MALAT1通过竞争miR-124上调SMYD3并促进乳腺癌细胞增殖与迁移
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摘要
为了探究lncRNA MALAT1是否会通过与组蛋白甲基化酶SMYD3间的相互调控对乳腺癌细胞的增殖和迁移产生影响,采用siRNA敲低MCF-7、MDA-MB-231乳腺癌细胞中的内源性MALAT1后,然后应用划痕和MTT法检测细胞迁移和增殖情况;实时定量PCR(Real time PCR)、免疫蛋白印迹(Western blot)等方法检测si-MALAT1对miRNA-124、SMYD3及其下游基因mRNA及蛋白质水平的影响。结果显示:siRNA靶向敲低MALAT1后可降低乳腺癌细胞的迁移和增殖速度,同时抑制SMYD3及其下游靶基因N-cadherin、MYL9、MMP9、CYR61等的转录表达,并上调miR-124。miR-124的过表达可降低SMYD3在乳腺癌细胞中的表达,且MALAT1的敲低可以缓解miR-124抑制剂对SMYD3蛋白质水平表达的促进作用。此外,转染外源质粒使SMYD3过表达可激活MALAT1转录,反之siRNA干扰SMYD3后则会下调MALAT1。研究结果表明:lncRNA MALAT1可以作为miR-124的ceRNA调控SMYD3,同时SMYD3可反馈性激活MALAT1的转录,这一相互调控作用将影响到乳腺癌细胞的增殖与迁移能力。
To investigate whether lncRNA MALAT1 affects the migration and proliferation of breast cancer cells through the regulation with histone methyltransferase SMYD3,the endogenous MALAT1 in the MCF-7 and MDA-MB-231 breast cancer cells were knocked down by siRNA,and then the migration and proliferation of cells were detected by wound healing migration and MTT assay.The effects of si-MALAT1 on the mRNA and protein levels of miRNA-124,SMYD3 and its downstream genes were detected by Real time PCR and Western blot.The results showed that siRNA-targeted knockdown of MALAT1 reduced the migration and proliferation of breast cancer cells,and inhibited the transcriptional expression of SMYD3 and its downstream genes,including N-cadherin,MYL9,MMP9 and CYR61,and up-regulated miR-124.Overexpression of miR-124 reduced the expression of SMYD3 in breast cancer cells,and knockdown of MALAT1 attenuated the promotion of SMYD3 protein expression by miR-124 inhibitors.In addition,SMYD3 overexpression activated MALAT1 transcription,whereas siRNA interference with SMYD3 downregulated MALAT1.These results indicate that LncRNA MALAT1 acted as a competing endogenous RNA(ceRNA) of miR-124 to regulate expression of SMYD3 in breast cancer cells,and SMYD3 can activate the transcription of MALAT1,which will affect the proliferation and migration of breast cancer cells.
To investigate whether lncRNA MALAT1 affects the migration and proliferation of breast cancer cells through the regulation with histone methyltransferase SMYD3,the endogenous MALAT1 in the MCF-7 and MDA-MB-231 breast cancer cells were knocked down by siRNA,and then the migration and proliferation of cells were detected by wound healing migration and MTT assay.The effects of si-MALAT1 on the mRNA and protein levels of miRNA-124,SMYD3 and its downstream genes were detected by Real time PCR and Western blot.The results showed that siRNA-targeted knockdown of MALAT1 reduced the migration and proliferation of breast cancer cells,and inhibited the transcriptional expression of SMYD3 and its downstream genes,including N-cadherin,MYL9,MMP9 and CYR61,and up-regulated miR-124.Overexpression of miR-124 reduced the expression of SMYD3 in breast cancer cells,and knockdown of MALAT1 attenuated the promotion of SMYD3 protein expression by miR-124 inhibitors.In addition,SMYD3 overexpression activated MALAT1 transcription,whereas siRNA interference with SMYD3 downregulated MALAT1.These results indicate that LncRNA MALAT1 acted as a competing endogenous RNA(ceRNA) of miR-124 to regulate expression of SMYD3 in breast cancer cells,and SMYD3 can activate the transcription of MALAT1,which will affect the proliferation and migration of breast cancer cells.
引文
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[26] Jiang Y,Li YH,Fang SJ,et al.The role of MALAT1 correlates with HPV in cervical cancer[J].Oncol Lett,2014,7(6):2135-2141.
