SNHG16通过Wnt/β-catenin信号通路促进肝细胞癌细胞增殖和迁移
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摘要
目的 :检测长链非编码RNA小核仁RNA宿主基因16(small nucleolar RNA host gene 16,SNHG16)在肝细胞癌(hepatocellular carcinoma,HCC)组织及细胞中的表达情况,探讨SNHG16表达调控对HCC细胞增殖和迁移的影响及其分子机制。方法 :采用实时荧光定量PCR法检测38例HCC患者的肝癌及癌旁组织,以及4种肝癌细胞株和正常肝细胞株中SNHG16的表达水平。分析SNHG16表达与HCC患者临床病理特征的关系。将SNHG16过表达或SNHG16-shRNA重组慢病毒分别感染肝癌Hep-3B或SK-Hep-1细胞,采用实时荧光定量PCR法验证细胞中SNHG16表达被调控后,采用CCK-8和Transwell小室法分别检测肝癌细胞增殖和迁移能力的变化,并采用蛋白质印迹法检测细胞中Wnt/β-catenin信号通路关键蛋白表达的变化。通过裸鼠成瘤实验检测SNHG16表达调控对裸鼠体内肝癌细胞成瘤能力的影响。结果 :HCC组织和细胞中SNHG16表达水平分别高于癌旁组织和正常肝细胞(P <0.001,P <0.05),而且HCC组织中SNHG16表达与肿瘤大小(P <0.01)、TNM分期(P <0.01)和谷丙转氨酶表达水平(P <0.05)密切相关。感染SNHG16过表达重组慢病毒后的Hep-3B细胞中SNHG16表达明显上调(P <0.001),细胞增殖和迁移能力明显增强(P值均<0.01),而且细胞中c-myc和β-catenin表达明显上调(P值均<0.01)。感染SNHG16-shRNA重组慢病毒后的SK-Hep-1细胞中SNHG16表达明显下调(P <0.001),细胞增殖和迁移能力明显减弱(P值均<0.001),而且细胞中c-myc和β-catenin表达明显下调(P <0.05,P <0.01)。另外,SNHG16过表达的Hep-3B细胞在裸鼠体内的成瘤能力明显增强(P <0.01),而SNHG16被敲低的SKHep-1细胞在裸鼠体内的成瘤能力明显减弱(P <0.05)。结论 :SNHG16在HCC组织和细胞系中表达水平升高,并可能通过Wnt/β-catenin信号通路促进肝癌细胞的增殖和迁移。
Objective: To investigate the expression of long non-coding RNA(lncRNA) small nucleolar RNA host gene 16(SNHG16) in hepatocellular carcinoma(HCC) tissues and cells, and to explore the effects of SNHG16 expression regulation on the proliferation and migration of HCC cells as well as the underlying molecular mechanisms.Methods: The cancer tissues and adjacent tissues were collected from 38 patients with HCC. The real-time fluorescent quantitative PCR was used to detected the expression of SNHG16 in 38 cases of clinical HCC tissue samples and their adjacent tissues, 4 kinds of HCC cell lines and normal hepatocellular cell line. The relationship between SNHG16 expression and the clinicopathological features of HCC patients was analyzed. The SNHG16 overexpression or SNHG16-shRNA recombinant lentivirus was infected into Hep-3B or SK-Hep-1 cells, respectively. The up-or down-regulation of SNHG16 expression in Hep-3B or SK-Hep-1 cells was verified by real-time fluorescent quantitative PCR. The effects of SNHG16 expression regulation on the proliferation and migration of HCC cells were determined by CCK-8 and Transwell chamber experiments, respectively. The expressions of key proteins in Wnt/β-catenin signaling pathway were detected by Western blotting. Xenograft tumor experiment was used to determine the effect of SNHG16 on the tumorigenic ability of HCC cells in nude mice.Results: The expression level of SNHG16 in HCC tissues and HCC cells was significantly higher than that in the adjacent tissues(P < 0.001) and normal hepatocellular cells(P < 0.05), respectively. The expression of SNHG16 in HCC tissues was associated with tumor size(P < 0.01), TNM stage(P < 0.01) and alanine aminotransaminase(ALT) expression level(P < 0.05). After the infection with SNHG16 overexpression recombinant lentivirus, the expression of SNHG16 was significantly up-regulated in Hep-3B cells(P < 0.001), the proliferation and migration of Hep-3B cells were significantly promoted(both P < 0.01), and the expressions of β-catenin and c-myc proteins were up-regulated(both P < 0.01). After the infection with SNHG16-shRNA recombinant lentivirus, the expression of SNHG16 was dramatically downregulated in SK-Hep-1 cells(P < 0.001), the proliferation and migration of SK-Hep-1 cells were significantly inhibited(both P < 