Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells
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摘要
BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.
BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.
BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.
引文
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3 Llovet JM,Zucman-Rossi J,Pikarsky E,Sangro B,Schwartz M,Sherman M,Gores G.Hepatocellular carcinoma.Nat Rev Dis Primers 2016;2:16018[PMID:27158749 DOI:10.1038/nrdp.2016.18]
4 Tunissiolli NM,Castanhole-Nunes MMU,Biselli-Chicote PM,Pavarino EC,da Silva RF,da Silva RC,Goloni-Bertollo EM.Hepatocellular Carcinoma:A Comprehensive Review of Biomarkers,Clinical Aspects,and Therapy.Asian Pac J Cancer Prev 2017;18:863-872[PMID:28545181 DOI:10.22034/APJCP.2017.18.4.863]
5 Medavaram S,Zhang Y.Emerging therapies in advanced hepatocellular carcinoma.Exp Hematol Oncol2018;7:17[PMID:30087805 DOI:10.1186/s40164-018-0109-6]
6 Kobayashi A,Ito E,Toki T,Kogame K,Takahashi S,Igarashi K,Hayashi N,Yamamoto M.Molecular cloning and functional characterization of a new Cap'n'collar family transcription factor Nrf3.J Biol Chem1999;274:6443-6452[PMID:10037736 DOI:10.1074/jbc.274.10.6443]
7 Chénais B,Derjuga A,Massrieh W,Red-Horse K,Bellingard V,Fisher SJ,Blank V.Functional and placental expression analysis of the human NRF3 transcription factor.Mol Endocrinol 2005;19:125-137[PMID:15388789 DOI:10.1210/me.2003-0379]
8 Zhang Y,Kobayashi A,Yamamoto M,Hayes JD.The Nrf3 transcription factor is a membrane-bound glycoprotein targeted to the endoplasmic reticulum through its N-terminal homology box 1 sequence.JBiol Chem 2009;284:3195-3210[PMID:19047052 DOI:10.1074/jbc.M805337200]
9 Chevillard G,Blank V.NFE2L3(NRF3):The Cinderella of the Cap'n'Collar transcription factors.Cell Mol Life Sci 2011;68:3337-3348[PMID:21687990 DOI:10.1007/s00018-011-0747-x]
10 Chevillard G,Paquet M,Blank V.Nfe2l3(Nrf3)deficiency predisposes mice to T-cell lymphoblastic lymphoma.Blood 2011;117:2005-2008[PMID:21148084 DOI:10.1182/blood-2010-02-271460]
11 Wang C,Saji M,Justiniano SE,Yusof AM,Zhang X,Yu L,Fernández S,Wakely P,La Perle K,Nakanishi H,Pohlman N,Ringel MD.RCAN1-4 is a thyroid cancer growth and metastasis suppressor.JCIInsight 2017;2:e90651[PMID:28289712 DOI:10.1172/jci.insight.90651]
12 Chowdhury AMMA,Katoh H,Hatanaka A,Iwanari H,Nakamura N,Hamakubo T,Natsume T,Waku T,Kobayashi A.Multiple regulatory mechanisms of the biological function of NRF3(NFE2L3)control cancer cell proliferation.Sci Rep 2017;7:12494[PMID:28970512 DOI:10.1038/s41598-017-12675-y]
13 Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative C(T)method.Nat Protoc2008;3:1101-1108[PMID:18546601 DOI:10.1038/nprot.2008.73]
14 Sengupta S,Parikh ND.Biomarker development for hepatocellular carcinoma early detection:Current and future perspectives.Hepat Oncol 2017;4:111-122[PMID:30191058 DOI:10.2217/hep-2017-0019]
15 Scalera A,Tarantino G.Could metabolic syndrome lead to hepatocarcinoma via non-alcoholic fatty liver disease?World J Gastroenterol 2014;20:9217-9228[PMID:25071314 DOI:10.3748/wjg.v20.i28.9217]
16 De Stefano F,Chacon E,Turcios L,Marti F,Gedaly R.Novel biomarkers in hepatocellular carcinoma.Dig Liver Dis 2018;50:1115-1123[PMID:30217732 DOI:10.1016/j.dld.2018.08.019]
