miR-142-3p通过调控S1PR3影响膀胱癌干细胞的干性
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摘要
背景:研究发现miR-142-3p低表达于多种肿瘤组织和细胞系中,并且有研究发现TUG1/miR-142/ZEB2轴可抑制膀胱癌增殖并诱导其凋亡。目的:观察miR-142-3p在膀胱癌干细胞中的表达水平,探讨其对S1PR3的靶向调控作用及对膀胱癌干细胞干性的影响。方法:从新鲜人膀胱癌组织中分离培养原代膀胱癌细胞,采用流式细胞仪筛选出CD44~+细胞与CD44~-细胞,Western-blot检测2种细胞中Nanog、Oct4蛋白的表达,qRT-PCR检测2种细胞中miR-142-3p基因的表达。将miR-142-3p mimic(实验组)、miR对照质粒(对照组)分别转染至CD44~+细胞,48 h后,采用MTS实验检测细胞增殖能力,软琼脂克隆实验检测细胞克隆形成能力,Transwell小室实验检测各组细胞转移能力,Western-blot检测细胞中pPi3k和pAkt蛋白表达,qRT-PCR检测细胞中S1PR3基因表达。将稳定转染的2种细胞分别注射至裸鼠(购自北京维通利华实验动物技术有限公司)右侧腋下,4周后检测肿瘤体积。将S1PR3-突变型+miR-142-3p mimic、S1PR3-突变型+miR对照质粒、S1PR3-野生型+miR-142-3p mimic、S1PR3-野生型+miR对照质粒分别转染至CD44~+细胞,48h后检测各组荧光素酶活性。结果与结论:①CD44~+细胞中miR-142-3p基因表达显著低于CD44-细胞(P <0.05);CD44~+细胞中Nanog、Oct4蛋白表达显著高于CD44~-细胞,证实CD44~+细胞为膀胱癌干细胞;②实验组膀胱癌干细胞的增殖能力、克隆形成能力及转移能力明显低于对照组(P <0.05);③实验组膀胱癌干细胞中pPi3k和pAkt蛋白表达弱于对照组,S1PR3基因表达低于对照组(P <0.05);④实验组裸鼠体内肿瘤体积小于对照组(P <0.05);⑤与相比,S1PR3-突变型+miR-142-3p mimic组荧光素酶活性显著低于S1PR3-突变型+miR对照质粒组(P <0.05),S1PR3-野生型+miR对照质粒组和S1PR3-野生型+miR-142-3p mimic组荧光素酶活性无差异(P> 0.05);⑥结果表明,miR-142-3p在膀胱癌干细胞中低表达,其可能通过负调控S1PR3下调PI3K/AKT信号通路来抑制膀胱癌干细胞的干性。
BACKGROUND: Studies have found that miR-142-3p is underexpressed in a variety of tumor tissues and cell lines, and the TUG1/miR-142/ZEB2 axis can inhibit proliferation and induce apoptosis in bladder cancer.OBJECTIVE: To observe the expression level of miR-142-3p in bladder cancer stem cells, and to explore its effects on targeted regulation of S1PR3 and the stemness of bladder cancer stem cells.METHODS: Primary bladder cancer cells were isolated and cultured from fresh human bladder cancer tissues. CD44~+ and CD44~- cells were screened by flow cytometry. The expressions of stemness-related molecular markers Nanog and Oct4 in CD44~+ and CD44~- cells were detected by western blot. q RT-PCR was used to detect the expression of mi R-142-3 p in CD44~+ and CD44~- cells. miR-142-3p mimic(experimental group) and empty plasmid vector(control group) were transfected into CD44~+ cells. At 48 hours after transfection, MTS was used to detect the cell proliferation ability of bladder cancer stem cells in each group; soft agar cloning assay was used to detect the tumor formation ability; Transwell assay was used to detect the cell migration ability; western blot assay was used to detect the expression of pPi3 k and p Akt; and q RT-PCR was used to detect the m RNA expression of S1PR3. The cells transfected with miR-142-3 p mimic or empty plasmid vector were injected into the right armpit of nude mice(provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd.), and the tumor volume was detected at 4 weeks after injection. S1PR3-mutant+miR-142-3p mimic, S1PR3-mutant+empty plasmid vector, S1PR3-wild type+miR-142-3p mimic, S1PR3-wild type+empty plasmid vector were transfected into the CD44~+ cells. And then luciferase activity in each group was detected at 48 hours after transfection.RESULTS AND CONCLUSION:(1) The expression of miR-142-3p was significantly decreased in the CD44~+ cells as compared with the CD44~- cells(P < 0.05), and the expression of Nanog and Oct4 in CD44~+ cells was significantly higher than that in CD44~- cells(P < 0.05),indicating that the CD44~+ cells are identified as bladder cancer stem cells.(2) The mi R-142-3p mimic group showed a significant reduction in the proliferation ability, the tumor formation ability, and the migration ability as compared with the control group(P < 0.05).(3) In the miR-142-3p mimic group, p Pi3k and p Akt protein expression was significantly reduced, and the m RNA expression of S1PR3 was also decreased as compared with the control group(P < 0.05).(4) The tumor volume in the mi R-142-3 p nude mice was significantly smaller than that in the control mice(P < 0.05).(5) The luciferase activity in the S1PR3-mutant+miR-142-3 p mimic group was significantly lower than that in the S1PR3-wild type+empty plasmid vector group(P < 0.05), whereas there was no significant difference between the S1PR3-wild type+miR-142-3p mimic and S1PR3-wild type+empty plasmid vector groups(P > 0.05). To conclude, expression of miR-142-3p is down-regulated in bladder cancer stem cells, which may inhibit the stemness of bladder cancer stem cells by negatively regulating S1PR3 and suppressing PI3K/AKT signaling pathway.
