人LncRNA MIR31HG基因稳定转染PC9细胞系的构建及其对细胞增殖和迁移的影响

doi:10.13481/j.1671-587x.20190410

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (04): 801-806.doi: 10.13481/j.1671-587x.20190410

• 基础研究 • 上一篇

人LncRNA MIR31HG基因稳定转染PC9细胞系的构建及其对细胞增殖和迁移的影响

代娟娟¹, 杨丽娟¹, 杜静¹, 苗双¹, 席思川¹, 李晨², 武艳¹

1. 滨州医学院附属医院肿瘤研究实验室, 山东滨州 256603;
2. 滨州医学院附属医院代谢与神经精神疾病研究所, 山东滨州 256603

收稿日期:2018-09-26 发布日期:2019-08-02
通讯作者: 武艳,讲师(Tel:0543-3258276,E-mail:wuyan55@126.com);杨丽娟,副教授,硕士研究生导师(Tel:0543-3258276,E-mail:yljlw@163.com) E-mail:wuyan55@126.com;yljlw@163.com
作者简介:代娟娟(1984-),女,山东省德州市人,助教,医学硕士,主要从事肺癌致病基因和抗癌药物方面的研究。
基金资助:
山东省科技厅科学基金资助课题（ZR2017PH028，ZR2014HQ080，ZR2016HM57）；滨州医学院科研计划与科研启动基金资助课题（BY2015KJ10）；山东省滨州市科技局科技发展计划资助课题（2015ZC0314）

Construction of PC9 cell lines stably transfectedwith human LncRNAMIR31HG gene and its effects on cell proliferation and migration

DAI Juanjuan¹, YANG Lijuan¹, DU Jing¹, MIAO Shuang¹, XI Sichuan¹, LI Chen², WU Yan¹

1. Tumor Research Laboratory, Affiliated Hospital, Binzhou Medical University, Binzhou 256603, China;
2. Institute for Metabolic and Neuropsychiatric Disorders, Affiliated Hospital, Binzhou Medical University, Binzhou 256603, China

Received:2018-09-26 Published:2019-08-02

摘要/Abstract

摘要： 目的：检测过表达人MIR31HG基因对PC9细胞增殖和迁移的影响，阐明促癌基因MIR31HG的作用机制。方法：通过RT-PCR法扩增长链非编码RNA（LncRNA）MIR31HG全长序列，分子克隆至pcDNA3.1（-）真核表达载体，将pcDNA3.1-MIR31HG过表达质粒和对照质粒pcDNA3.1分别转染人肺癌PC9细胞。采用酶切鉴定法检测MIR31HG过表达载体的构建情况。通过G418药物筛选建立稳定过表达MIR31HG的PC9细胞系（PC9-pcDNA3.1-MIR31HG，稳定转染组）和对照细胞系（PC9-pcDNA3.1，空载体组）。采用RT-PCR法检测稳定转染细胞系中MIR31HG基因表达水平，采用CCK-8法和划痕愈合实验检测PC9细胞增殖活性和迁移能力。结果：琼脂糖凝胶电泳，成功扩增得到了MIR31HG的特异基因片段；MIR31HG真核表达载体经双酶切后分别产生目的基因和载体2个片段。RT-PCR法检测，稳定转染组细胞中MIR31HG mRNA表达水平明显高于空载体组（P<0.05）。CCK-8检测，在培养24、36和48h时，稳定转染组细胞增殖活性明显高于空载体组（P<0.01）。划痕愈合实验，在培养48h时，稳定转染组细胞划痕愈合率明显高于空载体组（P<0.05）。结论：成功构建了人LncRNA MIR31HG基因的真核过表达载体和过表达MIR31HG的稳定转染PC9细胞系，且MIR31HG过表达能够促进肺癌PC9细胞增殖和迁移。

