miRNA-126对非小细胞肺癌细胞功能的影响及相关机制研究
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摘要
目的观察miRNA-126对非小细胞肺癌A549细胞增殖、凋亡侵袭以及迁移能力的影响及相关机制。方法 2015年3-5月在辽宁省葫芦岛市中心医院实验室进行实验。将非小细胞肺癌A549细胞转染,建立稳定转染的miRNA-126组和空白对照组。采用RT-PCR检测miRNA-126基因的表达,MTT比色法检测细胞增殖能力,倒置显微镜观察细胞侵袭能力、细胞迁移能力,Western Blot检测表皮生长因了-受体(EGFR)、AKT、哺乳动物雷帕霉素靶蛋白(mTOR)表达。结果 miRNA-126转染组24 h、48 h、72 h时miRNA-126水平均高于空白对照组(t/P=15.112/0.000、8.714/0.012、7.537/0.013),而空白对照组各时间段miRNA-126的相对表达量比较差异无统计学意义(P> 0. 05);miRNA-126转染组72 h miRNA-126的相对表达量显著高于48 h和24 h(F/P=4.575/0.043)。miRNA-126转染组细胞24 h、48 h、72 h增殖率均低于空白对照组(t/P=6.280/0.000,6.804/0.000,6.401/0.000),凋亡率均高于空白对照组(t/P=19.726/0.000,22. 052/0. 000,5.170/0. 000);空白对照组各时间段的细胞增殖率和凋亡率差异无统计学意义(P> 0.05)。miRNA-126转染组72 h的细胞增殖率显著低于48 h和24 h (F/P=5.738/0.001),miRNA-126转染组72 h的细胞凋亡率显著高于48 h和24 h(F/P=2. 681/0. 045); miRNA-126转染组划痕宽度宽于空白对照组(t/P=8. 318/0.000),侵袭细胞数显著低于空白对照组(t/P=24. 394/0. 000)。miRNA-126转染组EGFR、AKT、mTOR蛋白表达量均低于空白对照组(t/P=4.093/0.000、6.325/0.000、3.061/0.000)。结论转染的miRNA-126通过调控EGFR、AKT、mTOR表达,抑制非小细胞肺癌细胞增殖、凋亡、侵袭以及迁移能力。
Objective To investigate the effect of miRNA-126 on the proliferation,apoptosis,migration and related mechanism of non-small cell lung cancer(NSCLC) A549 cells. Methods From March 2015 to May 2015, experiments were conducted in the laboratory of our hospital, and non-small cell lung cancer(NSCLC) A549 cells were transfected to establish a stable transfected miRNA-126 group and a blank control group. The expression of miRNA-126 gene was detected by RTPCR, the proliferation capacity of cells was detected by MTT colorimetry, the invasion capacity of cells was observed by inverted microscope, and the expression of Epidermal growth factor receptor( EGFR) AKT, mammalian target of rapamycin(mTOR) was detected by Western Blotting. Results The miRNA-126 level in the miRNA-126 transfection group was significantly higher than that in the blank control group(t/P = 15. 112/0. 000). The miRNA-126 level at 48 h was significantly higher than that of the blank control group(t/P = 8.714/0.000). The miRNA-126 level at 72 h was significantly higher than that of the blank control group(t/P =7. 537/0. 000). The relative expression of miRNA-126 in the blank control group showed no significant difference(P >0.05). The relative expression of miRNA-126 at 72 h was significantly higher than that at 48 h and 24 h(t/P = 4. 575/0. 000), and the difference was statistically significant. The cell proliferation rate at 24 h,48 h,72 h in the miRNA-126 transfection group was lower than that in the blank control group(t/P = 6.280/0.000,6. 804/0.000,6.401/0.000). and the apoptosis rate was higher than that in the blank control group(t/P = 19.726/0.000,22.052/0. 000,5. 170/0. 000). There was no significant difference in cell proliferation rate and apoptosis rate at each time period in the blank control group( P > 0. 05). The cell proliferation rate at 72 h in the miRNA-126 transfection group was significantly lower than that at 48 h and 24 h( t/P = 2. 355/< 0. 05), and the apoptosis rate at 72 h in the miRNA-126 transfection group was significantly higher than that at 48 h and 24 h(t/P = 2. 681/0. 045). The difference was statistically significant. The scratch width of the miRNA-126 transfection group was wider than that of the blank control group(t/P = 8. 318/0. 000), and the number of invasive cells was significantly lower than that of the blank control group(t/P = 24. 394/0. 000). The difference was statistically significant. EGFR protein expression in the miRNA-126 transfection group was significantly lower than that of the blank control group(t/P = 4. 093/0. 000). AKT was significantly lower than that of the blank control group(t/P = 6. 325,0. 000), and mTOR protein expression was significantly lower than that of the blank control group(t/P =3.061/0.000), with statistically significant differences(P< 0. 01). Conclusion The transfected miRNA-126 inhibits the proliferation, apoptosis, invasion and migration of non-small cell lung cancer cells by regulating the expression of EGFR, AKT and mTOR.