BACKGROUND: Studies have found that miR-142-3p is underexpressed in a variety of tumor tissues and cell lines, and the TUG1/miR-142/ZEB2 axis can inhibit proliferation and induce apoptosis in bladder cancer.OBJECTIVE: To observe the expression level of miR-142-3p in bladder cancer stem cells, and to explore its effects on targeted regulation of S1PR3 and the stemness of bladder cancer stem cells.METHODS: Primary bladder cancer cells were isolated and cultured from fresh human bladder cancer tissues. CD44~+ and CD44~- cells were screened by flow cytometry. The expressions of stemness-related molecular markers Nanog and Oct4 in CD44~+ and CD44~- cells were detected by western blot. q RT-PCR was used to detect the expression of mi R-142-3 p in CD44~+ and CD44~- cells. miR-142-3p mimic(experimental group) and empty plasmid vector(control group) were transfected into CD44~+ cells. At 48 hours after transfection, MTS was used to detect the cell proliferation ability of bladder cancer stem cells in each group; soft agar cloning assay was used to detect the tumor formation ability; Transwell assay was used to detect the cell migration ability; western blot assay was used to detect the expression of pPi3 k and p Akt; and q RT-PCR was used to detect the m RNA expression of S1PR3. The cells transfected with miR-142-3 p mimic or empty plasmid vector were injected into the right armpit of nude mice(provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd.), and the tumor volume was detected at 4 weeks after injection. S1PR3-mutant+miR-142-3p mimic, S1PR3-mutant+empty plasmid vector, S1PR3-wild type+miR-142-3p mimic, S1PR3-wild type+empty plasmid vector were transfected into the CD44~+ cells. And then luciferase activity in each group was detected at 48 hours after transfection.RESULTS AND CONCLUSION:(1) The expression of miR-142-3p was significantly decreased in the CD44~+ cells as compared with the CD44~- cells(P < 0.05), and the expression of Nanog and Oct4 in CD44~+ cells was significantly higher than that in CD44~- cells(P < 0.05),indicating that the CD44~+ cells are identified as bladder cancer stem cells.(2) The mi R-142-3p mimic group showed a significant reduction in the proliferation ability, the tumor formation ability, and the migration ability as compared with the control group(P < 0.05).(3) In the miR-142-3p mimic group, p Pi3k and p Akt protein expression was significantly reduced, and the m RNA expression of S1PR3 was also decreased as compared with the control group(P < 0.05).(4) The tumor volume in the mi R-142-3 p nude mice was significantly smaller than that in the control mice(P < 0.05).(5) The luciferase activity in the S1PR3-mutant+miR-142-3 p mimic group was significantly lower than that in the S1PR3-wild type+empty plasmid vector group(P < 0.05), whereas there was no significant difference between the S1PR3-wild type+miR-142-3p mimic and S1PR3-wild type+empty plasmid vector groups(P > 0.05). To conclude, expression of miR-142-3p is down-regulated in bladder cancer stem cells, which may inhibit the stemness of bladder cancer stem cells by negatively regulating S1PR3 and suppressing PI3K/AKT signaling pathway.
