长链非编码RNA LINC00324对非小细胞肺癌细胞生物学功能的影响
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摘要
目的观测长链非编码RNA LINC00324在非小细胞肺癌中的表达情况,探讨其对非小细胞肺癌细胞系生物学功能的影响。方法通过qRT-PCR检测LINC00324在65对配对肺癌组织及癌旁正常组织中的表达,检测不同非小细胞肺癌细胞系(4株非小细胞肺癌细胞系:A549、SPCA1、H460和H1299; 1株支气管上皮细胞系:HBE)中LINC00324基因的表达;构建LINC00324基因的过表达载体,构建LINC00324过表达细胞系;采用CCK-8、Transwell、流式细胞术检测过表达LINC00324基因后对非小细胞肺癌细胞增殖、侵袭、迁移及凋亡的影响。结果 LINC00324在肺癌组织中较癌旁正常组织低表达(P <0. 05);以HBE作为对照,LINC00324在A549和H460细胞系中相对低表达(P <0. 05),在H1299和SPCA1细胞系中相对高表达(P <0. 05);成功构建LINC00324过表达A549和H460细胞系,LINC00324基因在A549和H460过表达细胞系中表达均升高(P <0. 05); CCK-8实验显示,过表达LINC00324基因能够抑制H460和A549肺癌细胞的增殖(P <0. 05); Transwell实验显示,过表达LINC00324基因较空质粒对照组能够显著降低H460和A549肺癌细胞的侵袭和迁移(P <0. 05);流式细胞术检测结果显示,过表达LINC00324基因较空质粒对照组能够显著增加H460和A549肺癌细胞的凋亡(P <0. 05)。结论长链非编码RNA LINC00324可能通过调节肺癌细胞的增殖、迁移、侵袭和凋亡,在肺癌的发生、发展中起着重要的抑癌基因作用。
Objective To investigate the expression of long non-coding RNA LINC00324 in non-small cell lung cancer( NSCLC) and explore its effect on the biological functions of the tumor cells. Methods q RTPCR was used to detect the expression of LINC00324 in 65 paired lung cancer tissues and adjacent normal tissues. The expression of LINC00324 was also detected in 4 different non-small cell lung cancer cell lines( A549,SPCA1,H460 and H1299) and human bronchial epithelial cell line HBE by qRT-PCR. The overexpression vector of LINC00324 gene was designed and constructed,and the LINC00324 overexpression cell lines were constructed through transfection. The effects of overexpression of LINC00324 on the proliferation,invasion, migration and apoptosis of NSCLC cells were measured by cell counting kit-8( CCK-8) assay,Transwell assay,and flow cytometry respectively. Results LINC00324 was down-regulated in the lung cancer tissues than adjacent normal tissues( P < 0. 05). The expression of LINC00324 was observed in 4 NSCLC lines and HBE cells. The expression of LINC00324 was lowest in A549 and H460 cells( P < 0. 05),and highest in H1299 and SPCA1 cells( P < 0. 05). LINC00324 overexpression was successfully constructed in A549 and H460 cells. The expression of LINC00324 gene was increased in both A549 and H460 overexpressed cell lines( P < 0. 05). The overexpression of LINC00324 gene inhibited the proliferation( P < 0. 05),reduced the invasion and migration( P < 0. 05),and promoted apoptosis( P < 0. 05)in the H460 and A549 cells,when compared with the cells transfected with blank plasmids. Conclusion Long non-coding RNA LINC00324 may play an important anti-oncogene role in the development and progression of lung cancer by regulating the proliferation,migration,invasion,and apoptosis of lung cancer cells.
Objective To investigate the expression of long non-coding RNA LINC00324 in non-small cell lung cancer( NSCLC) and explore its effect on the biological functions of the tumor cells. Methods q RTPCR was used to detect the expression of LINC00324 in 65 paired lung cancer tissues and adjacent normal tissues. The expression of LINC00324 was also detected in 4 different non-small cell lung cancer cell lines( A549,SPCA1,H460 and H1299) and human bronchial epithelial cell line HBE by qRT-PCR. The overexpression vector of LINC00324 gene was designed and constructed,and the LINC00324 overexpression cell lines were constructed through transfection. The effects of overexpression of LINC00324 on the proliferation,invasion, migration and apoptosis of NSCLC cells were measured by cell counting kit-8( CCK-8) assay,Transwell assay,and flow cytometry respectively. Results LINC00324 was down-regulated in the lung cancer tissues than adjacent normal tissues( P < 0. 05). The expression of LINC00324 was observed in 4 NSCLC lines and HBE cells. The expression of LINC00324 was lowest in A549 and H460 cells( P < 0. 05),and highest in H1299 and SPCA1 cells( P < 0. 05). LINC00324 overexpression was successfully constructed in A549 and H460 cells. The expression of LINC00324 gene was increased in both A549 and H460 overexpressed cell lines( P < 0. 05). The overexpression of LINC00324 gene inhibited the proliferation( P < 0. 05),reduced the invasion and migration( P < 0. 05),and promoted apoptosis( P < 0. 05)in the H460 and A549 cells,when compared with the cells transfected with blank plasmids. Conclusion Long non-coding RNA LINC00324 may play an important anti-oncogene role in the development and progression of lung cancer by regulating the proliferation,migration,invasion,and apoptosis of lung cancer cells.
