Integrin β4 induced epithelial-to-mesenchymal transition involves miR-383 mediated regulation of GATA6 levels.
Humans
beta Catenin
/ genetics
Cell Line, Tumor
Cell Movement
Epithelial-Mesenchymal Transition
GATA6 Transcription Factor
/ genetics
Gene Expression Regulation, Neoplastic
HeLa Cells
Integrin beta4
/ genetics
MicroRNAs
/ genetics
Proto-Oncogene Proteins c-akt
/ genetics
RNA, Small Interfering
/ metabolism
Epithelial-to-mesenchymal transition
GATA6
ITGB4 signaling
miR-383
β-catenin
Journal
Molecular biology reports
ISSN: 1573-4978
Titre abrégé: Mol Biol Rep
Pays: Netherlands
ID NLM: 0403234
Informations de publication
Date de publication:
Oct 2023
Oct 2023
Historique:
received:
27
05
2023
accepted:
16
07
2023
medline:
23
10
2023
pubmed:
1
9
2023
entrez:
1
9
2023
Statut:
ppublish
Résumé
The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin β4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT. The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. β-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and β catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA. The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/β-catenin.
Sections du résumé
BACKGROUND
BACKGROUND
The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin β4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT.
METHODS AND RESULTS
RESULTS
The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. β-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and β catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA.
CONCLUSION
CONCLUSIONS
The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/β-catenin.
Identifiants
pubmed: 37656269
doi: 10.1007/s11033-023-08682-0
pii: 10.1007/s11033-023-08682-0
doi:
Substances chimiques
beta Catenin
0
GATA6 protein, human
0
GATA6 Transcription Factor
0
Integrin beta4
0
MicroRNAs
0
MIRN383 microRNA, human
0
Proto-Oncogene Proteins c-akt
EC 2.7.11.1
RNA, Small Interfering
0
ITGB4 protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
8623-8637Subventions
Organisme : Department of Biotechnology, Ministry of Science and Technology, India
ID : 6242-P48/RGCB/PMD/DBT/VBSK/2015
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature B.V.
Références
Grigore AD, Jolly MK, Jia D, Farach-Carson MC, Levine H (2016) Tumor budding: the name is EMT. Partial EMT. J Clin Med 5:51. https://doi.org/10.3390/jcm5050051
doi: 10.3390/jcm5050051
pubmed: 27136592
pmcid: 4882480
Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat 154:8–20. https://doi.org/10.1159/000147748
doi: 10.1159/000147748
pubmed: 8714286
Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454. https://doi.org/10.1038/nrc822
doi: 10.1038/nrc822
pubmed: 12189386
Kalluri R, Neilson EG (2003) Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 112:1776–1784. https://doi.org/10.1172/JCI20530
doi: 10.1172/JCI20530
pubmed: 14679171
pmcid: 297008
Yoshimatsu Y, Watabe T (2011) Roles of TGF-β signals in endothelial-mesenchymal transition during cardiac fibrosis. Int J Inflam 2011:724080. https://doi.org/10.4061/2011/724080
doi: 10.4061/2011/724080
pubmed: 22187661
pmcid: 3235483
Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7:415–428. https://doi.org/10.1038/nrc2131
doi: 10.1038/nrc2131
pubmed: 17508028
Wang W, Wang Z, Tian D, Zeng X, Liu Y, Fu Q, Liang A, Zhang Y, Gao Q, Cheng J, Wang Y (2018) Integrin β3 mediates the endothelial-to-mesenchymal transition via the Notch Pathway. Cell Physiol Biochem 49:985–997. https://doi.org/10.1159/000493229