引文
[1]Yan M,Jing X,Liu Y.Screening and identification of key biomarkers in bladder carcinoma:Evidence from bioinformatics analysis.Oncol Lett.2018;16(3):3092-3100.
[2]Siegel RL,Miller KD.Cancer statistics,2018.CA Cancer J Clin.2018;68(1):7-30.
[3]Liu M,Tu J,Gingold JA,et al.Cancer in a dish:progress using stem cells as a platform for cancer research.Am J Cancer Res.2018;8(6):944-954.
[4]Naik PP,Panda PK.Oral Cancer Stem Cells Microenvironment.Adv Exp Med Biol.2017;1041(1):207-233.
[5]Akbarzadeh M,Movassaghpour AA,Ghanbari H,et al.The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells.Sci Rep,2017;7(1):17062.
[6]Li Y,Lin K,Yang Z,et al.Bladder cancer stem cells:clonal origin and therapeutic perspectives.Oncotarget.2017;8(39):66668-66679.
[7]Zhu F,Qian W,Zhang H,et al.SOX2 Is a Marker for Stem-like Tumor Cells in Bladder Cancer.Stem Cell Reports.2017;9(2):429-437.
[8]Pan JH,Zhou H,Zhao XX,et al.Role of exosomes and exosomal micro RNAs in hepatocellular carcinoma:Potential in diagnosis and antitumour treatments(Review).Int J Mol Med.2018;41(4):1809-1816.
[9]王明阳,王光磊,陈新亮,等.Micro RNA的生物学功能及其与肿瘤诊断和治疗的研究进展[J].动物医学进展,2018,39(1):95-98.
[10]Zhang Y,Xu B.Effects of mi RNAs on functions of breast cancer stem cells and treatment of breast cancer.Onco Targets Ther.2018;11(1):4263-4270.
[11]Lv M,Zhong Z,Huang M,et al.lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by mi R-29b-3p as competing endogenous RNA.Biochim Biophys Acta.2017;1864(10):1887-1899.
[12]Xu R,Zhu X,Chen F,et al.Lnc RNA XIST/miR-200c regulates the stemness properties and tumourigenicity of human bladder cancer stem cell-like cells.Cancer Cell Int.2018;18(2):41-53.
[13]Hua S,Liu C,Liu L.miR-142-3p inhibits aerobic glycolysis and cell proliferation in hepatocellular carcinoma via targeting LDHA.Biochem Biophys Res Commun.2018;496(3):947-954.
[14]Chen Y,Zhou X,Qiao J.Mi R-142-3p Overexpression Increases Chemo-Sensitivity of NSCLC by Inhibiting HMGB1-Mediated Autophagy.Cell Physiol Biochem.2017;41(4):1370-1382.
[15]Liu Q,Liu H,Cheng H,et al.Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells.Onco Targets Ther.2017;10(1):2461-2471.
[16]Antoni S,Ferlay J,Soerjomataram I,et al.Bladder Cancer Incidence and Mortality:A Global Overview and Recent Trends.Eur Urol.2017;71(1):96-108.
[17]朱峰宇,梁瑜,李洋.膀胱癌肿瘤干细胞研究进展[J].转化医学电子杂志,2017,4(5):4-10.
[18]杨德林,霍倩,王乙水,等.基质细胞衍生因子-1及其受体CXCR4对膀胱癌细胞侵袭能力及腔内种植的影响[J].医学研究生学报,2014,27(10):1028-1032.
[19]Prieto-Vila M,Takahashi RU,Usuba W,et al.Drug Resistance Driven by Cancer Stem Cells and Their Niche.Int J Mol Sci.2017;18(12):98-106.
[20]Ayob AZ.Cancer stem cells as key drivers of tumour progression.J Biomed Sci.2018;25(1):20.
[21]Zhang Q,Zhuang J,Deng Y,et al.mi R34a/GOLPH3 Axis abrogates Urothelial Bladder Cancer Chemoresistance via Reduced Cancer Stemness.Theranostics.2017;7(19):4777-4790.
[22]Luo H,Yang R,Li C,et al.MicroRNA-139-5p inhibits bladder cancer proliferation and self-renewal by targeting the Bmi1oncogene.Tumour Biol.2017;39(7):1010428317718414.
[23]Troschel FM,B?hly N,Borrmann K,et al.mi R-142-3p attenuates breast cancer stem cell characteristics and decreases radioresistance in vitro.Tumour Biol.2018;40(8):1010428318791887.