[27] Zuo YG,Li Y,Zhou ZY,et al.Long non-coding RNA MALAT1 promotes proliferation and invasion via targeting miR-129-5p in triple-negative breast cancer[J].Biomed Pharmacother,2017,95:922-928.
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[29] Bamodu OA,Huang WC,Lee WH,et al.Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448[J].BMC Cancer,2016,16:160.
[30] Chou JJ,Wang BY,Zheng TJ,et al.MALAT1 induced migration and invasion of human breast cancer cells by competitively binding miR-1 with cdc42[J].Biochem Biophys Res Commun,2016,472(1):262-269.
[31] Zheng LH,Zhang YH,Fu YJ,et al.Long non-coding RNA MALAT1 regulates BLCAP mRNA expression through binding to miR-339-5p and promotes poor prognosis in breast cancer[J].Biosci Rep,2019,39(2):1-12.
[32] Luo XG,Lin C,Lu YH,et al.Correlation between SMYD3 and cell proliferation and its effect on cell cycle[J].J China Pharm Univ(中国药科大学学报),2007,38(3):277-282.
[33] Luo XG,Pan H,Liu ZP,et al.The role of SMYD3 in tumorigenesis and development[J].Chem Life(生命的化学),2009,29(2):247-249.
[34] Chen DJ,Liu L,Luo XG,et al.Effect of SMYD3 on the microRNA expression profile of MCF-7 breast cancer cells[J].Oncol Lett,2017,14(2):1831-1840.
[2] Hamamoto R,Furukawa Y,Morita M,et al.SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells[J].Nat Cell Biol,2004,6(8):731-740.
[3] Liu Y,Luo XG,Deng JY,et al.SMYD3 overexpression was a risk factor in the biological behavior and prognosis of gastric carcinoma[J].Tumour Biol,2015,36(4):2685-2694.
[4] Luo XG,Zhang CL,Zhao WW,et al.Histone methyltransferase SMYD3 promotes MRTF-A-mediated transactivation of MYL9 and migration of MCF-7 breast cancer cells[J].Cancer Lett,2014,344(1):129-137.
[5] Fuji T,Tsunesumi S,Yamaguchi K,et al.SMYD3 is required for the development of cardiac and skeletal muscle in zebrafish[J].PLoS One,2011,6(8):e23491.
[6] Tracy CM,Warren JS,Szulik M,et al.The Smyd family of methyltransferases:role in cardiac and skeletal muscle physiology and pathology[J].Curr Opin Physiol,2018,1:140-152.
[7] Zeng B,Li ZH,Chen RF,et al.Epigenetic regulation of miR-124 by hepatitis C virus core protein promotes migration and invasion of intrahepatic cholangiocarcinoma cells by targeting SMYD3[J].FEBS Lett,2012,586(19):3271-3278.
[8] Zhang H,Chen ZH,Wang XX,et al.Long non-coding RNA:a new player in cancer[J].J Hematol Oncol,2013,6:37.
[9] Chen HY,Meng T.Effect of LncRNA MALAT1 on proliferation and apoptosis of human umbilical vein endothelial cells[J].Chin J Public Health(中国公共卫生),2016,32(10):1340-1342.
[10] Zhao ZY,Chen CJ,Liu Y,et al.17beta-Estradiol treatment inhibits breast cell proliferation,migration and invasion by decreasing MALAT-1 RNA level[J].Biochem Biophys Res Commun,2014,445(2):388-393.
[11] Arun G,Spector DL.MALAT1 long non-coding RNA and breast cancer[J].RNA Biol,2019.doi:10.1080/15476286.2019.1592072.
[12] Luo JH,Ren B,Keryanov S,et al.Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas[J].Hepatology,2006,44(4):1012-1024.
[13] Abbastabar M,Sarfi M,Golestani A,et al.lncRNA involvement in hepatocellular carcinoma metastasis and prognosis[J].Excli J,2018,17:900-913.
[14] Wang JQ,Su LP,Chen XH,et al.MALAT1 promotes cell proliferation in gastric cancer by recruiting SF2/ASF[J].Biomed Pharmacother,2014,68(5):557-564.
[15] Chang Y,Nu CY.The effect of LncRNA-MALAT1 on the hypoxia-induced gastric cancer metastasis and invasion[J].Mod Oncol(现代肿瘤医学),2015,23(24):3569-3573.
