制，调节细胞凋亡、增殖、迁移等基本过程，进而参与肿瘤的发生发展，在肿瘤的个体化诊疗及耐药干预策略中具有重要的价值。因此我们推测miRNAs亦可能在NSCLC顺铂耐药中存在潜在作用。
目的：明确miRNA在NSCLC顺铂耐药中的作用，进而探讨miRNA及其关键靶点作为预测含铂方案疗效的生物标记物的可行性。
方法：miRNA芯片检测NSCLC耐顺铂CDDP细胞株A549/CDDP及对应的亲体细胞A549细胞中miRNA表达差异；采用定量PCR验证结果，经文献检索、结合芯片结果和我们第一部分实验结论，我们挑选miR-21进行进一步的研究。我们运用细胞转染，细胞增殖和凋亡检测，验证其在肺癌细胞耐药中的作用。进一步运用文本挖掘和生物信息学方法分析miR-21在肺癌耐药中的潜在靶点和信号通路，之后在体外功能实验中我们验证了部分可能的作用靶点，探究其在顺铂耐药机制中的潜在作用。最后我们还运用Real-time PCR检测了58例接受了顺铂为基础的术后辅助化疗的NSCLC患者癌组织中miR-21的表达，分析miR-21的表达和患者术后含铂辅助化疗的疗效相关性；在入组患者中，32例患者有对应的血浆标本，我们亦对其血浆标本进行了了miR-21表达的检测，比较血浆miR-21与癌组织miR-21表达的相关性，并分析其与患者治疗疗效之间的关联。
结果：芯片结果显示，A549/CDDP与亲本细胞系A459比较有21条差异表达的miRNA（p <0.05,差异比值>1.5倍）。体外药物敏感试验及凋亡检测实验发现，抑制miR-21表达可以增加顺铂诱导的肺癌细胞凋亡，阻滞细胞增殖，从而使肺癌细胞对顺铂的敏感性明显增加，减轻了肿瘤耐药；而过表达miR-21抑制了顺铂诱导的肺癌细胞凋亡，促进细胞增殖，从而可导致细胞对顺铂的耐药性增加，提示miR-21参与了NSCLC顺铂耐药。我们进而运用生物信息学系统的分析了与miR-21潜在靶点和肺癌耐药相关的信号通路及其网络结构，初步筛选出肺癌细胞耐药相关的miR-21可能的24个枢纽靶点，并推测miR-21是通过调控这些靶点相关的信号通路：JAK/STAT信号通路；MAPK信号通路；Wnt信号通路；细胞周期相关通路；PPAR信号通路；凋亡相关通路及其他一些通路参与了肺癌细胞耐药。之后我们运用体外细胞转染，蛋白检测等方法证实了部分靶点：在A549/CDDP中转染Anti-miR-21可以增加抑癌基因PTEN和RECK蛋白的表达；可以增加促凋亡因子FASLG的表达而降低凋亡抵抗因子BCL-2的表达；相反地，转染pre-miR-21可以降低A549细胞PTEN、RECK、FASLG蛋白的表达而增加BCL-2蛋白的表达。进一步的临床分析研究表明：在接受术后以铂类为基础的化疗的NSCLC患者中，与铂类治疗敏感组患者相比，肺癌组织miR-21在耐药组患者中显著升高(n=58, p=0.000)。并且增高的miR-21与较短的无疾病生存期DFS相关(p=0.008)，是独立的预测因子(HR,2.820;95%CI,1.091-7.285; p=0.032)。58例接受了顺铂为基础的术后辅助化疗患者中，32例患者存有配对的血浆标本，大部分（68.75%）患者血浆标本miR-21的表达与癌组织miR-21的表达趋势一致，相关系数为0.375（p=0.034）。结论：本文识别了NSCLC细胞对铂类耐药相关的miRNA表达谱，并初步证实miR-21可以通过调控一系列重要的靶点介导了NSCLC的顺铂耐药，miR-21不仅可以作为候选的生物标记物预测NSCLC患者辅助含铂化疗的疗效和治疗转归，并且很可能可以作为逆转化疗耐药新策略中的一个潜在靶标。
Context: MicroRNAs (miRNAs) represent a class of small non-coding RNAs thatregulate the gene expressions at the posttranscriptional level, subsequently controlcrucial physiological processes. Recent evidence demonstrates that some miRNAshave the functions similar to oncogene or tumor suppressors, it may play importantroles in tumorigenesis. MiRNA expression profiles may become useful biomarkersfor cancer diagnostics, prognosis and prediction of response to treatment, and it couldbe a powerful tool for cancer prevention and therapeutics.
Objective: To explore the global expression profile of miRNAs in Non Small CellLung Cancer (NSCLC) and its potential relevance to clinicopathologicalcharacteristics and prognosis.