[24]Li H,Li F.Exosomes from BM-MSCs increase the population of CSCs via transfer of mi R-142-3p.Br J Cancer.2018;119(6):744-755.
[25]尹彦斌,王建杰,刘祎,等.肿瘤干细胞分离研究进展[J].现代生物医学进展,2016,16(2):382-385.
[26]Hatina J,Parmar HS,Kripnerova M,et al.Urothelial Carcinoma Stem Cells:Current Concepts,Controversies,and Methods.Methods Mol Biol.2018;1655(2):121-136.
[27]Tomiyama N,Ikeda R,Nishizawa Y,et al.S100A16 up-regulates Oct4 and Nanog expression in cancer stem-like cells of Yumoto human cervical carcinoma cells.Oncol Lett.2018;15(6):9929-9933.
[28]Adada MM,Canals D,Jeong N,et al.Intracellular sphingosine kinase 2-derived sphingosine-1-phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion.FASEB J.2015;29(11):4654-4669.
[29]Patmanathan SN,Wang W,Yap LF,et al.Mechanisms of sphingosine 1-phosphate receptor signalling in cancer.Cell Signal.2017;34(2):66-75.
[30]Zhao J,Liu J,Lee JF,et al.TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression:implication of sphingosine-1 phosphate receptor 3 in lung adenocarcinoma progression.J Biol Chem.2016;291(53):27343-27353.
[31]Wang S,Liang Y,Chang W,et al.Triple Negative Breast Cancer Depends on Sphingosine Kinase 1(SphK1)/Sphingosine-1-Phosphate(S1P)/Sphingosine 1-Phosphate Receptor 3(S1PR3)/Notch Signaling for Metastasis.Med Sci Monit.2018;24(1):1912-1923.
[32]Wang H,Huang H.Sphingosine-1-phosphate promotes the proliferation and attenuates apoptosis of Endothelial progenitor cells via S1PR1/S1PR3/PI3K/Akt pathway.Cell Biol Int.2018;(1):169-175.
[33]张保豫,侯博,何海勇,等.内质网应激通过抑制PI3K/AKT/m TOR信号通路诱导多形性胶质母细胞瘤干细胞凋亡的机制[J].中山大学学报(医学科学版),2016,37(2):183-189.
[2]Siegel RL,Miller KD.Cancer statistics,2018.CA Cancer J Clin.2018;68(1):7-30.
[3]Liu M,Tu J,Gingold JA,et al.Cancer in a dish:progress using stem cells as a platform for cancer research.Am J Cancer Res.2018;8(6):944-954.
[4]Naik PP,Panda PK.Oral Cancer Stem Cells Microenvironment.Adv Exp Med Biol.2017;1041(1):207-233.
[5]Akbarzadeh M,Movassaghpour AA,Ghanbari H,et al.The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells.Sci Rep,2017;7(1):17062.
[6]Li Y,Lin K,Yang Z,et al.Bladder cancer stem cells:clonal origin and therapeutic perspectives.Oncotarget.2017;8(39):66668-66679.
[7]Zhu F,Qian W,Zhang H,et al.SOX2 Is a Marker for Stem-like Tumor Cells in Bladder Cancer.Stem Cell Reports.2017;9(2):429-437.
[8]Pan JH,Zhou H,Zhao XX,et al.Role of exosomes and exosomal micro RNAs in hepatocellular carcinoma:Potential in diagnosis and antitumour treatments(Review).Int J Mol Med.2018;41(4):1809-1816.
[9]王明阳,王光磊,陈新亮,等.Micro RNA的生物学功能及其与肿瘤诊断和治疗的研究进展[J].动物医学进展,2018,39(1):95-98.
[10]Zhang Y,Xu B.Effects of mi RNAs on functions of breast cancer stem cells and treatment of breast cancer.Onco Targets Ther.2018;11(1):4263-4270.
[11]Lv M,Zhong Z,Huang M,et al.lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by mi R-29b-3p as competing endogenous RNA.Biochim Biophys Acta.2017;1864(10):1887-1899.
[12]Xu R,Zhu X,Chen F,et al.Lnc RNA XIST/miR-200c regulates the stemness properties and tumourigenicity of human bladder cancer stem cell-like cells.Cancer Cell Int.2018;18(2):41-53.
