微小RNA-218通过靶向调控LASP1影响食管癌细胞的生物学行为
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摘要
[目的 ]微小RNA-218(miR-218)作为一种肿瘤抑制因子,参与多种肿瘤的发生与进展。肌动蛋白骨架蛋白1(LASP1)是一种新的肌动蛋白结合蛋白,参与细胞骨架的重组调控及细胞迁移,与肿瘤的增殖、侵袭和转移密切相关。食管癌细胞中miR-218与LASP1基因间的调控关系尚未有报道,本研究对食管癌细胞中miR-218是否可以通过靶向调控LASP1而发挥抑癌作用进行初步探讨。[方法 ]应用实时定量逆转录聚合酶链反应(qRT-PCR)检测3株食管癌细胞(EC109、EC9706、KYSE510)和1株永生化食管上皮细胞(Het-1A)中miR-218的表达水平。以EC109为靶细胞,分别转染miR-218模拟物(miR-218 mimic)、miR-218 mimic的空载体对照(miR-NC)、LASP1的小干扰RNA(si-LASP1)、miR-218 mimic和si-LASP1(miR-218 mimic+si-LASP1)以及si-LASP1的空载体对照(si-NC)。采用qRT-PCR和Western blot法检测细胞的转染效果,采用细胞增殖与毒性检测试剂盒观察各组细胞生长曲线,采用细胞平板克隆形成实验、Transwell小室法和流式细胞术检测细胞增殖、迁移、侵袭和凋亡,通过生物信息学分析和双荧光素酶报告基因实验对miR-218的靶基因进行预测及验证。[结果] KYSE510、EC109和EC9706中miR-218表达水平分别为Het-1A的0.32%、1.81%和2.15%(均P <0.05)。生物信息学分析结果显示LASP1的3’非编码区有miR-218的结合位点,miR-218 mimic转染组EC109中LASP1 mRNA表达水平是miR-NC转染组的28.20%(P <0.05),Western blot结果显示过表达miR-218可下调LASP1蛋白表达水平。双荧光素酶报告基因实验结果显示LASP1-WT(野生型)+miR-218 mimic组的荧光素酶活性为0.31±0.02,低于LASP1-WT+miR-NC组(0.56±0.04)和LASP1-MUT(突变型)+miR-218 mimic组(0.49±0.07)(均P <0.05)。与miR-NC组相比,转染miR-218 mimic可抑制EC109的生长速率、迁移和侵袭能力(均P <0.05),促进细胞凋亡(P <0.05)。转染si-LASP1的EC109观察到相同的结果趋势。与siLASP1单独转染组比较,miR-218 mimic和si-LASP1共转染组细胞生长速率下降,平板克隆形成率降低(P <0.05),迁移和侵袭能力降低(P <0.05),凋亡增加(P <0.05)。[结论 ] miR-218在食管癌细胞中低表达;在EC109中miR-218可通过直接靶向调控LASP1基因的表达,抑制细胞的增殖、迁移和侵袭,促进凋亡,从而在食管癌的发展进程中发挥抑癌作用。
[Objective] As a tumor suppressor, microRNA-218(miR-218) is involved in the occurrence and progression of various tumors. Actin cytoskeleton protein 1(LIM and SH3 domain structure protein 1, LIM and SH3 protein 1, LASP1) is a newly identified actin binding protein, participating in cytoskeleton reorganization regulation and cell migration, and closely related to the proliferation, invasion, and metastasis of tumor. The regulatory relationship between miR-218 and LASP1 in esophageal cancer cells has not been reported. Therefore, this study aims to investigate whether miR-218 functions as a tumor suppressor by targeting LASP1.[Methods] The expression levels of miR-218 in three esophageal cancer cells(EC109, EC9706, and KYSE510) and one permanent esophageal epithelial cell(Het-1 A) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR). EC109 was separately transfected with miR-218 mimic, negative control of miR-218 mimic(miR-NC), small interfering RNA of LASP1(siLASP1), miR-218 mimic + si-LASP1, and negative control of si-LASP1(si-NC). The cell transfection efficiency was detected by qRT-PCR and Western blot; the cell growth curve of each group was observed by cell proliferation and toxicity assay kit(Cell Counting Kit-8, CCK-8); the cell proliferation, migration, invasion, and apoptosis were detected by plate clone formation experiment, Transwell assay, and flow cytometry; the target genes of miR-218 were predicted and verified by bioinformatics analysis and dual-luciferase reporter gene experiment.[Results] The expressions of miR-218 in KYSE510, EC109, and EC9706 were 0.32%, 1.81%, and 2.15% of that in Het-1 A respectively(Ps < 0.05). The results of bioinformatics analysis showed that 3' untranslated regions(3'UTR) of LASP1 had binding sites of miR-218, and LASP1 mRNA expression level in EC109 transfected with miR-218 mimic was 28.20% of that in miR-NC transfection group(P < 0.05); the Western blot results showed that overexpression of miR-218 down-regulated LASP1 protein expression level. The dual-luciferase reporter gene experiment showed that the luciferase activity in the LASP1-WT(wild type) + miR-218 mimic group was 0.31±0.02, lower than those in the LASP1-WT + miR-NC group(0.56±0.04) and the LASP1-MUT(mutant) + miR-218 mimic group(0.49±0.07)(Ps < 0.05). Compared with the miR-NC group, transfection with miR-218 mimic inhibited the growth rate, cell plate clone formation rate, cell migration, and cell invasion(Ps < 0.05), as well as promoted cell apoptosis rate(P < 0.05). The same trend pattern was observed in EC109 transfected with siLASP1. Compared with the EC109 transfected with si-LASP1 alone, co-transfection with miR-218 mimic and si-LASP1 lowered the cell growth rate, plate clone formation rate(P < 0.05), migration(P < 0.05), and invasion(P < 0.05), as well as elevated cell apoptosis rate(P < 0.05).