骨形态发生蛋白9调节乳腺癌细胞中长链非编码RNA的表达
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摘要
目的:筛选出与骨形态发生蛋白9(bone morphogenetic protein-9,BMP-9)表达相关的长链非编码RNA(long non-coding RNA,LncRNA),为探讨BMP-9通过调节LncRNA表达水平参与乳腺癌细胞生长、分化、迁移和凋亡等的作用机制提供实验依据。方法 :将携带有BMP-9全基因的重组腺病毒Ad-BMP-9和携带有绿色荧光蛋白(greenuorescent protein,GFP)基因的空载体腺病毒Ad-GFP(对照组)分别感染MDA-MB-231细胞。应用微阵列芯片技术分别检测BMP-9过表达组(MDA-MB-231/BMP-9细胞)和对照组(MDA-MB-231/GFP细胞)中Lnc RNA表达谱的差异,并通过实时荧光定量PCR法验证筛选获得的14条LncRNA(LINC00443、LINC00638、LINC00486、RHNO1、SERHL、HOXA11-AS、IQCA1、LINC00461、LOC440173、LHFPL、ANKRD36BP2、BVES-AS1、LINC00937和LINC00608)在MDA-MB-231/BMP-9和MDA-MB-231/GFP细胞中的表达情况。通过基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析发生表达水平变化的Lnc RNA涉及的功能及通路。结果 :重组BMP-9腺病毒感染的MDA-MB-231细胞中BMP-9 mRNA的表达水平较对照组明显升高。与对照组相比,BMP-9过表达的MDA-MB-231/BMP-9细胞中LncRNA表达水平发生明显变化。实时荧光定量PCR检测结果显示,LINC00443、LINC00638和LINC00486的表达水平上调(P值均<0.05),IQCA1、LINC00461、LOC440173、LHFPL和ANKRD36BP2的表达水平下调(P值均<0.05)。发生表达改变的LncRNA参与了细胞骨架和细胞膜等的形成,或作为细胞间信号转导的信号分子发挥作用。结论 :BMP-9过表达的MDA-MB-231细胞中LncRNA表达谱较对照组出现显著变化,提示部分LncRNA可能参与了BMP-9调控乳腺癌MDAMB-231细胞增殖和侵袭等生物学过程,并发挥关键作用。
Objective: To screen out the long non-coding RNAs(LncRNAs) related to bone morphogenetic protein-9(BMP-9) expression, and to investigate the role of BMP-9 in the growth, differentiation, migration and apoptosis of breast cancer cells by regulating the expression of LncRNA.Methods: MDA-MB-231 cells were infected with the recombinant adenovirus Ad-BMP-9 carrying whole BMP-9 gene(named as MDA-MB-231/BMP-9 cells) or the empty vehicle adenovirus Ad-GFP carrying green fluorescent protein(GFP) gene(named as MDA-MB-231/GFP cells), respectively. Microarray technology was used to detect the difference in LncRNAs expression between MDAMB-231/BMP-9 and MDA-MB-231/GFP cells. The changes of LncRNA LINC00443, LINC00638, LINC00486, RHNO1, SERHL, HOXA11-AS, IQCA1, LINC00461, LOC440173, LHFPL, ANKRD36 BP2, BVES-AS1, LINC00937 and LINC00608 were validated by real-time fluorescent quantitative PCR. The biological funcation and related pathways of the above LncRNAs were analyzed by gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases.Results: The expression level of BMP-9 mRNA in MDA-MB-231/BMP-9 cells was significantly higher than that in MDA-MB-231/GFP cells(as the control). Compared with the control group, the expression levels of LncRNAs in MDA-MB-231/BMP-9 cells changed significantly. The expression levels of LINC00443, LINC00638 and LINC00486 were up-regulated, while the expression levels of IQCA1, LINC00461, LOC440173, LHFPL and ANKRD36 BP2 were down-regulated. The altered LncRNAs participated in the formation of cytoskeleton, cell membrane and so on, or played roles as signal molecules in intercellular signal transduction.Conclusion: The expression profile of LncRNAs in MDA-MB-231 cells with BMP-9 overexpression is significantly changed, indicating that LncRNAs may play key roles in the proliferation and invasion of breast cancer MDA-MB-231 cells regulated by BMP-9.