0.001), and the expressions of β-catenin(P < 0.05) and c-myc(P < 0.01) proteins were down-regulated. In addition, the tumorigenic ability of Hep-3B cells with SNHG16 over-expression was significantly enhanced in nude mice(P < 0.01), while the tumorigenic ability of SK-Hep-1 cells with SNHG16 knock-down was significantly weakened in nude mice(P < 0.05).Conclusion: SNHG16 is highly expressed in HCC tissues and cell lines, and may promote the proliferation and migration of HCC cells through Wnt/β-catenin signaling pathway.
Objective: To investigate the expression of long non-coding RNA(lncRNA) small nucleolar RNA host gene 16(SNHG16) in hepatocellular carcinoma(HCC) tissues and cells, and to explore the effects of SNHG16 expression regulation on the proliferation and migration of HCC cells as well as the underlying molecular mechanisms.Methods: The cancer tissues and adjacent tissues were collected from 38 patients with HCC. The real-time fluorescent quantitative PCR was used to detected the expression of SNHG16 in 38 cases of clinical HCC tissue samples and their adjacent tissues, 4 kinds of HCC cell lines and normal hepatocellular cell line. The relationship between SNHG16 expression and the clinicopathological features of HCC patients was analyzed. The SNHG16 overexpression or SNHG16-shRNA recombinant lentivirus was infected into Hep-3B or SK-Hep-1 cells, respectively. The up-or down-regulation of SNHG16 expression in Hep-3B or SK-Hep-1 cells was verified by real-time fluorescent quantitative PCR. The effects of SNHG16 expression regulation on the proliferation and migration of HCC cells were determined by CCK-8 and Transwell chamber experiments, respectively. The expressions of key proteins in Wnt/β-catenin signaling pathway were detected by Western blotting. Xenograft tumor experiment was used to determine the effect of SNHG16 on the tumorigenic ability of HCC cells in nude mice.Results: The expression level of SNHG16 in HCC tissues and HCC cells was significantly higher than that in the adjacent tissues(P < 0.001) and normal hepatocellular cells(P < 0.05), respectively. The expression of SNHG16 in HCC tissues was associated with tumor size(P < 0.01), TNM stage(P < 0.01) and alanine aminotransaminase(ALT) expression level(P < 0.05). After the infection with SNHG16 overexpression recombinant lentivirus, the expression of SNHG16 was significantly up-regulated in Hep-3B cells(P < 0.001), the proliferation and migration of Hep-3B cells were significantly promoted(both P < 0.01), and the expressions of β-catenin and c-myc proteins were up-regulated(both P < 0.01). After the infection with SNHG16-shRNA recombinant lentivirus, the expression of SNHG16 was dramatically downregulated in SK-Hep-1 cells(P < 0.001), the proliferation and migration of SK-Hep-1 cells were significantly inhibited(both P < 0.001), and the expressions of β-catenin(P < 0.05) and c-myc(P < 0.01) proteins were down-regulated. In addition, the tumorigenic ability of Hep-3B cells with SNHG16 over-expression was significantly enhanced in nude mice(P < 0.01), while the tumorigenic ability of SK-Hep-1 cells with SNHG16 knock-down was significantly weakened in nude mice(P < 0.05).Conclusion: SNHG16 is highly expressed in HCC tissues and cell lines, and may promote the proliferation and migration of HCC cells through Wnt/β-catenin signaling pathway.