[Conclusion] The findings show low miR-218 expression in esophageal cancer cells. In addition, miR-218 may inhibit the proliferation, migration, and invasion and promote the apoptosis of esophageal cancer cells by directly targeting LASP1 gene expression in EC109, playing a cancer suppressive role in the development of esophageal cancer.
[Objective] As a tumor suppressor, microRNA-218(miR-218) is involved in the occurrence and progression of various tumors. Actin cytoskeleton protein 1(LIM and SH3 domain structure protein 1, LIM and SH3 protein 1, LASP1) is a newly identified actin binding protein, participating in cytoskeleton reorganization regulation and cell migration, and closely related to the proliferation, invasion, and metastasis of tumor. The regulatory relationship between miR-218 and LASP1 in esophageal cancer cells has not been reported. Therefore, this study aims to investigate whether miR-218 functions as a tumor suppressor by targeting LASP1.[Methods] The expression levels of miR-218 in three esophageal cancer cells(EC109, EC9706, and KYSE510) and one permanent esophageal epithelial cell(Het-1 A) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR). EC109 was separately transfected with miR-218 mimic, negative control of miR-218 mimic(miR-NC), small interfering RNA of LASP1(siLASP1), miR-218 mimic + si-LASP1, and negative control of si-LASP1(si-NC). The cell transfection efficiency was detected by qRT-PCR and Western blot; the cell growth curve of each group was observed by cell proliferation and toxicity assay kit(Cell Counting Kit-8, CCK-8); the cell proliferation, migration, invasion, and apoptosis were detected by plate clone formation experiment, Transwell assay, and flow cytometry; the target genes of miR-218 were predicted and verified by bioinformatics analysis and dual-luciferase reporter gene experiment.[Results] The expressions of miR-218 in KYSE510, EC109, and EC9706 were 0.32%, 1.81%, and 2.15% of that in Het-1 A respectively(Ps < 0.05). The results of bioinformatics analysis showed that 3' untranslated regions(3'UTR) of LASP1 had binding sites of miR-218, and LASP1 mRNA expression level in EC109 transfected with miR-218 mimic was 28.20% of that in miR-NC transfection group(P < 0.05); the Western blot results showed that overexpression of miR-218 down-regulated LASP1 protein expression level. The dual-luciferase reporter gene experiment showed that the luciferase activity in the LASP1-WT(wild type) + miR-218 mimic group was 0.31±0.02, lower than those in the LASP1-WT + miR-NC group(0.56±0.04) and the LASP1-MUT(mutant) + miR-218 mimic group(0.49±0.07)(Ps < 0.05). Compared with the miR-NC group, transfection with miR-218 mimic inhibited the growth rate, cell plate clone formation rate, cell migration, and cell invasion(Ps < 0.05), as well as promoted cell apoptosis rate(P < 0.05). The same trend pattern was observed in EC109 transfected with siLASP1. Compared with the EC109 transfected with si-LASP1 alone, co-transfection with miR-218 mimic and si-LASP1 lowered the cell growth rate, plate clone formation rate(P < 0.05), migration(P < 0.05), and invasion(P < 0.05), as well as elevated cell apoptosis rate(P < 0.05).[Conclusion] The findings show low miR-218 expression in esophageal cancer cells. In addition, miR-218 may inhibit the proliferation, migration, and invasion and promote the apoptosis of esophageal cancer cells by directly targeting LASP1 gene expression in EC109, playing a cancer suppressive role in the development of esophageal cancer.