17 Siegenthaler B,Defila C,Muzumdar S,Beer HD,Meyer M,Tanner S,Bloch W,Blank V,Sch?fer M,Werner S.Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion.Cell Death Differ 2018;25:1749-1765[PMID:29487353 DOI:10.1038/s41418-018-0074-y]
18 Schwenk RW,Vogel H,Schürmann A.Genetic and epigenetic control of metabolic health.Mol Metab2013;2:337-347[PMID:24327950 DOI:10.1016/j.molmet.2013.09.002]
19 Dongre A,Weinberg RA.New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat Rev Mol Cell Biol 2019;20:69-84[PMID:30459476 DOI:10.1038/s41580-018-0080-4]
20 Prieto-García E,Díaz-García CV,García-Ruiz I,Agulló-Ortu?o MT.Epithelial-to-mesenchymal transition in tumor progression.Med Oncol 2017;34:122[PMID:28560682 DOI:10.1007/s12032-017-0980-8]
21 Gheldof A,Berx G.Cadherins and epithelial-to-mesenchymal transition.Prog Mol Biol Transl Sci 2013;116:317-336[PMID:23481201 DOI:10.1016/B978-0-12-394311-8.00014-5]
22 Wang Y,Shi J,Chai K,Ying X,Zhou BP.The Role of Snail in EMT and Tumorigenesis.Curr Cancer Drug Targets 2013;13:963-972[PMID:24168186 DOI:10.2174/15680096113136660102]
23 Huang BP,Lin CS,Wang CJ,Kao SH.Upregulation of heat shock protein 70 and the differential protein expression induced by tumor necrosis factor-alpha enhances migration and inhibits apoptosis of hepatocellular carcinoma cell HepG2.Int J Med Sci 2017;14:284-293[PMID:28367089 DOI:10.7150/ijms.17861]
24 Jing Y,Sun K,Liu W,Sheng D,Zhao S,Gao L,Wei L.Tumor necrosis factor-αpromotes hepatocellular carcinogenesis through the activation of hepatic progenitor cells.Cancer Lett 2018;434:22-32[PMID:29981431 DOI:10.1016/j.canlet.2018.07.001]
25 Xiao Q,Pepe AE,Wang G,Luo Z,Zhang L,Zeng L,Zhang Z,Hu Y,Ye S,Xu Q.Nrf3-Pla2g7interaction plays an essential role in smooth muscle differentiation from stem cells.Arterioscler Thromb Vasc Biol 2012;32:730-744[PMID:22247257 DOI:10.1161/ATVBAHA.111.243188]
26 Smith MW,Yue ZN,Geiss GK,Sadovnikova NY,Carter VS,Boix L,Lazaro CA,Rosenberg GB,Bumgarner RE,Fausto N,Bruix J,Katze MG.Identification of novel tumor markers in hepatitis C virusassociated hepatocellular carcinoma.Cancer Res 2003;63:859-864[PMID:12591738 DOI:10.1002/cncr.11257]
27 Vainio P,Lehtinen L,Mirtti T,Hilvo M,Sepp?nen-Laakso T,Virtanen J,Sankila A,Nordling S,Lundin J,Rannikko A,Ore?i?M,Kallioniemi O,Iljin K.Phospholipase PLA2G7,associated with aggressive prostate cancer,promotes prostate cancer cell migration and invasion and is inhibited by statins.Oncotarget 2011;2:1176-1190[PMID:22202492 DOI:10.18632/oncotarget.397]
28 Low HB,Png CW,Li C,Wang Y,Wong SB,Zhang Y.Monocyte-derived factors including PLA2G7induced by macrophage-nasopharyngeal carcinoma cell interaction promote tumor cell invasiveness.Oncotarget 2016;7:55473-55490[PMID:27487154 DOI:10.18632/oncotarget.10980]
29 Chowdhury I,Mo Y,Gao L,Kazi A,Fisher AB,Feinstein SI.Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element.Free Radic Biol Med 2009;46:146-153[PMID:18973804 DOI:10.1016/j.freeradbiomed.2008.09.027]
30 Xu X,Lu D,Zhuang R,Wei X,Xie H,Wang C,Zhu Y,Wang J,Zhong C,Zhang X,Wei Q,He Z,Zhou L,Zheng S.The phospholipase A2 activity of peroxiredoxin 6 promotes cancer cell death induced by tumor necrosis factor alpha in hepatocellular carcinoma.Mol Carcinog 2016;55:1299-1308[PMID:26293541 DOI:10.1002/mc.22371]