Objective To investigate the effect of miRNA-126 on the proliferation,apoptosis,migration and related mechanism of non-small cell lung cancer(NSCLC) A549 cells. Methods From March 2015 to May 2015, experiments were conducted in the laboratory of our hospital, and non-small cell lung cancer(NSCLC) A549 cells were transfected to establish a stable transfected miRNA-126 group and a blank control group. The expression of miRNA-126 gene was detected by RTPCR, the proliferation capacity of cells was detected by MTT colorimetry, the invasion capacity of cells was observed by inverted microscope, and the expression of Epidermal growth factor receptor( EGFR) AKT, mammalian target of rapamycin(mTOR) was detected by Western Blotting. Results The miRNA-126 level in the miRNA-126 transfection group was significantly higher than that in the blank control group(t/P = 15. 112/0. 000). The miRNA-126 level at 48 h was significantly higher than that of the blank control group(t/P = 8.714/0.000). The miRNA-126 level at 72 h was significantly higher than that of the blank control group(t/P =7. 537/0. 000). The relative expression of miRNA-126 in the blank control group showed no significant difference(P >0.05). The relative expression of miRNA-126 at 72 h was significantly higher than that at 48 h and 24 h(t/P = 4. 575/0. 000), and the difference was statistically significant. The cell proliferation rate at 24 h,48 h,72 h in the miRNA-126 transfection group was lower than that in the blank control group(t/P = 6.280/0.000,6. 804/0.000,6.401/0.000). and the apoptosis rate was higher than that in the blank control group(t/P = 19.726/0.000,22.052/0. 000,5. 170/0. 000). There was no significant difference in cell proliferation rate and apoptosis rate at each time period in the blank control group( P > 0. 05). The cell proliferation rate at 72 h in the miRNA-126 transfection group was significantly lower than that at 48 h and 24 h( t/P = 2. 355/< 0. 05), and the apoptosis rate at 72 h in the miRNA-126 transfection group was significantly higher than that at 48 h and 24 h(t/P = 2. 681/0. 045). The difference was statistically significant. The scratch width of the miRNA-126 transfection group was wider than that of the blank control group(t/P = 8. 318/0. 000), and the number of invasive cells was significantly lower than that of the blank control group(t/P = 24. 394/0. 000). The difference was statistically significant. EGFR protein expression in the miRNA-126 transfection group was significantly lower than that of the blank control group(t/P = 4. 093/0. 000). AKT was significantly lower than that of the blank control group(t/P = 6. 325,0. 000), and mTOR protein expression was significantly lower than that of the blank control group(t/P =3.061/0.000), with statistically significant differences(P< 0. 01). Conclusion The transfected miRNA-126 inhibits the proliferation, apoptosis, invasion and migration of non-small cell lung cancer cells by regulating the expression of EGFR, AKT and mTOR.
引文
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[2]伍梅校,刘东华.miRNA-126与非小细胞肺癌关系的研究进展[J].沈阳医学院学报,2016,18(6):472-475. DOI:10. 16753/j.cnki. 1008-2344.2016.06.017.
[3]黄辰,刘利英,倪磊,等.肿瘤miRNAs调控机制的研究进展与展望[J].西安交通大学学报:医学版,2015,36(4):429-434.DOI:10.7652/jdyxb201504001.
[4]魏西翠,王惠霞,贾汝臻,等.DC-CIK细胞免疫疗法治疗非小细胞肺癌患者的效果及对相关指标的影响[J].疑难病杂志,2018,17(5):476-479,485. DOI:10. 3969/j. issn. 1671-6450. 2018. 05.011.
[5]周艳珍,李红霞,李爱华,等.多种肿瘤标志物联合诊断肺癌的价值分析[J].疑难病杂志,2018,17(1):18-21. DOI:10. 3969/j.issn. 1671-6450. 2018. 01.005.
[6]管洁,韩文慧,王辉.SBRT联合化疗治疗老年非小细胞肺癌的临床疗效[J].癌症进展,2017,15(12):1453-1456.
