长链非编码RNA H19促进胃癌干细胞的增殖和转移
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摘要
背景:研究发现,干扰长链非编码RNA H19的表达,可以降低胃癌细胞系MGC-803肿瘤球细胞的成瘤能力、增殖和克隆形成能力,而H19在胃癌中是否能通过影响肿瘤干细胞关键标志物Sox2的表达进而调控胃癌干细胞尚不清楚。目的:研究长链非编码RNA H19在胃癌组织及胃癌干细胞中的表达水平,并探讨其对胃癌干细胞增殖和转移的影响及作用机制。方法:qRT-PCR检测H19在38例胃癌组织、癌旁组织、胃癌干细胞CSC-G、人永生化正常胃上皮细胞GES-1中的表达;经脂质体分别转染H19 siRNA(si-H19)和阴性对照siRNA(si-NC)48 h后,软琼脂克隆形成实验、MTS实验、Transwell实验检测CSC-G细胞干性、增殖和转移能力,Western blot检测CSC-G细胞中Sox2蛋白的表达。结果与结论:①qRT-PCR结果显示H19在胃癌组织中的表达显著高于其配对的癌旁组织,且在肿瘤T分期高及有淋巴结转移的组织中高表达(P均<0.05),同时H19在CSC-G细胞中的表达显著高于GES-1细胞(P <0.05);②转染H19 siRNA后,CSC-G细胞软琼脂克隆球形成的数目显著减少,细胞增殖能力抑制、细胞侵袭能力减弱、Sox2蛋白表达减少(P均<0.05);③结果表明,H19在胃癌组织和胃癌干细胞中高表达,H19可能通过作用于干细胞相关蛋白Sox2促进胃癌干细胞的增殖和转移。
BACKGROUND: Studies have shown that long non-coding RNA H19 can reduce the tumorigenic ability, proliferation and clonality of gastric cancer cell line MGC-803, and it is unclear whether H19 can regulate gastric cancer stem cells through changing the expression of Sox2, a key marker of tumor stem cells.OBJECTIVE: To study the expression levels of long non-coding RNA H19 in gastric cancer tissues and gastric cancer stem cells, and to explore its effect on the proliferation and invasion of gastric cancer stem cells and its mechanism of action.METHODS: q RT-PCR was used to detect the expression of H19 in lung cancer tissues and paracancerous tissues in 38 cases as well as in gastric cancer stem cells CSC-G and human immortalized normal gastric epithelial cells GES-1. After transfection of H19 siRNA(si-H19) and control(si-NC) for 48 hours by liposome, soft agar cloning assay was used to detect the effect of H19 expression on the stemness of CSC-G cells, MTS assay used to detect the effect of H19 downregulation on CSC-G cell proliferation, and Transwell assay used to detect the effect of H19 downregulation on CSC-G cell metastasis, and western blot assay used to detect the effect of H19 downregulation on Sox2 protein in CSC-G cells.RESULTS AND CONCLUSION:(1) The results of q RT-PCR showed that the expression of H19 in gastric cancer tissues was significantly higher than that in the matched paracancerous tissues, and it was highly expressed in tissues with high tumor T stage and lymph node metastasis(P < 0.05), while the expression of H19 in CSC-G cells was significantly higher than that in GES-1 cells(P < 0.05).(2) After transfection of H19 si RNA, the number of soft agar cloned spheres in CSC-G cells was significantly decreased, the cell proliferation and invasion abilities were inhibited and the expression of Sox2 protein was decreased(P < 0.05). In conclusion, H19 is highly expressed in gastric cancer tissues and gastric cancer stem cells. H19 may promote the proliferation and metastasis of gastric cancer stem cells by acting on the stem cell-associated protein Sox2.Subject headings:
BACKGROUND: Studies have shown that long non-coding RNA H19 can reduce the tumorigenic ability, proliferation and clonality of gastric cancer cell line MGC-803, and it is unclear whether H19 can regulate gastric cancer stem cells through changing the expression of Sox2, a key marker of tumor stem cells.OBJECTIVE: To study the expression levels of long non-coding RNA H19 in gastric cancer tissues and gastric cancer stem cells, and to explore its effect on the proliferation and invasion of gastric cancer stem cells and its mechanism of action.METHODS: q RT-PCR was used to detect the expression of H19 in lung cancer tissues and paracancerous