引文
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[18]ZHU H,ZENG Y,ZHOU CC,et al.SNHG16/miR-216-5p/ZEB1 signal pathway contributes to the tumorigenesis of cervical cancer cells[J].Arch Biochem Biophys,2018,637:1-8.
[19]WANG Y,HE L,DU Y,et al.The long noncoding RNA lncTCF7 promotes selfrenewal of human liver cancer stem cells through activation of Wnt signaling[J].Cell Stem Cell,2015,16(4):413-425.
[20]HAN GH,LU KJ,WANG P,et al.LncRNASNHG16 predicts poor prognosis in ESCC and promotes cell proliferation and invasion by regulating Wnt/β-catenin signaling pathway[J].Eur Rev Med Pharmacol Sci,2018,22(12):3795-3803.
[21]YANG XS,WANG GX,LUO L.Long noncoding RNA SNHG16 promotes cell growth and metastasis in ovarian cancer[J].Eur Rev Med Pharmacol Sci,2018,22(3):616-622.
[22]LI B,YU F,WU F,et al.EZH2 impairs human dental pulp cell mineralization via the Wnt/β-catenin pathway[J].J Dent Res,2018,97(5):571-579.
[23]SEBIO A,KAHN M,LENZ HJ.The potential of targeting Wnt/beta-catenin in colon cancer[J].Expert Opin Ther Targets,2014,18(6):611-615.
[24]RUSSELL JO,MONGA SP.Wnt/β-catenin signaling in liver development,homeostasis,and pathobiology[J].Annu Rev Pathol,2018,13:351-378.
[25]刘莉娟,邢燕,岳青芬.PDCD4通过Wnt/β-catenin信号通路抑制宫颈癌细胞增殖的基础研究[J].癌症进展,2018,16(9):1099-1102.
[26]彭伟辉,黄江生,段伦喜,等.miRNA-145调控靶基因c-Myc抑制胃癌增殖、侵袭[J].医学研究杂志,2018,47(5):60-64.
[27]ATTALLAH AM,EL-FAR M,ABDELRAZEKMA,et al.Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients[J].Br J Biomed Sci,2017,74(4):170-175.
[2]FORNER A,LLOVET J M,BRUIX J.Hepatocellular carcinoma[J].Lancet,2012,379(9822):1245-1255.
[3]GHOURI YA,MIAN I,ROWE JH.Review of hepatocellular carcinoma:Epidemiology,etiology,and carcinogenesis[J].J Carcinog,2017,6:1.
[4]KLINGENBERG M,MATSUDA A,DIEDERICHSS,et al.Non-coding RNA in hepatocellular carcinoma:Mechanisms,biomarkers and therapeutic targets[J].J Hepatol,2017,67(3):603-618.
[5]WANG Y,HE L,DU Y,et al.The long noncoding RNA lncTCF7 promotes selfrenewal of human liver cancer stem cells through activation of Wnt signaling[J].Cell Stem Cell,2015,16(4):413-425.
[6]ZHANG A,ZHANG J,KAIPAINEN A,et al.Long non-coding RNA:A newly deciphered"code"in prostate cancer[J].Cancer Lett,2016,375(2):323-330.
[7]KUNEJ T,OBSTETER J,POGACAR Z,et al.The decalog of long non-coding RNAinvolvement in cancer diagnosis and monitoring[J].Crit Rev Clin Lab Sci,2014,51(6):344-357.
[8]HUANG G,KANG Y,HUANG Z,et al.Identification and characterization of long non-coding RNAs in osteogenic differentiation of human adipose-derived stem cells[J].Cell Physiol Biochem,2017,42(3):1037-1050.