关键词: 长链非编码RNA, MIR31HG, PC9细胞, 细胞增殖, 细胞迁移

Abstract: Objective:To detect the effect of over-expressed human MIR31HG gene on the proliferation and migration of PC9 cells, and to clarify the mechanism of oncogene MIR31HG. Methods:The full-length sequence of long non-coding RNA (LncRNA) MIR31HG was amplified by RT-PCR and cloned into the pcDNA3.1(-) eukaryotic expression vector. The PC9 cells were transfected with the pcDNA3.1-MIR31HG overexpression vector and control vector pcDNA3.1. The construction of MIR31HG overexpression vector was detected by enzymatic digestion identification. The stable cell lines with overexpression of MIR31HG (PC9-pcDNA3.1-MIR31HG,stable transfection group) and control cell lines (PC9-pcDNA3.1,empty vector group) were established by G418 drug screening, and the expression level of MIR31HG gene in stably transfected cell line was detected by RT-PCR. CCK-8 method and scratch healing assay were used to detect the proliferation activities and migration abilities of PC9 cells. Results:The agarose gel electrophoresis results showed that the specific gene fragment of MIR31HG was obtained by amplification successfully. The gene fragments of target gene and vector were produced by double enzyme digestion of MIR31HG eukaryotic expression vector. The RT-PCR results showed that the MIR31HG RNA expression level in the cells in stable transfection group was significantly higher than that in empty vector group(P<0.05).The results of CCK-8 test showed that the proliferation activities of the cells in stable transfection group were significantly higher than those in empty vector group at 24, 36 and 48 h after culture(P<0.01).The results of scratch healing assay showed that the scratch healing rate of cells in stable transfection group was significantly higher than that in empty vector group at 48 h after culture(P<0.05). Conclusion:The eukaryotic overexpression vector and the PC9 cell line stably transfected with human LncRNA MIR31HG gene are constructed successfully, and MIR31HG overexpression can promote the proliferation and migration of lung cancer PC9 cells.

Key words: long non-coding RNA, MIR31HG, PC9 cell lines, cell proliferation, cell migration

中图分类号:

代娟娟, 杨丽娟, 杜静, 苗双, 席思川, 李晨, 武艳. 人LncRNA MIR31HG基因稳定转染PC9细胞系的构建及其对细胞增殖和迁移的影响[J]. 吉林大学学报(医学版), 2019, 45(04): 801-806.

DAI Juanjuan, YANG Lijuan, DU Jing, MIAO Shuang, XI Sichuan, LI Chen, WU Yan. Construction of PC9 cell lines stably transfectedwith human LncRNAMIR31HG gene and its effects on cell proliferation and migration[J]. Journal of Jilin University(Medicine Edition), 2019, 45(04): 801-806.