引文
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[3] ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome[J]. Nature,2012,489(7414):57-74.
[4] XIE S S,JIN J,XU X,et al. Emerging roles of non-coding RNAs in gastric cancer:Pathogenesis and clinical implications[J]. World J Gastroenterol,2016,22(3):1213-1223. DOI:10. 3748/wjg. v22. i3. 1213.
[5] LUO S,LU J Y,LIU L,et al. Divergent lnc RNAs regulate gene expression and lineage differentiation in pluripotent cells[J]. Cell Stem Cell,2016,18(5):637-652. DOI:10.1016/j. stem. 2016. 01. 024.
[6] TAO H,YANG J J,ZHOU X,et al. Emerging role of long noncoding RNAs in lung cancer:Current status and future prospects[J]. Respir Med,2016,110:12-19. DOI:10.1016/j. rmed. 2015. 10. 006.
[7]鲍卫. Linc00324在自身免疫疾病中的表达及临床意义[D].南宁:广西医科大学,2018.BAO W. Expression and clinical significance of linc00324 in autoimmune diseases[D]. Nanning:Guangxi Medical University,2018.
[8] ZOU Z,MA T,HE X,et al. Long intergenic non-coding RNA 00324 promotes gastric cancer cell proliferation via binding with Hu R and stabilizing FAM83B expression[J]. Cell Death Dis,2018,9(7):717. DOI:10. 1038/s41419-018-0758-8.
[9]刘芳尘.荧光定量PCR检测Linc00324在白血病患者白细胞中表达及其临床意义[D].南宁:广西医科大学,2017.LIU F C. Detection of Linc00324 expression in leukocytes of patients with leukemia by fluorescence quantitation[D]. Nanning:Guangxi Medical University,2017.
[10]王贵露,袁帅,胡泽曜,等.长链非编码RNA MUC5BAS1在人肺腺癌细胞系中的功能研究[J].第三军医大学学报,2017,39(21):2072-2077. DOI:10. 16016/j.1000-5404. 201705020.WANG G L,YUAN S,HU Z Y,et al. Functional study of Lnc RNA MUC5B-AS1 in lung adenocarcinoma cell lines[J]. J Third Mil Med Univ,2017,39(21):2072-2078. DOI:10.16016/j. 1000-5404. 201705020.
[11] OKAZAKI Y,FURUNO M,KASUKAWA T,et al. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length c DNAs[J]. Nature,2002,420(6915):563-573. DOI:10. 1038/nature01266.
[12] JI P,DIEDERICHS S,WANG W,et al. MALAT-1,a novel noncoding RNA,and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer[J].Oncogene,2003,22(39):8031-8041. DOI:10. 1038/sj.onc. 1206928.
[13] LIU G,XIANG T,WU Q F,et al. Long noncoding RNA H19-Derived mi R-675 enhances proliferation and invasion via RUNX1 in gastric cancer cells[J]. Oncol Res,2016,23(3):99-107. DOI:10. 3727/096504015X14496932933575.
[14] YAO Y,LI J,WANG L. Large intervening Non-Coding RNA HOTAIR Is an indicator of poor prognosis and a therapeutic target in human cancers[J]. Int J Mol Sci,2014,15(10):18985-18999. DOI:10. 3390/ijms151018985.
[15] VILLEGAS V E,ZAPHIROPOULOS P G. Neighboring gene regulation by antisense long non-coding RNAs[J]. Int J Mol Sci,2015,16(2):3251-3266. DOI:10. 3390/ijms16023251.
[16] JOHNSSON P,ACKLEY A,VIDARSDOTTIR L,et al. A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells[J]. Nat Struc Mol Biol,2013,20(4):440.
[17] FAGHIHI M A,MODARRESI F,KHALIL A M,et al.Expression of a noncoding RNA is elevated in alzheimer’s disease and drives rapid feed-forward regulation of betasecretase[J]. Nat Med,2008,14(7):723-730. DOI:10. 1038/nm1784.
[18] LIU X H,SUN M,NIE F Q,et al. Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging mi R-331-3p in gastric cancer[J]. Mol Cancer,2014,13:92. DOI:10. 1186/1476-4598-13-92.
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[20] YUAN T,HUANG X,WOODCOCK M,et al. Plasma extracellular RNA profiles in healthy and cancer patients[J]. Sci Rep,2016,6:19413. DOI:10. 1038/srep19413.
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