[13]Hua S,Liu C,Liu L.miR-142-3p inhibits aerobic glycolysis and cell proliferation in hepatocellular carcinoma via targeting LDHA.Biochem Biophys Res Commun.2018;496(3):947-954.
[14]Chen Y,Zhou X,Qiao J.Mi R-142-3p Overexpression Increases Chemo-Sensitivity of NSCLC by Inhibiting HMGB1-Mediated Autophagy.Cell Physiol Biochem.2017;41(4):1370-1382.
[15]Liu Q,Liu H,Cheng H,et al.Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells.Onco Targets Ther.2017;10(1):2461-2471.
[16]Antoni S,Ferlay J,Soerjomataram I,et al.Bladder Cancer Incidence and Mortality:A Global Overview and Recent Trends.Eur Urol.2017;71(1):96-108.
[17]朱峰宇,梁瑜,李洋.膀胱癌肿瘤干细胞研究进展[J].转化医学电子杂志,2017,4(5):4-10.
[18]杨德林,霍倩,王乙水,等.基质细胞衍生因子-1及其受体CXCR4对膀胱癌细胞侵袭能力及腔内种植的影响[J].医学研究生学报,2014,27(10):1028-1032.
[19]Prieto-Vila M,Takahashi RU,Usuba W,et al.Drug Resistance Driven by Cancer Stem Cells and Their Niche.Int J Mol Sci.2017;18(12):98-106.
[20]Ayob AZ.Cancer stem cells as key drivers of tumour progression.J Biomed Sci.2018;25(1):20.
[21]Zhang Q,Zhuang J,Deng Y,et al.mi R34a/GOLPH3 Axis abrogates Urothelial Bladder Cancer Chemoresistance via Reduced Cancer Stemness.Theranostics.2017;7(19):4777-4790.
[22]Luo H,Yang R,Li C,et al.MicroRNA-139-5p inhibits bladder cancer proliferation and self-renewal by targeting the Bmi1oncogene.Tumour Biol.2017;39(7):1010428317718414.
[23]Troschel FM,B?hly N,Borrmann K,et al.mi R-142-3p attenuates breast cancer stem cell characteristics and decreases radioresistance in vitro.Tumour Biol.2018;40(8):1010428318791887.
[24]Li H,Li F.Exosomes from BM-MSCs increase the population of CSCs via transfer of mi R-142-3p.Br J Cancer.2018;119(6):744-755.
[25]尹彦斌,王建杰,刘祎,等.肿瘤干细胞分离研究进展[J].现代生物医学进展,2016,16(2):382-385.
[26]Hatina J,Parmar HS,Kripnerova M,et al.Urothelial Carcinoma Stem Cells:Current Concepts,Controversies,and Methods.Methods Mol Biol.2018;1655(2):121-136.
[27]Tomiyama N,Ikeda R,Nishizawa Y,et al.S100A16 up-regulates Oct4 and Nanog expression in cancer stem-like cells of Yumoto human cervical carcinoma cells.Oncol Lett.2018;15(6):9929-9933.
[28]Adada MM,Canals D,Jeong N,et al.Intracellular sphingosine kinase 2-derived sphingosine-1-phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion.FASEB J.2015;29(11):4654-4669.
[29]Patmanathan SN,Wang W,Yap LF,et al.Mechanisms of sphingosine 1-phosphate receptor signalling in cancer.Cell Signal.2017;34(2):66-75.
[30]Zhao J,Liu J,Lee JF,et al.TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression:implication of sphingosine-1 phosphate receptor 3 in lung adenocarcinoma progression.J Biol Chem.2016;291(53):27343-27353.
[31]Wang S,Liang Y,Chang W,et al.Triple Negative Breast Cancer Depends on Sphingosine Kinase 1(SphK1)/Sphingosine-1-Phosphate(S1P)/Sphingosine 1-Phosphate Receptor 3(S1PR3)/Notch Signaling for Metastasis.Med Sci Monit.2018;24(1):1912-1923.
[32]Wang H,Huang H.Sphingosine-1-phosphate promotes the proliferation and attenuates apoptosis of Endothelial progenitor cells via S1PR1/S1PR3/PI3K/Akt pathway.Cell Biol Int.2018;(1):169-175.
[33]张保豫,侯博,何海勇,等.内质网应激通过抑制PI3K/AKT/m TOR信号通路诱导多形性胶质母细胞瘤干细胞凋亡的机制[J].中山大学学报(医学科学版),2016,37(2):183-189.