doi: 10.1159/000493229
pubmed: 30196283
Li XL, Liu L, Li DD, He YP, Guo LH, Sun LP, Liu LN, Xu HX, Zhang XP (2017) ITGB4 promotes cell invasion and epithelial-mesenchymal transition through the modulation of slug expression in hepatocellular carcinoma. Sci Rep 7:40464. https://doi.org/10.1038/srep40464
Masugi Y, Yamazaki K, Emoto K, Effendi K, Tsujikawa H, Kitago M, Itano O, Kitagawa Y, Sakamoto M (2015) Upregulation of ITGB4 promotes epithelial-mesenchymal transition and is a novel prognostic marker in pancreatic ductal adenocarcinoma. Lab Invest 95:308–319. https://doi.org/10.1038/labinvest.2014.166
doi: 10.1038/labinvest.2014.166
pubmed: 25599535
Gerson KD, Maddula VS, Seligmann BE, Shearstone JR, Khan A, Mercurio AM (2012) Effects of β4 integrin expression on microRNA patterns in breast cancer. Biol Open 1:658–666. https://doi.org/10.1242/bio.20121628
doi: 10.1242/bio.20121628
pubmed: 23213459
pmcid: 3507297
Chen Y, Zhang J, Wang H, Zhao J, Xu C, Du Y, Luo X, Zheng F, Liu R, Zhang H, Ma D (2012) miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10. BMC Cancer 12:111. https://doi.org/10.1186/1471-2407-12-111
doi: 10.1186/1471-2407-12-111
pubmed: 22439757
pmcid: 3350382
Zhang C, Ge S, Hu C, Yang N, Zhang J (2013) MiRNA-218, a new regulator of HMGB1, suppresses cell migration and invasion in non-small cell lung cancer. Acta Biochim Biophys Sin 45:1055–1061. https://doi.org/10.1093/abbs/gmt109
doi: 10.1093/abbs/gmt109
pubmed: 24247270
Edatt L, Maurya AK, Raji G, Kunhiraman H, Kumar S (2018) MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin-1 dependent mechanism. J Cell Physiol 233:238–248. https://doi.org/10.1002/jcp.25870
doi: 10.1002/jcp.25870
pubmed: 28233301
Cha ST, Chen PS, Johansson G, Chu CY, Wang MY, Jeng YM, Yu SL, Chen JS, Chang KJ, Jee SH, Tan CT, Lin MT, Kuo ML (2010) MicroRNA-519c suppresses hypoxia-inducible factor-1α expression and tumor angiogenesis. Cancer Res 70:2675–2685. https://doi.org/10.1158/0008-5472.CAN-09-2448
doi: 10.1158/0008-5472.CAN-09-2448
pubmed: 20233879
Fang JH, Zhou HC, Zeng C, Yang J, Liu Y, Huang X, Zhang JP, Guan XY, Zhuang SM (2011) MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression. Hepatology 54:1729–1740. https://doi.org/10.1002/hep.24577
doi: 10.1002/hep.24577
pubmed: 21793034
Sruthi TV, Edatt L, Raji GR, Kunhiraman H, Shankar SS, Shankar V, Ramachandran V, Poyyakkara A, Kumar SVB (2018) Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis. J Cell Physiol 233:3498–3514. https://doi.org/10.1002/jcp.26202
doi: 10.1002/jcp.26202
pubmed: 28929578
Hao M, Zhang L, An G, Sui W, Yu Z, Zou D, Xu Y, Chang H, Qiu L (2011) Suppressing miRNA-15a/-16 expression by interleukin-6 enhances drug-resistance in myeloma cells. J Hematol Oncol 4:1–3. https://doi.org/10.1186/1756-8722-4-37
doi: 10.1186/1756-8722-4-37
Raji GR, Sruthi TV, Edatt L, Haritha K, Sharath Shankar S, Sameer Kumar VB (2017) Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin. Cell Signal 38:146–158. https://doi.org/10.1016/j.cellsig.2017.07.005
doi: 10.1016/j.cellsig.2017.07.005
pubmed: 28709644
Raji GR, Poyyakkara A, Krishnan AK, Maurya AK, Changmai U, Shankar SS, Kumar VBS (2021) Horizontal transfer of miR-643 from cisplatin-resistant cells confers Chemoresistance to Recipient Drug-Sensitive cells by targeting APOL6. Cells 10:1341. https://doi.org/10.3390/cells10061341