[7]杨慧,吴玉芬,黄勇,等.非小细胞肺癌脑转移的危险因素分析[J].山东医药,2015,55(7):41-43. DOI:10. 3969/j. issn. 1002-266X.2015.07.015.
[8]凌云,邱田,李卓,等.非小细胞肺癌中EGFR和KRAS基因突变的特点及与临床病理特征的关系[J].临床与实验病理学杂志,2015,31(5):536-541. DOI:10. 13315/j. cnki. cjcep. 2015. 05.013.
[9]官廷华,姜建青,俞永康,毛平.胸腔镜手术治疗非小细胞肺癌患者的近期疗效及对血清Pentraxin-3、HE4水平的影响[J].疑难病杂志,2017,16(3):246-250. DOI:10. 3969/j. issn. 1671-6450.2017.03.008.
[10]姜博伦,赵晨光,郭惠琴.PD-1/PD-L1抑制剂及其疗效预测标志物在非小细胞肺癌中的研究进展更新[J].癌症进展,2017,15(12):1369-1374.
[11]燕翔,焦顺昌.肿瘤浸润淋巴细胞与非小细胞肺癌预后关系的Meta分析[J].中国医学科学院学报,2015,37(4):406-414.DOI:10. 3881/j. issn. 1000-503X. 2015. 04.007.
[12] Kuraoka N,Hara K,Terai S,et al. Peroral cholangioscopy of nivolumab-related(induced)ulcerative cholangitis in a patient with nonsmall cell lung cancer[J]. Endoscopy,2018,50(9):259-261. DOI:10.1055/a-0640-2392. PubMed PMID:29969801.
[13]张潍,张伟,王辉,等.miRNA-141、miRNA-145在非小细胞肺癌中的表达及与临床病理的关系[J].西安交通大学学报:医学版,2015,36(3):368-372. DOI:10.7652/jdyxb201503016.
[14] Feng B,Zhang K,Wang R,et al. Non-small-cell lung cancer and miRNAs:novel biomarkers and promising tools for treatment[J].Clin Sci,2015,128(10):619-634.DOI:10.1042/CS20140530.
[15]刘朝富,林益坤,符剑鸣,等.非小细胞肺癌中微RNA的功能及临床价值的研究进展[J].医学综述,2018,24(4):693-697. DOI:10.3969/j. issn. 1006-2084.2018.04.013.
[16]张玉洁,王微微,商安全,等.非小细胞肺癌患者血清miR-22和miR-126的检测及其临床意义[J].中国肿瘤生物治疗杂志,2017,24(12):1403-1408. DOI:10. 3872/j. issn. 1007-385x. 2017.12.011.
[17]李玺,荣福.非小细胞肺癌经支气管针吸活检标本microRNA-126和microRNA-205的表达及意义[J].广东医学,2016,37(22):3393-3397. DOI:10. 3969/j. issn. 1001-9448.2016.22.025.
[18] Wozniak MB, Scelo G, Muller DC, et. al. Circulating MicroRNAs as Non-Invasive Biomarkers for Early Delection of Non-Small-Cell Lung Cancer[J]. PLoS One, 2015,10(5):e0125026. DOI:10. 1371/journal. pone. 0125026. eCollection 2015.
[19]张玉洁,王微微,商安全,等.非小细胞肺癌患者血清miR-22和miR-126的检测及其临床意义[J].中国肿瘤生物治疗杂志,2017,24(12):1403-1408. DOI:10. 3872/j. issn. 1007-385x. 2017.12.011.
[20]张美玲,李春,叶茂松,等.改良Blocker PCR检测EGFR-TKI耐药后非小细胞肺癌血浆EGFR T790M突变的价值[J].复旦大学学报:医学版,2018,45(1):45-51. DOI:10. 3969/j. issn. 1672-8467.2018.01.007.
[21]侯爱画,刘伟,张金波,等.清热散结方对非小细胞肺癌化疗患者免疫功能的影响研究[J].癌症进展,2017,15(12):1477-1479.
[22]高世乐,胡宗涛.血管内皮生长因子和黏附分子蛋白CD44变异体6与非小细胞肺癌同步放化疗近期疗效的相关性[J].中国医药,2017,12(10):1507-1510. DOI:10. 3760/cma. j. issn. 1673-4777.2017.10.017.
[23]闫斌,王靖博,张宏,等.槲皮素对高糖培养海马神经元凋亡及Akt、p-Akt、Bcl-2、Bax蛋白表达的影响[J].中国康复理论与实践,2017,23(12):1390-1396. DOI:10. 3969/j. issn. 1006-9771.2017.12.005.
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