2 Waller LP,Deshpande V,Pyrsopoulos N.Hepatocellular carcinoma:A comprehensive review.World JHepatol 2015;7:2648-2663[PMID:26609342 DOI:10.4254/wjh.v7.i26.2648]
3 Llovet JM,Zucman-Rossi J,Pikarsky E,Sangro B,Schwartz M,Sherman M,Gores G.Hepatocellular carcinoma.Nat Rev Dis Primers 2016;2:16018[PMID:27158749 DOI:10.1038/nrdp.2016.18]
4 Tunissiolli NM,Castanhole-Nunes MMU,Biselli-Chicote PM,Pavarino EC,da Silva RF,da Silva RC,Goloni-Bertollo EM.Hepatocellular Carcinoma:A Comprehensive Review of Biomarkers,Clinical Aspects,and Therapy.Asian Pac J Cancer Prev 2017;18:863-872[PMID:28545181 DOI:10.22034/APJCP.2017.18.4.863]
5 Medavaram S,Zhang Y.Emerging therapies in advanced hepatocellular carcinoma.Exp Hematol Oncol2018;7:17[PMID:30087805 DOI:10.1186/s40164-018-0109-6]
6 Kobayashi A,Ito E,Toki T,Kogame K,Takahashi S,Igarashi K,Hayashi N,Yamamoto M.Molecular cloning and functional characterization of a new Cap'n'collar family transcription factor Nrf3.J Biol Chem1999;274:6443-6452[PMID:10037736 DOI:10.1074/jbc.274.10.6443]
7 Chénais B,Derjuga A,Massrieh W,Red-Horse K,Bellingard V,Fisher SJ,Blank V.Functional and placental expression analysis of the human NRF3 transcription factor.Mol Endocrinol 2005;19:125-137[PMID:15388789 DOI:10.1210/me.2003-0379]
8 Zhang Y,Kobayashi A,Yamamoto M,Hayes JD.The Nrf3 transcription factor is a membrane-bound glycoprotein targeted to the endoplasmic reticulum through its N-terminal homology box 1 sequence.JBiol Chem 2009;284:3195-3210[PMID:19047052 DOI:10.1074/jbc.M805337200]
9 Chevillard G,Blank V.NFE2L3(NRF3):The Cinderella of the Cap'n'Collar transcription factors.Cell Mol Life Sci 2011;68:3337-3348[PMID:21687990 DOI:10.1007/s00018-011-0747-x]
10 Chevillard G,Paquet M,Blank V.Nfe2l3(Nrf3)deficiency predisposes mice to T-cell lymphoblastic lymphoma.Blood 2011;117:2005-2008[PMID:21148084 DOI:10.1182/blood-2010-02-271460]
11 Wang C,Saji M,Justiniano SE,Yusof AM,Zhang X,Yu L,Fernández S,Wakely P,La Perle K,Nakanishi H,Pohlman N,Ringel MD.RCAN1-4 is a thyroid cancer growth and metastasis suppressor.JCIInsight 2017;2:e90651[PMID:28289712 DOI:10.1172/jci.insight.90651]
12 Chowdhury AMMA,Katoh H,Hatanaka A,Iwanari H,Nakamura N,Hamakubo T,Natsume T,Waku T,Kobayashi A.Multiple regulatory mechanisms of the biological function of NRF3(NFE2L3)control cancer cell proliferation.Sci Rep 2017;7:12494[PMID:28970512 DOI:10.1038/s41598-017-12675-y]
13 Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative C(T)method.Nat Protoc2008;3:1101-1108[PMID:18546601 DOI:10.1038/nprot.2008.73]
14 Sengupta S,Parikh ND.Biomarker development for hepatocellular carcinoma early detection:Current and future perspectives.Hepat Oncol 2017;4:111-122[PMID:30191058 DOI:10.2217/hep-2017-0019]
15 Scalera A,Tarantino G.Could metabolic syndrome lead to hepatocarcinoma via non-alcoholic fatty liver disease?World J Gastroenterol 2014;20:9217-9228[PMID:25071314 DOI:10.3748/wjg.v20.i28.9217]
16 De Stefano F,Chacon E,Turcios L,Marti F,Gedaly R.Novel biomarkers in hepatocellular carcinoma.Dig Liver Dis 2018;50:1115-1123[PMID:30217732 DOI:10.1016/j.dld.2018.08.019]