引文
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[13]TAKESHITA N,MORI M,KANO M,et al.miR-203 inhibits the migration and invasion of esophageal squamous cell carcinoma by regulating LASP1[J].Int J Oncol,2012,41(5):1653-1661.
[14]何彬.MicroRNA-133b在食管鳞状细胞癌中的表达及调控[D].长沙:中南大学,2012.
[15]DU Y Y,ZHAO L M,CHEN L,et al.The tumor-suppressive function of miR-1 by targeting LASP1 and TAGLN2 in esophageal squamous cell carcinoma[J].J Gastroenterol Hepatol,2016,31(2):384-393.
[16]CHIYOMARU T,ENOKIDA H,KAWAKAMI K,et al.Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer[J].Urol Oncol,2012,30(4):434-443.
[17]NISHIKAWA R,GOTO Y,SAKAMOTO S,et al.Tumorsuppressive microRNA-218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer[J].Cancer Sci,2014,105(7):802-811.
[18]TU Y,GAO X,LI G,et al.MicroRNA-218 inhibits glioma invasion,migration,proliferation,and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1[J].Cancer Res,2013,73(19):6046-6055.
[19]YU T,CAO R,LI S,et al.MiR-130b plays an oncogenic role by repressing PTEN expression in esophageal squamous cell carcinoma cells[J].BMC Cancer,2015,15:29.
[20]LI B,WANG L,LI Z,et al.miR-3174 contributes to apoptosis and autophagic cell death defects in gastric cancer cells by targeting ARHGAP10[J].Mol Ther Nucleic Acids,2017,9:294-311.
[21]YANG M,LIU R,SHENG J,et al.Differential expression profiles of microRNAs as potential biomarkers for the early diagnosis of esophageal squamous cell carcinoma[J].Oncol Rep,2013,29(1):169-176.
[22]YANG M,LIU R,LI X,et al.Epigenetic repression of miR-218promotes esophageal carcinogenesis by targeting ROBO1[J].Int J Mol Sci,2015,16(11):27781-27795.
[23]杨森.miR-183和miR-218参与调控食管癌发生发展的机制研究[D].南京:东南大学,2016.
[24]WANG T,CHEN T,NIU H,et al.MicroRNA-218 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma cells by targeting BMI1[J].Int J Mol Med,2015,36(1):93-102.
[25]SUI Y,ZHANG X,YANG H,et al.MicroRNA-133a acts as a tumour suppressor in breast cancer through targeting LASP1[J].Oncol Rep,2018,39(2):473-482.
[26]GAO Q,TANG L,WU L,et al.LASP1 promotes nasopharyngeal carcinoma progression through negatively regulation of the tumor suppressor PTEN[J].Cell Death Dis,2018,9(3):393.
[27]HU Z,CUI Y,ZHOU Y,et al.MicroRNA-29a plays a suppressive role in non-small cell lung cancer cells via targeting LASP1[J].Onco Targets Ther,2016,9:6999-7009.
[28]邱瑜,黄建平,周勤仙,等.Hsa-miR-218靶向调控LASP1对宫颈癌HeLa细胞生长的影响[J].中国病理生理杂志,2015,31(9):1572-1577.
[2]ZHOU P,DONG H,HE S,et al.miR612 is associated with esophageal squamous cell carcinoma development and metastasis,mediated through TP53[J].Mol Med Rep,2017,16(2):1855-1863.
[3]YANG H,WEI Y N,ZHOU J,et al.MiR-455-3p acts as a prognostic marker and inhibits the proliferation and invasion of esophageal squamous cell carcinoma by targeting FAM83F[J].Eur Rev Med Pharmacol Sci,2017,21(14):3200-3206.
[4]YEH Y M,CHUANG C M,CHAO K C,et al.MicroRNA-138suppresses ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1α[J].Int J Cancer,2013,133(4):867-878.
[5]ZHENG B,LIANG L,WANG C,et al.MicroRNA-148a suppresses tumor cell invasion and metastasis by downregulating ROCK1 in gastric cancer[J].Clin Cancer Res,2011,17(24):7574-7583.