Objective: To screen out the long non-coding RNAs(LncRNAs) related to bone morphogenetic protein-9(BMP-9) expression, and to investigate the role of BMP-9 in the growth, differentiation, migration and apoptosis of breast cancer cells by regulating the expression of LncRNA.Methods: MDA-MB-231 cells were infected with the recombinant adenovirus Ad-BMP-9 carrying whole BMP-9 gene(named as MDA-MB-231/BMP-9 cells) or the empty vehicle adenovirus Ad-GFP carrying green fluorescent protein(GFP) gene(named as MDA-MB-231/GFP cells), respectively. Microarray technology was used to detect the difference in LncRNAs expression between MDAMB-231/BMP-9 and MDA-MB-231/GFP cells. The changes of LncRNA LINC00443, LINC00638, LINC00486, RHNO1, SERHL, HOXA11-AS, IQCA1, LINC00461, LOC440173, LHFPL, ANKRD36 BP2, BVES-AS1, LINC00937 and LINC00608 were validated by real-time fluorescent quantitative PCR. The biological funcation and related pathways of the above LncRNAs were analyzed by gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) databases.Results: The expression level of BMP-9 mRNA in MDA-MB-231/BMP-9 cells was significantly higher than that in MDA-MB-231/GFP cells(as the control). Compared with the control group, the expression levels of LncRNAs in MDA-MB-231/BMP-9 cells changed significantly. The expression levels of LINC00443, LINC00638 and LINC00486 were up-regulated, while the expression levels of IQCA1, LINC00461, LOC440173, LHFPL and ANKRD36 BP2 were down-regulated. The altered LncRNAs participated in the formation of cytoskeleton, cell membrane and so on, or played roles as signal molecules in intercellular signal transduction.Conclusion: The expression profile of LncRNAs in MDA-MB-231 cells with BMP-9 overexpression is significantly changed, indicating that LncRNAs may play key roles in the proliferation and invasion of breast cancer MDA-MB-231 cells regulated by BMP-9.
引文
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[25] HUANG J, ZHOU N, WATABE K, et al.Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27(Kipl)[J]. Cell Death Dis, 2014,5:el008.
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[27] VILLEGAS VE, ZAPHIROPOULOS PG.Neighboring gene regulation by antisense long non-coding RNAs[J].Int J Mol Sci, 201 5, 16(2):3251-66.
[28] CHENG SY, SEO J, HUANG BT, et al.Mitomycin C and decarbamoyl mitomycin C induce p53-independent p21WAF1/CIP1 activation[J]. Int J Oncol,2016, 49(5):1815-1824.
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[2] LI CS, TIAN H, ZOU M, et al. Secreted phosphoprotein 24kD(Spp24)inhibits growth of human pancreaticcancer cells caused by BMP-2[J].Biochem Biophys Res Commun, 2015,466(2):167-172.
[3] HU F, ZHANG Y, LI M, et al. BMP-6inhibits the metastasis of MDAMB-231 breast cancer cells byregulating MMP-1 expression[J]. Oncol Rep, 2016, 35(3):1 823-1 830.
[4] YOKOYAMA Y, WATANABE T. Autocrine BMP-4 signaling is a therapeutic target in colorectal cancer[J]. Cancer Res, 2017, 77(1 5):4026-4038.
[5] OWENS P, PICKUP MW, NOVITSKIY SV,et al. Inhibition of BMP signaling suppresses metastasis in mammary cancer[J]. Oncogene, 2015,34(1 9):2437-2449.
[6] WANG K, FENG H, REN W, et al.BMP9 inhibits the proliferation and invasiveness of breast cancer cells MDA-MB-231[J].J Cancer Res Clin Oncol, 201 1, 137(1 1):1687-1696.
[7] YANG G, LU X, YUAN L, et al. LncRNA:a link between RNA and cancer[J].Biochim Biophys Acta,2014,1839(11):1097-1 109.
[8] JIANG YJ, BIKLE DD. LncRNA profiling reveals new mechanism for VDR protection against skin cancer formation[J]. J Steroid Biochem Mol Biol, 201 4, 144(Pt A):87-90.
[9] LI C, LIANG G, YANG S, et al.Dysregulated IncRNA-UCA1contributes to the progression of gastric cancer through regulation of the PI3K-Akt-mTOR signaling pathway[J]. Oncotarget, 2017,8(55):93476-93491.