Methods: LNA microRNA microarrays were utilized to detect miRNA expressionlevels in eight surgically removed lung carcinoma tissues (LCT) and theircorresponding normal lung tissues (NT). After initial microarray validation byquantitative real-time reverse transcription polymerase chain reaction assays(qRT-PCR), miR-21, miR-143and miR-181a were selected for further study inanother47paired LCTs and matched NTs by qRT-PCR using Taqman microRNAassay.
Result: Twenty-seven microRNAs were observed to be deregulated greater than two-fold in LCT compared with NT by microarray. Consistenting with the microarray,the expression level of miR-21by qRT-PCR was significantly higher in tumor tissuesthan in adjacent normal tissues (p=0.026); while miR-143(p=0.000) and miR-181a(p=0.000) were downregulated in LCT. Interestingly, among the47NSCLC cases,low level expression of miR-143was significantly correlated with smoking status(p=0.026), high miR-21expression (hazard ratio,5.993;95%confidence interval,2.518–14.264; p=0.000) and low miR-181a expression (hazard ratio,0.328;95%confidence interval0.142–0.756; p=0.009) were associated with poor survival,independent of clinical covariates, including TNM staging, lymph note status.
Conclusion： Our data thus indicating the potential of miR-21, miR-143andmiR-181a as a novel diagnostic or prognostic biomarker for NSCLC. Besides, thesedata will guide further studies of specific microRNAs might become potential targetsfor therapeutic intervention.
Context: Recent developments in clinical trials for adjuvant chemotherapy usingplatinum based regimens have proven to prolong survival after surgery of NSCLC.However, the ability of cancer cells to become resistant to platinum remains asignificant impediment to successful chemotherapy, which usually leads to a relapseand worsening of prognosis, even though such treatment is associated with seriousadverse effects. The mechanisms regulating lung cancer resistance to chemotherapyagents are poorly understood and biomarkers which can predict a good outcome fromadjuvant platinum based chemotherapy are required. A better understanding of theprocesses and mechanisms leading to platinum resistance of NSCLC is necessary todevelop effective therapies that can improve the prognosis of patients with this deadlydisease. Recent evidence has indicated that miRNAs are involved in tumor formationand progression by serving as either oncogenes or tumor suppressor genes, as well asby offering resistance to cytotoxic anticancer therapy. Considering the critical role ofmiRNA in cancer, it was hypothesized that miRNAs could also affect the response toplatinum by regulating the biological processes which are relevant to lung cancerchemoresistance.
Purpose: To investigate the possible role of microRNAs in the resistance toplatinum based chemotherapy in Non Small Cell Lung Cancer (NSCLC), explore their potential role and find potential biomarkers for prediction of the response toplatinum.
Methods: Microarray was employed to compare the expression of miRNAs betweenA549and A549/CDDP cells. The effect of a differently expressed miRNA (miR-21)was examined on the sensitivity of cells to platinumAfter a systematic review ofEnglish language studies of lung cancer-related molecules were pooled; genes wereclassified in three functional groups by gene ontology (GO) analysis. The keymolecules were indentified by establishing lung cancer related networks andpathways. MiR-21targets were predicted by computational method, followed byscreening for matched gene symbols in NCBI human sequences and GO、pathwayand network analysis. MiR-21targets and their network, which are involved in theresistant mechanisms of lung cancer, were obtained by the final integrative analysis.In vitro study such as trasfection and western blot were used to validate some ofpotential targets. Moreover, miR-21expression in NSCLC tumor tissues and matchedplasma sample was also analyzed by Real-time PCR.
Results:21miRNAs were deregulated in A549/CDDP. Increased miR-21expression significantly increased the resistance of A549cell to platinum, whereasreduced miR-21decreased the resistance of A549/CDDP cell. In thesystematic-analysis of lung cancer related miR-21-targets analysis,24Hub geneswere identified by overlap calculation, suggesting that miR-21may play an importantrole in the development and progression of lung cancer through JAK/STAT signalpathway, MAPK signaling pathway, Wnt signaling pathway, cell cycle, PPARsignaling pathway, apoptosis pathway and other pathways. Finally, transfection ofA549/CDDP with anti-miR-21increased the expression of PTEN, RECK, FASLGand decreased BCL-2expression. In contrast, pre-miR-21decreased the expression ofPTEN, RECK, FASLG and increased BCL-2in A549. This finding was further validated in the tissue samples of58patients and it was found that miR-21expressionwas significantly increased in platinum based chemotherapy-resistant patients (n=58,p=0.000). And increased miR-21expression was associated with the shorter DFS (p=0.008). Among these58patients,32had the corresponding plasma samples andsimilar tendencies were detected in68.75%patients.
Conclusion: Our data suggests that the expression level of miR-21in tumor tissueand plasma might be used as a biomarker to predict adjuvant platinum basedchemotherapy response and disease free survival in patients with NSCLC. Thus, itmay serve as a novel therapeutic target to modulate platinum-based chemotherapy.
引文
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