tissues in 38 cases as well as in gastric cancer stem cells CSC-G and human immortalized normal gastric epithelial cells GES-1. After transfection of H19 siRNA(si-H19) and control(si-NC) for 48 hours by liposome, soft agar cloning assay was used to detect the effect of H19 expression on the stemness of CSC-G cells, MTS assay used to detect the effect of H19 downregulation on CSC-G cell proliferation, and Transwell assay used to detect the effect of H19 downregulation on CSC-G cell metastasis, and western blot assay used to detect the effect of H19 downregulation on Sox2 protein in CSC-G cells.RESULTS AND CONCLUSION:(1) The results of q RT-PCR showed that the expression of H19 in gastric cancer tissues was significantly higher than that in the matched paracancerous tissues, and it was highly expressed in tissues with high tumor T stage and lymph node metastasis(P < 0.05), while the expression of H19 in CSC-G cells was significantly higher than that in GES-1 cells(P < 0.05).(2) After transfection of H19 si RNA, the number of soft agar cloned spheres in CSC-G cells was significantly decreased, the cell proliferation and invasion abilities were inhibited and the expression of Sox2 protein was decreased(P < 0.05). In conclusion, H19 is highly expressed in gastric cancer tissues and gastric cancer stem cells. H19 may promote the proliferation and metastasis of gastric cancer stem cells by acting on the stem cell-associated protein Sox2.Subject headings:
引文
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[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015.CA Cancer J Clin.2016;66(2):115-132.
[3]Berger H,Marques MS,Zietlow R,et al.Gastric cancer pathogenesis.Helicobacter.2016;21 Suppl 1:34-38.
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[6]Malhotra S,Freeberg MA,Winans SJ,et al.A Novel Long Non-Coding RNA in the h TERT Promoter Region Regulates h TERT Expression.Noncoding RNA.2017;4(1):E1.
[7]Hagan M,Zhou M,Ashraf M,et al.Long noncoding RNAs and their roles in skeletal muscle fate determination.Noncoding RNA Investig.2017;pii:24.
[8]Pan B,Shi ZJ,Yan JY,et al.Long non-coding RNANONMMUG014387 promotes Schwann cell proliferation after peripheral nerve injury.Neural Regen Res.2017;12(12):2084-2091.
[9]Diamantopoulos MA,Tsiakanikas P,Scorilas A.Non-coding RNAs:the riddle of the transcriptome and their perspectives in cancer.Ann Transl Med.2018;6(12):241.
[10]Zhao M,Wang S,Li Q,et al.MALAT1:A long non-coding RNAhighly associated with human cancers.Oncol Lett.2018;16(1):19-26.
[11]Zhang L,Zhou Y Huang T,et al.The Interplay of Lnc RNA-H19and Its Binding Partners in Physiological Process and Gastric Carcinogenesis.Int J Mol Sci.2017;18(2):E450.
[12]Collette J,Le Bourhis X,Adriaenssens E.Regulation of Human Breast Cancer by the Long Non-Coding RNA H19.Int J Mol Sci.2017;18(11):E2319.
[13]Shima H,Kida K,Adachi S,et al.Lnc RNA H19 is associated with poor prognosis in breast cancer patients and promotes cancer stemness.Breast Cancer Res Treat.2018;170(3):507-516.
[14]Li W,Jiang P,Sun X,et al.Suppressing H19 Modulates Tumorigenicity and Stemness in U251 and U87MG Glioma Cells.Cell Mol Neurobiol.2016;36(8):1219-1227.
[15]钟李锋.长链非编码RNA H19在胃癌干细胞中的表达及功能研究[D].福州:福建医科大学,2015.
[16]白金铭,张炫,乔佳欢,等.胃癌的发病机制及其防治方法[J].吉林医药学院学报,2018,39(4):310-312.
[17]Kanaji S,Suzuki S,Matsuda Y,et al.Recent updates in perioperative chemotherapy and recurrence pattern of gastric cancer.Ann Gastroenterol Surg.2018;2(6):400-405.