[9]GUO G,KANG Q,ZHU X,et al.A long noncoding RNA critically regulates BcrAbl-mediated cellular transformation by acting as a competitive endogenous RNA[J].Oncogene,2015,34(14):1768-1779.
[10]CAI C,HUO Q,WANG X,et al.SNHG16contributes to breast cancer cell migration by competitively binding miR-98 with E2F5[J].Biochem Biophys Res Commun,2017,485(2):272-278.
[11]CHRISTENSEN LL,TTUE K,HAMILTON MP,et al.SNHG16 is regulated by the Wnt pathway in colorectal cancer and affects genes involved in lipid metabolism[J].Mol Oncol,2016,10(8):1266-1282.
[12]LU YF,CAI XL,LI ZZ,et al.LncRNA SNHG16functions as an oncogene by sponging miR-4518 and up-regulating PRMT5expression in glioma[J].Cell Physiol Biochem,2018,45(5):1975-1985.
[13]宋丹,王孜尧,黄平.微RNA-186在人肝细胞肝癌中的表达及作用[J].肿瘤,2017,37(7):750-761.
[14]YANG Y,CHEN L,GU J,et al.Recurrently deregulated lncRNAs in hepatocellular carcinoma[J].Nat Commun,2017,8:14421.
[15]THOMSON DW,DINGER ME.Endogenous microRNA sponges:evidence and controversy[J].Nat Rev Genet,2016,17(5):272-283.
[16]YUAN J,YUE H,ZHANG M,et al.Transcriptional profiling analysis and functional prediction of long noncoding RNAs in cancer[J].Oncotarget,2016,7(7):8131-8142.
[17]LIAN D,AMIN B,DU D,et al.Enhanced expression of the long non-coding RNASNHG16 contributes to gastric cancer progression and metastasis[J].Cancer Biomark,2017,21(1):151-160.
[18]ZHU H,ZENG Y,ZHOU CC,et al.SNHG16/miR-216-5p/ZEB1 signal pathway contributes to the tumorigenesis of cervical cancer cells[J].Arch Biochem Biophys,2018,637:1-8.
[19]WANG Y,HE L,DU Y,et al.The long noncoding RNA lncTCF7 promotes selfrenewal of human liver cancer stem cells through activation of Wnt signaling[J].Cell Stem Cell,2015,16(4):413-425.
[20]HAN GH,LU KJ,WANG P,et al.LncRNASNHG16 predicts poor prognosis in ESCC and promotes cell proliferation and invasion by regulating Wnt/β-catenin signaling pathway[J].Eur Rev Med Pharmacol Sci,2018,22(12):3795-3803.
[21]YANG XS,WANG GX,LUO L.Long noncoding RNA SNHG16 promotes cell growth and metastasis in ovarian cancer[J].Eur Rev Med Pharmacol Sci,2018,22(3):616-622.
[22]LI B,YU F,WU F,et al.EZH2 impairs human dental pulp cell mineralization via the Wnt/β-catenin pathway[J].J Dent Res,2018,97(5):571-579.
[23]SEBIO A,KAHN M,LENZ HJ.The potential of targeting Wnt/beta-catenin in colon cancer[J].Expert Opin Ther Targets,2014,18(6):611-615.
[24]RUSSELL JO,MONGA SP.Wnt/β-catenin signaling in liver development,homeostasis,and pathobiology[J].Annu Rev Pathol,2018,13:351-378.
[25]刘莉娟,邢燕,岳青芬.PDCD4通过Wnt/β-catenin信号通路抑制宫颈癌细胞增殖的基础研究[J].癌症进展,2018,16(9):1099-1102.
[26]彭伟辉,黄江生,段伦喜,等.miRNA-145调控靶基因c-Myc抑制胃癌增殖、侵袭[J].医学研究杂志,2018,47(5):60-64.
[27]ATTALLAH AM,EL-FAR M,ABDELRAZEKMA,et al.Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients[J].Br J Biomed Sci,2017,74(4):170-175.