[16] Zhu MH,Zhang CY,Chen DH,et al.lncRNA MALAT1 potentiates the progression of tongue squamous cell carcinoma through regulating miR-140-5p-PAK1 pathway [J].Onco Targets Ther,2019,12:1365-1377.
[17] Zhuang M,Zhao SL,Jiang Z,et al.MALAT1 sponges miR-106b-5p to promote the invasion and metastasis of colorectal cancer via SLAIN2 enhanced microtubules mobility[J].EBioMedicine,2019,41:286-298.
[18] Xi ZW,Si JC,Nan J.LncRNA MALAT1 potentiates autophagyassociated cisplatin resistance by regulating the microRNA30b/autophagyrelated gene 5 axis in gastric cancer[J].Int J Oncol,2019,54(1):239-248.
[19] Dong YQ,Liang GJ,Yuan BQ,et al.MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the PI3K/Akt pathway[J].Tumour Biol,2015,36(3):1477-1486.
[20] Pang EJ,Yang R,Fu XB,et al.Overexpression of long non-coding RNA MALAT1 is correlated with clinical progression and unfavorable prognosis in pancreatic cancer[J].Tumour Biol,2015,36(4):2403-2407.
[21] Cock-Rada AM,Medjkane S,Janski N,et al.SMYD3 promotes cancer invasion by epigenetic upregulation of the metalloproteinase MMP-9[J].Cancer Res,2012,72(3):810-820.
[22] Fenizia C,Bottino C,Corbetta S,et al.SMYD3 promotes the epithelial-mesenchymal transition in breast cancer[J].Nucleic Acids Res,2019,47(3):1278-1293.
[23] Wang Y,Wu CQ,Zhang C,et al.TGF-beta-induced STAT3 overexpression promotes human head and neck squamous cell carcinoma invasion and metastasis through malat1/miR-30a interactions[J].Cancer Lett,2018,436:52-62.
[24] Duan GC,Zhang CL,Xu CK,et al.Knockdown of MALAT1 inhi-bits osteosarcoma progression via regulating the miR34a/cyclin D1 axis[J].Int J Oncol,2019,54(1):17-28.
[25] Chang SM,Hu WW.Long non-coding RNA MALAT1 promotes oral squamous cell carcinoma development via microRNA-125b/STAT3 axis[J].J Cell Physiol,2018,233(4):3384-3396.
[26] Jiang Y,Li YH,Fang SJ,et al.The role of MALAT1 correlates with HPV in cervical cancer[J].Oncol Lett,2014,7(6):2135-2141.
[27] Zuo YG,Li Y,Zhou ZY,et al.Long non-coding RNA MALAT1 promotes proliferation and invasion via targeting miR-129-5p in triple-negative breast cancer[J].Biomed Pharmacother,2017,95:922-928.
[28] Liu S,Zhou X,Wang XF,et al.Metastasis-associated lung adenocarcinoma transcript 1 modulates oral squamous cell carcinoma invasion in vitro and in vivo[J].Chin J Clin Oncol(中国肿瘤临床),2015,42(9):460-465.
[29] Bamodu OA,Huang WC,Lee WH,et al.Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448[J].BMC Cancer,2016,16:160.
[30] Chou JJ,Wang BY,Zheng TJ,et al.MALAT1 induced migration and invasion of human breast cancer cells by competitively binding miR-1 with cdc42[J].Biochem Biophys Res Commun,2016,472(1):262-269.
[31] Zheng LH,Zhang YH,Fu YJ,et al.Long non-coding RNA MALAT1 regulates BLCAP mRNA expression through binding to miR-339-5p and promotes poor prognosis in breast cancer[J].Biosci Rep,2019,39(2):1-12.
[32] Luo XG,Lin C,Lu YH,et al.Correlation between SMYD3 and cell proliferation and its effect on cell cycle[J].J China Pharm Univ(中国药科大学学报),2007,38(3):277-282.
[33] Luo XG,Pan H,Liu ZP,et al.The role of SMYD3 in tumorigenesis and development[J].Chem Life(生命的化学),2009,29(2):247-249.
[34] Chen DJ,Liu L,Luo XG,et al.Effect of SMYD3 on the microRNA expression profile of MCF-7 breast cancer cells[J].Oncol Lett,2017,14(2):1831-1840.
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