参考文献

[1] RINN J L, CHANG H Y. Genome regulation by long noncoding RNAs[J]. Annu Rev Biochem, 2012, 81:145-166.
[2] YER M K, NIKNAFS Y S, MALIK R, et al. The landscape of long noncoding RNAs in the human transcriptome[J]. Nat Genet, 2015, 47(3):199-208.
[3] WANG K C, YANG Y W, LIU B, et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression[J]. Nature, 2011, 472(7341):120-124.
[4] HARRIES L W. Long non-coding RNAs and human disease[J]. Biochem Soc Trans, 2012, 40(4):902-906.
[5] SHIH J W, CHIANG W F, WU A T, et al. Long noncoding RNA LncHIFCAR/MIR31HG is a HIF-1α co-activator driving oral cancer progression[J]. Nat Commun, 2017, 8:15874.
[6] LIU C J, TSAI M M, HUNG P S, et al. miR-31 ablates expression of the HIF regulatory factor FIH to activate the HIF pathway in head and neck carcinoma[J]. Cancer Res, 2010, 70(4):1635-1644.
[7] SUN K, ZHAO X, WAN J, et al. The diagnostic value of long non-coding RNA MIR31HG and its role in esophageal squamous cell carcinoma[J]. Life Sci, 2018, 202:124-130.
[8] SHI Y, LU J, ZHOU J, et al. Long non-coding RNA Loc554202 regulates proliferation and migration in breast cancer cells[J]. Biochem Biophys Res Commun, 2014, 446(2):448-453.
[9] GUO J, MIAO Y, XIAO B, et al. Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues[J]. J Gastroen Hepatol, 2009, 24(4):652-657.
[10] SCHAEFER A, JUNG M, MOLLENKOPF H J, et al. Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma[J]. Int J Cancer, 2010, 126(5):1166-1176.
[11] AHERI M, OMRANI M D, GHAFOURI-FARD S. Long non-coding RNA expression in bladder cancer[J]. Biophys Rev, 2018, 10(4):1205-1213.
[12] NIE F Q, MA S, XIE M, et al. Decreased long noncoding RNA MIR31HG is correlated with poor prognosis and contributes to cell proliferation in gastric cancer[J]. Tumor Biol, 2016, 37(6):7693-7701.
[13] YAN S, TANG Z, CHEN K, et al. Long noncoding RNA MIR31HG inhibits hepatocellular carcinoma proliferation and metastasis by sponging microRNA-575 to modulate ST7L expression[J]. J Exp Clin Canc Res, 2018, 37(1):214.
[14] SUN D, YU F, MA Y, et al. MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1(RASA1)[J]. J Biol Chem, 2013, 288(13):9508-9518.
[15] WANG R, MA Z, FENG L, et al. LncRNA MIR31HG targets HIF1A and P21 to facilitate head and neck cancer cell proliferation and tumorigenesis by promoting cell-cycle progression[J]. Mol Cancer, 2018, 17(1):162.
[16] LIU X, SEMPERE L F, OUYANG H, et al. MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors[J]. J Clin Invest, 2010, 120(4):1298-1309.
[17] ZHENG S, ZHANG X, WANG X, et al. MIR31HG promotes cell proliferation and invasion by activating the Wnt/β-catenin signaling pathway in non-small cell lung cancer[J]. Oncol Lett, 2019, 17(1):221-229.
[18] XI S, YANG M, TAO Y, et al. Cigarette smoke induces C/EBP-β-mediated activation of miR-31 in normal human respiratory epithelia and lung cancer cells[J]. PLoS One, 2010, 5(10):e13764.
[19] BEMSTEIN B, BIMEY E, DUNHAM I. An integrated encyclopedia of DNA elements in the human genomc[J]. Nature, 2012, 489(7414):57-74.
[20] MUERS M. RNA:Genome-wide views of long non-coding RNAs[J]. Nat Rev Genetics, 2011, 12(11):742-743.
[21] WONG Q W L, LUNG R W M, LAW P T Y, et al. MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1[J]. Gastroenterology, 2008, 135(1):257-269.
[22] LIU B, CHEN Y, YANG J. LncRNAs are altered in lung squamous cell carcinoma and lung adenocarcinoma[J]. Oncotarget, 2017, 8(15):24275.
[23] WANG B, JIANG H, WANG L, et al. Increased MIR31HG lncRNA expression increases gefitinib resistance in non-small cell lung cancer cell lines through the EGFR/PI3K/AKT signaling pathway[J]. Oncol Lett, 2017, 13(5):3494-3500.
[24] LIU X, LI D, ZHANG W, et al. Long non-coding RNA gadd7 interacts with TDP-43 and regulates Cdk6 mRNA decay[J]. EMBO J, 2012, 31(23):4415-4427.
[25] WANG K C, CHANG H Y. Molecular mechanisms of long noncoding RNAs[J]. Mol Cell, 2011, 43(6):904-914.
[26] 段艺菲,赵清喜,荆雪.长链非编码RNA对原发性肝癌中糖酵解途径的影响机制[J]. 临床肝胆病杂志,2019,35(6):1374-1376.

人LncRNA MIR31HG基因稳定转染PC9细胞系的构建及其对细胞增殖和迁移的影响

Construction of PC9 cell lines stably transfectedwith human LncRNAMIR31HG gene and its effects on cell proliferation and migration

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