Study on the mechanism of proliferation of human lung cancer A549 cells regulated by hsa-miRNA-30a-5p
-
摘要: 目的 研究hsa-miRNA-30a-5p促进肺癌细胞A549增殖的调控机制。 方法 收集临床肺癌标本及癌旁组织5对,用荧光定量法(real time-PCR)和蛋白质印迹法(Western blotting)分别检测肿瘤及其癌旁组织中hsa-miRNA-30a-5p和SCARA5的蛋白含量;生物信息学预测hsa-miRNA-30a-5p与SCARA5基因3'UTR区结合位点,并通过荧光素酶报告基因法进行结合位点验证;构建SCARA5基因沉默表达载体pshRNA-SCARA5,脂质体瞬时转染pshRNA-SCARA5及hsa-miRNA-30a-5p阻遏物(miRNA-30a-5p inhibitor)到A549细胞,转染后48 h,real time-PCR检测细胞内hsa-miRNA-30a-5p含量,Western blotting检测细胞中SCARA5蛋白含量;MTT法检测转染后A549细胞对数期增殖活性。 结果 肺癌组织中SCARA5蛋白表达量明显低于癌旁组织,组间差异显著(P<0.05);肺癌组织中hsa-miRNA-30a-5p含量则明显高于癌旁组织,组间差异显著(P<0.05)。荧光素酶实验显示,与报告基因表达载体单独转染组比较,miRNA-30a拟似物(miRNA-30a-5p mimics)和miRNA-30a-5p inhibitor与野生型荧光素酶报告基因共转染组可以明显抑制或增强荧光素酶活性(P<0.05);miRNA-30a-5p inhibitor转染细胞后48 h,与转染对照组比较,细胞内hsa-miRNA-30a-5p含量明显降低(P<0.05),阴性对照(miRNA-30a-5p NC)转染组和转染对照组细胞内hsa-miRNA-30a-5p含量无显著差异(P>0.05)。转染后24~72 h,miRNA-30a-5p inhibitor转染组A549细胞增值活性明显降低(P<0.05),而pshRNA-SCARA5转染能够逆转miRNA-30a-5p inhibitor增强A549细胞增殖活性的趋势,共转染组与miRNA-30a-5p inhibitor单独转染组及转染对照组比较,差异有统计学意义(P<0.05)。 结论 Hsa-miRNA-30a-5p通过抑制SCARA5基因表达,增强A549细胞的增殖活性,转染miRNA-30a-5p inhibitor,可以抑制A549细胞增殖活性。
-
关键词:
- SCARA5 /
- A549细胞 /
- hsa-miRNA-30a-5p /
- 增殖活性
Abstract: Objective To study the regulation and mechanism of hsa-miRNA-30a-5p on the proliferation of human lung cancer A549 cells. Methods Five pairs of lung cancer and para-carcinoma tissues were harvested in clinical and measured for hsa-miRNA-30a-5p and SCARA5 levels by real-time PCR and western blotting,respectively.The theoretical binding site of hsa-miRNA-30a-5p in SCARA5's 3'UTR was predicted by bioinformatics,and validated by luciferase report assay.A549 cells were transfected with pshRNA-SCARA5 and miRNA-30a-5p inhibitor and 48 h later,the proliferation of A549 cells after genetic intervention was assayed by the MTT method,and the cell growth curve was plotted to observe the effect of hsa-miRNA-30a-5p knockdown on cell proliferation. Results For all five pairs of samples tested,hsa-miRNA-30a-5p was higher in the cancer tissues than in the adjacent tissue(P<0.05),and SCARA5 protein was lower in the cancer tissues than in the adjacent tissue (P<0.05).The bioinformatics analysis showed that there was a theoretical binding site of hsa-miRNA-30a-5p in SCARA5's 3'UTR.The luciferase assay showed that miRNA-30a mimics inhibited the luciferase expressed by the luciferase reporter vector carrying a wild-type SCARA5's 3'UTR(pGL3-WT- SCARA5)(P<0.05,vs pGL3-WT-SCARA5 alone),and miRNA-30a-5p inhibitor enhanced the luciferase activity(P<0.05,vs pGL3-WT- SCARA5 alone).No change was observed when miRNA-30a-5p mimics or miRNA-30a-5p inhibitor was co-transfected with the luciferase reporter vector carrying a mutant SCARA5's 3'UTR(pGL3-MT- SCARA5)(P>0.05,vs pGL3-WT- SCARA5 alone).Hsa-miRNA-30a-5p level in A549 cells transfected with miRNA-30a inhibitor for 48 h was significantly decreased(P<0.05,vs cell control or NC),and there was no difference in hsa-miRNA-30a-5p between the negative control and the transfection control(P>0.05).The proliferation of A549 cells was suppressed by transfection with miRNA-30a-5p inhibitor 24-72 h after transfection(P<0.05,vs control or NC). Conclusion Hsa-miRNA-30a-5p could increase the proliferation in A549 cells via suppressing the expression of SCARA5,and transfection of miRNA-30a-5p inhibitor could inhibit the proliferation of A549 cells via up-regulating SCARA5 expression.-
Key words:
- SCARA5 /
- A549 /
- hsa-miRNA-30a-5p /
- proliferation activity
-