doi: 10.3390/cells10061341
pubmed: 34071504
pmcid: 8229894
Li LQ, Yang Y, Chen H, Zhang L, Pan D, Xie WJ (2016) MicroRNA-181b inhibits glycolysis in gastric cancer cells via targeting hexokinase 2 gene. Cancer Biomarkers 17:75–81. https://doi.org/10.3233/CBM-160619
doi: 10.3233/CBM-160619
pubmed: 27314295
Zhu W, Huang Y, Pan Q, Xiang P, Xie N, Yu H (2017) MicroRNA-98 suppress Warburg effect by targeting HK2 in colon cancer cells. Dig Dis Sci 62:660–668. https://doi.org/10.1007/s10620-016-4418-5
doi: 10.1007/s10620-016-4418-5
pubmed: 28025745
Poyyakkara A, Raji GR, Kunhiraman H, Edatt L, Kumar SVB (2018) ER stress mediated regulation of miR23a confer hela cells better adaptability to utilize glycolytic pathway. J Cell Biochem 119:4907–4917. https://doi.org/10.1002/jcb.26718
doi: 10.1002/jcb.26718
pubmed: 29377281
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297. https://doi.org/10.1016/s0092-8674(04)00045-5
doi: 10.1016/s0092-8674(04)00045-5
pubmed: 14744438
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. https://doi.org/10.1016/j.cell.2009.01.002
doi: 10.1016/j.cell.2009.01.002
pubmed: 19167326
pmcid: 3794896
Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874. https://doi.org/10.1016/0019-2791(71)90454-x
doi: 10.1016/0019-2791(71)90454-x
pubmed: 5135623
Sys GM, Lapeire L, Stevens N, Favoreel H, Forsyth R, Bracke M, De Wever O (2013) The in ovo CAM-assay as a xenograft model for sarcoma. J Vis Exp 77:e50522. https://doi.org/10.3791/50522
doi: 10.3791/50522
Kunhiraman H, Edatt L, Thekkeveedu S, Poyyakkara A, Raveendran V, Kiran MS, Sudhakaran P, Kumar SV (2016) 2-Deoxy glucose modulates expression and biological activity of VEGF in a SIRT-1 dependent mechanism. J Cell Biochem 118:252–262. https://doi.org/10.1002/jcb.25629
doi: 10.1002/jcb.25629
pubmed: 27302189
Yang X, Li L, Huang Q, Xu W, Cai X, Zhang J, Yan W, Song D, Liu T, Zhou W, Li Z, Yang C, Dang Y, Xiao J (2015) Wnt signaling through Snail1 and Zeb1 regulates bone metastasis in lung cancer. Am J Cancer Res 5:748–755
pubmed: 25973312
pmcid: 4396030
Gonzalez DM, Medici D (2014) Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 7:re8. https://doi.org/10.1126/scisignal.2005189
doi: 10.1126/scisignal.2005189
pubmed: 25249658
pmcid: 4372086
Zaravinos A (2015) The Regulatory Role of MicroRNAs in EMT and Cancer. J Oncol 2015:865816. https://doi.org/10.1155/2015/865816
Han RL, Wang FP, Zhang PA, Zhou XY, Li Y (2017) miR-383 inhibits ovarian cancer cell proliferation, invasion and aerobic glycolysis by targeting LDHA. Neoplasma 64:244–252. https://doi.org/10.4149/neo_2017_211
doi: 10.4149/neo_2017_211
pubmed: 28043152
Chen L, Guan H, Gu C, Cao Y, Shao J, Wang F (2016) miR-383 inhibits hepatocellular carcinoma cell proliferation via targeting APRIL. Tumour Biol 37:2497–2507. https://doi.org/10.1007/s13277-015-4071-1
doi: 10.1007/s13277-015-4071-1
pubmed: 26385772
Li KK, Pang JC, Lau KM, Zhou L, Mao Y, Wang Y, Poon WS, Ng HK (2013) MiR-383 is downregulated in medulloblastoma and targets peroxiredoxin 3 (PRDX3). Brain Pathol 23:413–425. https://doi.org/10.1111/bpa.12014
doi: 10.1111/bpa.12014
pubmed: 23227829
pmcid: 8029115
Xu Z, Zeng X, Tian D, Xu H, Cai Q, Wang J, Chen Q (2014) MicroRNA-383 inhibits anchorage-independent growth and induces cell cycle arrest of glioma cells by targeting CCND1. Biochem Biophys Res Commun 453:833–838. https://doi.org/10.1016/j.bbrc.2014.10.047