[18]Reid PA,Wilson P,Li Y,et al.Current understanding of cancer stem cells:Review of their radiobiology and role in head and neck cancers.Head Neck.2017;39(9):1920-1932.
[19]骆黎静,张宇迪,卢丹,等.肿瘤干细胞学说的应用前景与争议[J].中国临床医生杂志,2018,46(11):1274-1278.
[20]Feng Y,Spezia M,Huang S,et al.Breast cancer development and progression:Risk factors,cancer stem cells,signaling pathways,genomics,and molecular pathogenesis.Genes Dis.2018;5(2):77-106.
[21]Ren J,Ding L,Zhang D,et al.Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19.Theranostics.2018;8(14):3932-3948.
[22]Ding K,Liao Y,Gong D,et al.Effect of long non-coding RNAH19 on oxidative stress and chemotherapy resistance of CD133+cancer stem cells via the MAPK/ERK signaling pathway in hepatocellular carcinoma.Biochem Biophys Res Commun.2018;502(2):194-201.
[23]邹兴斌.胃癌干细胞制备方法及在胃癌侵袭转移中的机制[J].中国老年学杂志,2018,38(12):2968-2971.
[24]Peng X,Liu G,Peng H,et al.SOX4 contributes to TGF-β-induced epithelial-mesenchymal transition and stem cell characteristics of gastric cancer cells.Genes Dis.2017;5(1):49-61.
[25]Chen L,Zhou Y,Li H.Lnc RNA,mi RNA and lncRNA-miRNAinteraction in viral infection.Virus Res.2018;257:25-32.
[26]Chen C,Cui Q,Zhang X,et al.Long non-coding RNAs regulation in adipogenesis and lipid metabolism:Emerging insights in obesity.Cell Signal.2018;51:47-58.
[27]Zarkou V,Galaras A,Giakountis A,et al.Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs.Noncoding RNA Res.2018;3(2):42-53.
[28]Ji D,Zhong X,Jiang X,et al.The role of long non-coding RNAAFAP1-AS1 in human malignant tumors.Pathol Res Pract.2018;214(10):1524-1531.
[29]Li BL,Wan XP.The role of lnc RNAs in the development of endometrial carcinoma.Oncol Lett.2018;16(3):3424-3429.
[30]Brannan CI,Dees EC,Ingram RS,et al.The product of the H19 gene may function as an RNA.Mol Cell Biol.1990;10(1):28-36.
[31]Gabory A,Jammes H,Dandolo L.The H19 locus:role of an imprinted non-coding RNA in growth and development.Bioessays.2010;32(6):473-480.
[32]Ariel I,Sughayer M,Fellig Y,et al.The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma.Mol Pathol.2000;53(6):320-323.
[33]兰霞斌,张浩.长链非编码RNA H19在肿瘤中的研究进展[J].肿瘤学杂志,2017,23(12):1115-1120.
[34]Y?rüker EE,Keskin M,Kulle CB,et al.Diagnostic and prognostic value of circulating lncRNA H19 in gastric cancer.Biomed Rep.2018;9(2):181-186.
[35]Liu G,Xiang T,Wu QF,et al.Long Noncoding RNAH19-Derived mi R-675 Enhances Proliferation and Invasion via RUNX1 in Gastric Cancer Cells.Oncol Res.2016;23(3):99-107.
[36]刘志坤,祝淑钗,苏景伟,等.RNA干扰MDC1基因对食管癌细胞X线照后细胞周期及相关蛋白表达影响[J].中华放射肿瘤学杂志,2015,24(6):708-713.
[37]Ooki A,Dinalankara W,Marchionni L,et al.Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma.Oncogene.2018;37(45):5967-5981.
[38]Zayed H,Petersen I.Stem cell transcription factor SOX2 in synovial sarcoma and other soft tissue tumors.Pathol Res Pract.2018;214(7):1000-1007.
[39]Maehara R,Fujikura K,Takeuchi K,et al.SOX2-silenced squamous cell carcinoma:a highly malignant form of esophageal cancer with SOX2 promoter hypermethylation.Mod Pathol.2018;31(1):83-92.