[1] 陈万青,张思维,邹小农.中国肺癌发病死亡的估计和流行趋势研究[J].中国肺癌杂志,2010,13(5):488-493. [2] 李媛秋,代敏,陈元立,等.中国省区水平肺癌死亡率估计方法研究[J].中国肺癌杂志,2011,14(2):120-126. [3] BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297. [4] ISSAC B,GALOIAN K,GUETTOUCHE T,et al.Genome-wide mRNA and miRNA expression data analysis to screen for markers involved in sarcomagenesis in human chondrosarcoma cell lines[J].Genom Data,2014,2:320-324. [5] MISHRA S,YADAV T,RANI V.Exploring miRNA based approaches in cancer diagnostics and therapeutics[J].Crit Rev Oncol Hematol,2016,98:12-23. [6] WINTHER M,ALSNER J,TRAMM T,et al.Evaluation of miR-21 and miR-375 as prognostic biomarkers in esophageal cancer[J].Acta Oncol,2015,54(9):1582-1591. [7] WANG P,GUO X Y,ZONG W,et al.MicroRNA-128b suppresses tumor growth and promotes apoptosis by targeting A2bR in gastric cancer[J].Biochem Biophys Res Commun,2015,467(4):798-804. [8] EGELAND N G,LUNDE S R,JONSDOTTIR K,et al.The role of microRNAs as predictors of response to tamoxifen treatment in breast cancer patients[J].Int J Mol Sci,2015,16(10):24243-24275. [9] LI L P,GUO Y,CHEN Y Z,et al.The diagnostic efficacy and biological effects of microRNA-29b for colon cancer[J].Technol Cancer Res Treat,2016,15(6):772-779. [10] KNUDSEN K N,NIELSEN B S,LINDEBJERG J,et al.MicroRNA-17 is the most up-regulated member of the mir-17-92 cluster during early colon cancer evolution[J].PLoS One,2015,10(10):e0140503. [11] I H,CHO J Y.Lung cancer biomarkers[J].Adv Clin Chem,2015,72:107-170. [12] CHEN Z Z,WU Y,MENG Q T,et al.Elevated microRNA-25 inhibits cell apoptosis in lung cancer by targeting RGS3[J].In Vitro Cell Dev Biol Anim,2016,52(1):62-67. [13] DONG W,YAO C P,TENG X P,et al.MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer[J].Tumour Biol,2016,37(3):2973-2985. [14] SONG L,LI D,ZHAO Y K,et al.MiR-218 suppressed the growth of lung carcinoma by reducing MEF2D expression[J].Tumour Biol,2016,37(3):2891-2900. [15] LIN L,LIN H B,WANG L,et al.MiR-130a regulates macrophage polarization and is associated with non-small cell lung cancer[J].Oncol Rep,2015,34(6):3088-3096. [16] SONG Y F,HONG J F,LIU D L,et al.MiR-630 targets LMO3 to regulate cell growth and metastasis in lung cancer[J].Am J Transl Res,2015,7(7):1271-1279. [17] MA R Q,WANG C Y,WANG J J,et al.MiRNA-mRNA interaction network in non-small cell lung cancer[J].Interdiscip Sci,2016,8(3):209-219. [18] HUANG J,ZHENG D L,QIN F S,et al.Genetic and epigenetic silencing of SCARA5 may contribute to human hepatocellular carcinoma by activating FAK signaling[J].J Clin Invest,2010,120(1):223-241. [19] KHAMAS A,ISHIKAWA T,SHIMOKAWA K,et al.Screening for epigenetically masked genes in colorectal cancer Using 5-Aza-2'-deoxycytidine,microarray and gene expression profile[J].Cancer Genom Proteomics,2012,9(2):67-75. [20] YAN N,ZHANG S,YANG Y,et al.Therapeutic upregulation of class A scavenger receptor member 5 inhibits tumor growth and metastasis[J].Cancer Sci,2012,103(9):1631-1639. [21] LIU J,HU G,CHEN D,et al.Suppression of SCARA5 by Snail1 is essential for EMT-associated cell migration of A549 cells[J].Oncogenesis,2013,2:e73. [22] WEI W,YANG Y,CAI J,et al.MiR-30a-5p suppresses tumor metastasis of human colorectal cancer by targeting ITGB3[J].Cell Physiol Biochem,2016,39(3):1165-1176. [23] BARANISKIN A,BIRKENKAMP-DEMTRODER K,MAGHNOUJ A,et al.MiR-30a-5p suppresses tumor growth in colon carcinoma by targeting DTL[J].Carcinogenesis,2012,33(4):732-739. [24] FRANZETTI G A,LAUD-DUVAL K,BELLANGER D,et al.MiR-30a-5p connects EWS-FLI1 and CD99,two major therapeutic targets in Ewing tumor[J].Oncogene,2013,32(33):3915-3921. 期刊类型引用(4)
1. 钱辉,彭银花,景江. 负荷剂量氯吡格雷对急性进展型脑梗死患者血液流变学及神经功能的影响. 中国基层医药. 2021(07): 983-987 . 百度学术
2. 苏海霞. 阿托伐他汀联合氯吡格雷对脑梗死患者侧支循环建立及血管内皮功能的影响. 临床医学. 2019(06): 104-106 . 百度学术
3. 郑燕红,蔡海明,张旭. 不同剂量氯吡格雷对急性脑梗死患者神经功能及预后不良事件的影响. 实用临床医药杂志. 2019(22): 24-26 . 百度学术
4. 买雷. 联合阿司匹林短期治疗急性脑梗死的临床效果. 北方药学. 2018(12): 59-60 . 百度学术
其他类型引用(1)
-

计量
- 文章访问数: 4573
- HTML全文浏览量: 478
- PDF下载量: 315
- 被引次数: 5