doi: 10.1016/j.bbrc.2014.10.047
pubmed: 25450356
He Z, Cen D, Luo X, Li D, Li P, Liang L, Meng Z (2013) Downregulation of miR-383 promotes glioma cell invasion by targeting insulin-like growth factor 1 receptor. Med Oncol 30:557. https://doi.org/10.1007/s12032-013-0557-0
doi: 10.1007/s12032-013-0557-0
pubmed: 23564324
Han S, Cao C, Tang T, Lu C, Xu J, Wang S, Xue L, Zhang X, Li M (2015) ROBO3 promotes growth and metastasis of pancreatic carcinoma. Cancer Lett 366:61–70. https://doi.org/10.1016/j.canlet.2015.06.004
doi: 10.1016/j.canlet.2015.06.004
pubmed: 26070964
Ko LJ, Engel JD (1993) DNA-binding specificities of the GATA transcription factor family. Mol Cell Biol 13:4011–4022. https://doi.org/10.1128/mcb.13.7.4011-4022.1993
doi: 10.1128/mcb.13.7.4011-4022.1993
pubmed: 8321208
pmcid: 359950
Song Y, Tian T, Fu X, Wang W, Li S, Shi T, Suo A, Ruan Z, Guo H, Yao Y (2015) GATA6 is overexpressed in breast cancer and promotes breast cancer cell epithelial-mesenchymal transition by upregulating slug expression. Exp Mol Pathol 99:617–627. https://doi.org/10.1016/j.yexmp.2015.10.005
doi: 10.1016/j.yexmp.2015.10.005
pubmed: 26505174
Campbell K, Whissell G, Franch-Marro X, Batlle E, Casanova J (2011) Specific GATA factors act as conserved inducers of an endodermal-EMT. Dev Cell 21(6):1051–1061. https://doi.org/10.1016/j.devcel.2011.10.005
doi: 10.1016/j.devcel.2011.10.005
pubmed: 22172671
Martinelli P, Carrillo-de Santa Pau E, Cox T, Sainz B Jr, Dusetti N, Greenhalf W, Rinaldi L, Costello E, Ghaneh P, Malats N, Büchler M, Pajic M, Biankin AV, Iovanna J, Neoptolemos J, Real FX (2017) GATA6 regulates EMT and tumour dissemination, and is a marker of response to adjuvant chemotherapy in pancreatic cancer. Gut 66:1665–1676. https://doi.org/10.1136/gutjnl-2015-311256
doi: 10.1136/gutjnl-2015-311256
pubmed: 27325420
Zhong Y, Wang Z, Fu B, Pan F, Yachida S, Dhara M, Albesiano E, Li L, Naito Y, Vilardell F, Cummings C, Martinelli P, Li A, Yonescu R, Ma Q, Griffin CA, Real FX, Iacobuzio-Donahue CA (2011) GATA6 activates wnt signaling in pancreatic cancer by negatively regulating the wnt antagonist Dickkopf-1. PLoS ONE 6:e22129. https://doi.org/10.1371/journal.pone.0022129
doi: 10.1371/journal.pone.0022129
pubmed: 21811562
pmcid: 3139620
Li M, Pathak RR, Lopez-Rivera E, Friedman SL, Aguirre-Ghiso JA, Sikora AG (2015) The in Ovo Chick Chorioallantoic membrane (CAM) assay as an efficient xenograft model of Hepatocellular Carcinoma. J Vis Exp 104:52411. https://doi.org/10.3791/52411
doi: 10.3791/52411
Vähätalo R, Asikainen TM, Karikoski R, Kinnula VL, White CW, Andersson S, Heikinheimo M, Myllärniemi M (2011) Expression of transcription factor GATA-6 in alveolar epithelial cells is linked to neonatal lung disease. Neonatology 99:231–240. https://doi.org/10.1159/000317827
doi: 10.1159/000317827
pubmed: 21071980
Jung SH, Lee M, Park HA, Lee HC, Kang D, Hwang HJ, Park C, Yu DM, Jung YR, Hong MN, Kim YN, Park HJ, Ko YG, Lee JS (2019) Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues. Cell Death Differ 26:245–259. https://doi.org/10.1038/s41418-018-0114-7
doi: 10.1038/s41418-018-0114-7
pubmed: 29786073
Leng C, Zhang ZG, Chen WX, Luo HP, Song J, Dong W, Zhu XR, Chen XP, Liang HF, Zhang BX (2016) An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK-AKT pathway. Cancer Lett 376:188–196. https://doi.org/10.1016/j.canlet.2016.03.023
doi: 10.1016/j.canlet.2016.03.023
pubmed: 26996299