[40]Fujiki H,Sueoka E,Rawangkan A,et al.Human cancer stem cells are a target for cancer prevention using(-)-epigallocatechin gallate.J Cancer Res Clin Oncol.2017;143(12):2401-2412.
[41]Liu X,Qiao B,Zhao T,et al.Sox2 promotes tumor aggressiveness and epithelial?mesenchymal transition in tongue squamous cell carcinoma.Int J Mol Med.2018;42(3):1418-1426.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015.CA Cancer J Clin.2016;66(2):115-132.
[3]Berger H,Marques MS,Zietlow R,et al.Gastric cancer pathogenesis.Helicobacter.2016;21 Suppl 1:34-38.
[4]Leal MF,Assump??o PP,Smith MC,et al.Searching for Gastric Cancer Biomarkers Through Proteomic Approaches.Journal of Gastroenterology and Hepatology Research.2014;3(3):989-995.
[5]颜涛,王枭雄,王春雷,等.肿瘤干细胞及肿瘤可塑性和异质性的研究进展[J].中国微侵袭神经外科杂志,2017,22(3):142-144.
[6]Malhotra S,Freeberg MA,Winans SJ,et al.A Novel Long Non-Coding RNA in the h TERT Promoter Region Regulates h TERT Expression.Noncoding RNA.2017;4(1):E1.
[7]Hagan M,Zhou M,Ashraf M,et al.Long noncoding RNAs and their roles in skeletal muscle fate determination.Noncoding RNA Investig.2017;pii:24.
[8]Pan B,Shi ZJ,Yan JY,et al.Long non-coding RNANONMMUG014387 promotes Schwann cell proliferation after peripheral nerve injury.Neural Regen Res.2017;12(12):2084-2091.
[9]Diamantopoulos MA,Tsiakanikas P,Scorilas A.Non-coding RNAs:the riddle of the transcriptome and their perspectives in cancer.Ann Transl Med.2018;6(12):241.
[10]Zhao M,Wang S,Li Q,et al.MALAT1:A long non-coding RNAhighly associated with human cancers.Oncol Lett.2018;16(1):19-26.
[11]Zhang L,Zhou Y Huang T,et al.The Interplay of Lnc RNA-H19and Its Binding Partners in Physiological Process and Gastric Carcinogenesis.Int J Mol Sci.2017;18(2):E450.
[12]Collette J,Le Bourhis X,Adriaenssens E.Regulation of Human Breast Cancer by the Long Non-Coding RNA H19.Int J Mol Sci.2017;18(11):E2319.
[13]Shima H,Kida K,Adachi S,et al.Lnc RNA H19 is associated with poor prognosis in breast cancer patients and promotes cancer stemness.Breast Cancer Res Treat.2018;170(3):507-516.
[14]Li W,Jiang P,Sun X,et al.Suppressing H19 Modulates Tumorigenicity and Stemness in U251 and U87MG Glioma Cells.Cell Mol Neurobiol.2016;36(8):1219-1227.
[15]钟李锋.长链非编码RNA H19在胃癌干细胞中的表达及功能研究[D].福州:福建医科大学,2015.
[16]白金铭,张炫,乔佳欢,等.胃癌的发病机制及其防治方法[J].吉林医药学院学报,2018,39(4):310-312.
[17]Kanaji S,Suzuki S,Matsuda Y,et al.Recent updates in perioperative chemotherapy and recurrence pattern of gastric cancer.Ann Gastroenterol Surg.2018;2(6):400-405.
[18]Reid PA,Wilson P,Li Y,et al.Current understanding of cancer stem cells:Review of their radiobiology and role in head and neck cancers.Head Neck.2017;39(9):1920-1932.
[19]骆黎静,张宇迪,卢丹,等.肿瘤干细胞学说的应用前景与争议[J].中国临床医生杂志,2018,46(11):1274-1278.
[20]Feng Y,Spezia M,Huang S,et al.Breast cancer development and progression:Risk factors,cancer stem cells,signaling pathways,genomics,and molecular pathogenesis.Genes Dis.2018;5(2):77-106.