17 Siegenthaler B,Defila C,Muzumdar S,Beer HD,Meyer M,Tanner S,Bloch W,Blank V,Sch?fer M,Werner S.Nrf3 promotes UV-induced keratinocyte apoptosis through suppression of cell adhesion.Cell Death Differ 2018;25:1749-1765[PMID:29487353 DOI:10.1038/s41418-018-0074-y]
18 Schwenk RW,Vogel H,Schürmann A.Genetic and epigenetic control of metabolic health.Mol Metab2013;2:337-347[PMID:24327950 DOI:10.1016/j.molmet.2013.09.002]
19 Dongre A,Weinberg RA.New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat Rev Mol Cell Biol 2019;20:69-84[PMID:30459476 DOI:10.1038/s41580-018-0080-4]
20 Prieto-García E,Díaz-García CV,García-Ruiz I,Agulló-Ortu?o MT.Epithelial-to-mesenchymal transition in tumor progression.Med Oncol 2017;34:122[PMID:28560682 DOI:10.1007/s12032-017-0980-8]
21 Gheldof A,Berx G.Cadherins and epithelial-to-mesenchymal transition.Prog Mol Biol Transl Sci 2013;116:317-336[PMID:23481201 DOI:10.1016/B978-0-12-394311-8.00014-5]
22 Wang Y,Shi J,Chai K,Ying X,Zhou BP.The Role of Snail in EMT and Tumorigenesis.Curr Cancer Drug Targets 2013;13:963-972[PMID:24168186 DOI:10.2174/15680096113136660102]
23 Huang BP,Lin CS,Wang CJ,Kao SH.Upregulation of heat shock protein 70 and the differential protein expression induced by tumor necrosis factor-alpha enhances migration and inhibits apoptosis of hepatocellular carcinoma cell HepG2.Int J Med Sci 2017;14:284-293[PMID:28367089 DOI:10.7150/ijms.17861]
24 Jing Y,Sun K,Liu W,Sheng D,Zhao S,Gao L,Wei L.Tumor necrosis factor-αpromotes hepatocellular carcinogenesis through the activation of hepatic progenitor cells.Cancer Lett 2018;434:22-32[PMID:29981431 DOI:10.1016/j.canlet.2018.07.001]
25 Xiao Q,Pepe AE,Wang G,Luo Z,Zhang L,Zeng L,Zhang Z,Hu Y,Ye S,Xu Q.Nrf3-Pla2g7interaction plays an essential role in smooth muscle differentiation from stem cells.Arterioscler Thromb Vasc Biol 2012;32:730-744[PMID:22247257 DOI:10.1161/ATVBAHA.111.243188]
26 Smith MW,Yue ZN,Geiss GK,Sadovnikova NY,Carter VS,Boix L,Lazaro CA,Rosenberg GB,Bumgarner RE,Fausto N,Bruix J,Katze MG.Identification of novel tumor markers in hepatitis C virusassociated hepatocellular carcinoma.Cancer Res 2003;63:859-864[PMID:12591738 DOI:10.1002/cncr.11257]
27 Vainio P,Lehtinen L,Mirtti T,Hilvo M,Sepp?nen-Laakso T,Virtanen J,Sankila A,Nordling S,Lundin J,Rannikko A,Ore?i?M,Kallioniemi O,Iljin K.Phospholipase PLA2G7,associated with aggressive prostate cancer,promotes prostate cancer cell migration and invasion and is inhibited by statins.Oncotarget 2011;2:1176-1190[PMID:22202492 DOI:10.18632/oncotarget.397]
28 Low HB,Png CW,Li C,Wang Y,Wong SB,Zhang Y.Monocyte-derived factors including PLA2G7induced by macrophage-nasopharyngeal carcinoma cell interaction promote tumor cell invasiveness.Oncotarget 2016;7:55473-55490[PMID:27487154 DOI:10.18632/oncotarget.10980]
29 Chowdhury I,Mo Y,Gao L,Kazi A,Fisher AB,Feinstein SI.Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element.Free Radic Biol Med 2009;46:146-153[PMID:18973804 DOI:10.1016/j.freeradbiomed.2008.09.027]
30 Xu X,Lu D,Zhuang R,Wei X,Xie H,Wang C,Zhu Y,Wang J,Zhong C,Zhang X,Wei Q,He Z,Zhou L,Zheng S.The phospholipase A2 activity of peroxiredoxin 6 promotes cancer cell death induced by tumor necrosis factor alpha in hepatocellular carcinoma.Mol Carcinog 2016;55:1299-1308[PMID:26293541 DOI:10.1002/mc.22371]