[6]LIU L,ZHAO Z,ZHOU W,et al.Enhanced expression of miR-425 promotes esophageal squamous cell carcinoma tumorigenesis by targeting SMAD2[J].J Genet Genomics,2015,42(11):601-611.
[7]TIE J,PAN Y,ZHAO L,et al.MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor[J].PLoS Genet,2010,6(3):e1000879.
[8]ALAJEZ N M,LENARDUZZI M,ITO E,et al.MiR-218suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway[J].Cancer Res,2011,71(6):2381-2391.
[9]YU J,WANG Y,DONG R,et al.Circulating microRNA-218 was reduced in cervical cancer and correlated with tumor invasion[J].J Cancer Res Clin Oncol,2012,138(4):671-674.
[10]ZHANG C,GE S,HU C,et al.MiRNA-218,a new regulator of HMGB1,suppresses cell migration and invasion in non-small cell lung cancer[J].Acta Biochim Biophys Sin(Shanghai),2013,45(12):1055-1061.
[11]LIU Y,YAN W,ZHANG W,et al.MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1[J].Oncol Rep,2012,28(3):1013-1021.
[12]WANG L L,WANG L,WANG X Y,et al.MicroRNA-218inhibits the proliferation,migration,and invasion and promotes apoptosis of gastric cancer cells by targeting LASP1[J].Tumour Biol,2016,37(11):15241-15252.
[13]TAKESHITA N,MORI M,KANO M,et al.miR-203 inhibits the migration and invasion of esophageal squamous cell carcinoma by regulating LASP1[J].Int J Oncol,2012,41(5):1653-1661.
[14]何彬.MicroRNA-133b在食管鳞状细胞癌中的表达及调控[D].长沙:中南大学,2012.
[15]DU Y Y,ZHAO L M,CHEN L,et al.The tumor-suppressive function of miR-1 by targeting LASP1 and TAGLN2 in esophageal squamous cell carcinoma[J].J Gastroenterol Hepatol,2016,31(2):384-393.
[16]CHIYOMARU T,ENOKIDA H,KAWAKAMI K,et al.Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer[J].Urol Oncol,2012,30(4):434-443.
[17]NISHIKAWA R,GOTO Y,SAKAMOTO S,et al.Tumorsuppressive microRNA-218 inhibits cancer cell migration and invasion via targeting of LASP1 in prostate cancer[J].Cancer Sci,2014,105(7):802-811.
[18]TU Y,GAO X,LI G,et al.MicroRNA-218 inhibits glioma invasion,migration,proliferation,and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1[J].Cancer Res,2013,73(19):6046-6055.
[19]YU T,CAO R,LI S,et al.MiR-130b plays an oncogenic role by repressing PTEN expression in esophageal squamous cell carcinoma cells[J].BMC Cancer,2015,15:29.
[20]LI B,WANG L,LI Z,et al.miR-3174 contributes to apoptosis and autophagic cell death defects in gastric cancer cells by targeting ARHGAP10[J].Mol Ther Nucleic Acids,2017,9:294-311.
[21]YANG M,LIU R,SHENG J,et al.Differential expression profiles of microRNAs as potential biomarkers for the early diagnosis of esophageal squamous cell carcinoma[J].Oncol Rep,2013,29(1):169-176.
[22]YANG M,LIU R,LI X,et al.Epigenetic repression of miR-218promotes esophageal carcinogenesis by targeting ROBO1[J].Int J Mol Sci,2015,16(11):27781-27795.
[23]杨森.miR-183和miR-218参与调控食管癌发生发展的机制研究[D].南京:东南大学,2016.
[24]WANG T,CHEN T,NIU H,et al.MicroRNA-218 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma cells by targeting BMI1[J].Int J Mol Med,2015,36(1):93-102.
[25]SUI Y,ZHANG X,YANG H,et al.MicroRNA-133a acts as a tumour suppressor in breast cancer through targeting LASP1[J].Oncol Rep,2018,39(2):473-482.
[26]GAO Q,TANG L,WU L,et al.LASP1 promotes nasopharyngeal carcinoma progression through negatively regulation of the tumor suppressor PTEN[J].Cell Death Dis,2018,9(3):393.
[27]HU Z,CUI Y,ZHOU Y,et al.MicroRNA-29a plays a suppressive role in non-small cell lung cancer cells via targeting LASP1[J].Onco Targets Ther,2016,9:6999-7009.
[28]邱瑜,黄建平,周勤仙,等.Hsa-miR-218靶向调控LASP1对宫颈癌HeLa细胞生长的影响[J].中国病理生理杂志,2015,31(9):1572-1577.
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