[10] YANG Q, WAN Q, ZHANG L, et al.Analysis of LncRNA expression in cell differentiation[J]. RNA Biol, 2018,15(3):41 3-422.
[11] LI T, ZHU J, WANG X, et al. Long non-coding RNA lncTCF7 activates the Wnt/β-cateni n pathway to promote metastasis and invasion in colorectal cancer[J]. Oncol Lett, 201 7,14(6):7384-7390.
[12] KANG MH, OH SC, LEE HJ, et al.Metastatic function of BMP-2 in gastric cancer cells:The role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression[J]. Exp Cell Res,2011,317(12):1746-1 762.
[13] SHIMIZU T, KAYAMORI T, MURAYAMA C,et al. Bone morphogenetic protein(BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways:BMP-4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC[J].Biochem Biophys Res Commun, 2012,417(2):869-873.
[14] GARCIA-ALVARO M, ADDANTE A,RONCERO C,et al. BMP9-induced survival effect in liver tumor cells requires p38MAPK activation[J]. Int J Mol Sci, 201 5, 1 6(9):20431-20448.
[15] LV Z, WANG C, YUAN T, et al. Bone morphogenetic protein 9 regulates tumor growth of osteosarcoma cells through the Wnt/p-catenin pathway[J].Oncol Rep, 2014, 31(2):989-994.
[16] LI S, DAI H, HE Y, et al. BMP9 inhibits the growth of breast cancer cells by downregulation of the PI3K/Akt signaling pathway[J]. Oncol Rep, 2018,40(3):1 743-1 751.
[17] WEN YZ, ZHENG LL, QU LH, et al.Pseudogenes are not pseudo any more[J]. RNA Biol, 2012, 9(1):27-32.
[18] WANG D, CHEN Z, XU H, et al.Long noncoding RNA CCAT2 as a novel biomaker of metastasis and prognosis in human cancer:a meta-analysis[J]. Oncotarget, 201 7,8(43):75664-75674.
[19] DING X, ZHANG Y, YANG H, et al.Long non-coding RNAs may serve as biomarkers in breast cancer combined with primary lung cancer[J]. Oncotarget, 201 7,8(35):5821 0-58221.
[20] ZIDAN HE, KARAM RA, EI-SEIFI OS, et al.Circulating long non-coding RNA MALAT1 expression as molecular biomarker in Egyptian patients with breast cancer[J]. Cancer Genet, 2018,220:32-37.
[21] GOODING AJ, ZHANG B, JAHANBANI FK, et al.The IncRNA BORG drives breast cancer metastasis and disease recurrenceU].Sci Rep, 2017, 7(1):1 2698.
[22] VIKRAM R, RAMACHANDRAN R, ABDUL KS.Functional significance of long noncoding RNAs in breast cancer[J]. Breast Cancer, 2014, 21(5):51 5-521.
[23] GOU L, LIU M, XIA J, et al. BMP9promotes the proliferation and migration of bladder cancer cells through up-regulating Lncrna UCA1[J].Int J Mol Sci, 201 8, 19(4):E111 6.
[24] GUPTA RA, SHAH N, WANG KC, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastas is[J]. Nature, 2010,464(7291):1071-1148.
[25] HUANG J, ZHOU N, WATABE K, et al.Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27(Kipl)[J]. Cell Death Dis, 2014,5:el008.
[26] RASHID F, SHAH A, SHAN G, Long non-coding RNAs in the cytoplasm[J].Genomics Proteomics Bioinformatics,2016, 14(2):73-80.
[27] VILLEGAS VE, ZAPHIROPOULOS PG.Neighboring gene regulation by antisense long non-coding RNAs[J].Int J Mol Sci, 201 5, 16(2):3251-66.
[28] CHENG SY, SEO J, HUANG BT, et al.Mitomycin C and decarbamoyl mitomycin C induce p53-independent p21WAF1/CIP1 activation[J]. Int J Oncol,2016, 49(5):1815-1824.
[29] DASTJERDI MN, MEHDIABADY EM,IRANPOUR FG, et al. Effect of Thymoquinone on p 5 3 gene expression and consequence apoptosis in breast cancer cell line[J].Int J Prev Med, 2016, 7:66.