[21]Ren J,Ding L,Zhang D,et al.Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19.Theranostics.2018;8(14):3932-3948.
[22]Ding K,Liao Y,Gong D,et al.Effect of long non-coding RNAH19 on oxidative stress and chemotherapy resistance of CD133+cancer stem cells via the MAPK/ERK signaling pathway in hepatocellular carcinoma.Biochem Biophys Res Commun.2018;502(2):194-201.
[23]邹兴斌.胃癌干细胞制备方法及在胃癌侵袭转移中的机制[J].中国老年学杂志,2018,38(12):2968-2971.
[24]Peng X,Liu G,Peng H,et al.SOX4 contributes to TGF-β-induced epithelial-mesenchymal transition and stem cell characteristics of gastric cancer cells.Genes Dis.2017;5(1):49-61.
[25]Chen L,Zhou Y,Li H.Lnc RNA,mi RNA and lncRNA-miRNAinteraction in viral infection.Virus Res.2018;257:25-32.
[26]Chen C,Cui Q,Zhang X,et al.Long non-coding RNAs regulation in adipogenesis and lipid metabolism:Emerging insights in obesity.Cell Signal.2018;51:47-58.
[27]Zarkou V,Galaras A,Giakountis A,et al.Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs.Noncoding RNA Res.2018;3(2):42-53.
[28]Ji D,Zhong X,Jiang X,et al.The role of long non-coding RNAAFAP1-AS1 in human malignant tumors.Pathol Res Pract.2018;214(10):1524-1531.
[29]Li BL,Wan XP.The role of lnc RNAs in the development of endometrial carcinoma.Oncol Lett.2018;16(3):3424-3429.
[30]Brannan CI,Dees EC,Ingram RS,et al.The product of the H19 gene may function as an RNA.Mol Cell Biol.1990;10(1):28-36.
[31]Gabory A,Jammes H,Dandolo L.The H19 locus:role of an imprinted non-coding RNA in growth and development.Bioessays.2010;32(6):473-480.
[32]Ariel I,Sughayer M,Fellig Y,et al.The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma.Mol Pathol.2000;53(6):320-323.
[33]兰霞斌,张浩.长链非编码RNA H19在肿瘤中的研究进展[J].肿瘤学杂志,2017,23(12):1115-1120.
[34]Y?rüker EE,Keskin M,Kulle CB,et al.Diagnostic and prognostic value of circulating lncRNA H19 in gastric cancer.Biomed Rep.2018;9(2):181-186.
[35]Liu G,Xiang T,Wu QF,et al.Long Noncoding RNAH19-Derived mi R-675 Enhances Proliferation and Invasion via RUNX1 in Gastric Cancer Cells.Oncol Res.2016;23(3):99-107.
[36]刘志坤,祝淑钗,苏景伟,等.RNA干扰MDC1基因对食管癌细胞X线照后细胞周期及相关蛋白表达影响[J].中华放射肿瘤学杂志,2015,24(6):708-713.
[37]Ooki A,Dinalankara W,Marchionni L,et al.Epigenetically regulated PAX6 drives cancer cells toward a stem-like state via GLI-SOX2 signaling axis in lung adenocarcinoma.Oncogene.2018;37(45):5967-5981.
[38]Zayed H,Petersen I.Stem cell transcription factor SOX2 in synovial sarcoma and other soft tissue tumors.Pathol Res Pract.2018;214(7):1000-1007.
[39]Maehara R,Fujikura K,Takeuchi K,et al.SOX2-silenced squamous cell carcinoma:a highly malignant form of esophageal cancer with SOX2 promoter hypermethylation.Mod Pathol.2018;31(1):83-92.
[40]Fujiki H,Sueoka E,Rawangkan A,et al.Human cancer stem cells are a target for cancer prevention using(-)-epigallocatechin gallate.J Cancer Res Clin Oncol.2017;143(12):2401-2412.
[41]Liu X,Qiao B,Zhao T,et al.Sox2 promotes tumor aggressiveness and epithelial?mesenchymal transition in tongue squamous cell carcinoma.Int J Mol Med.2018;42(3):1418-1426.