Regulation of Smad4 by miRNA-196a-5p influences gastric cancer stem cell characteristics

doi:10.3969/j.issn.2095-4344.0907

Abstract

Abstract:

BACKGROUND: miRNA-196a is a recently discovered microRNAs biotarget molecule associated with various tumors such as breast cancer and cervical cancer. Studies have confirmed that miRNA-196a-5p is upregulated in both gastric cancer cell line and gastric cancer tissues. However, little is known about the function and mechanism of miRNA-196a-5p in gastric cancer stem cells.
OBJECTIVE: To analyze the function of miRNA-196a-5p in the invasion of gastric cancer stem cells and to investigate the possible mechanism.
METHODS: CD44⁺ gastric cancer stem cells were sorted from the gastric cancer BGC-823 cell line using flow cytometry. The expression of miRNA-196a-5p was detected by quantitative real-time PCR (qRT-PCR) and the cell invasion was determined by Transwell assay. With reference to the specification of Lipofectamine 2000m kit, miRNA-196a-5p inhibitor (miRNA-196a-5p INH) was transferred into the sorted cells. Regulation of Smad4 gene by the miRNA-196a-5p was validated by dual-luciferase reporter gene assay. Expression of epithelial-mesenchymal transition-associated proteins (E-cadherin, N-cadherin and Vimentin) and Smad4 protein was evaluated by western blot assay.
RESULTS AND CONCLUSION: The miRNA-196a-5p was up-regulated in the gastric cancer stem cells (P < 0.05). Inhibiting the miRNA-196a-5p expression could reduce the invasion ability of gastric cancer stem cells (P < 0.05), increase the E-cadherin protein expression (P < 0.05), decrease the N-cadherin and Vimentin protein expression (P < 0.05), and enhance the Smad4 protein expression (P < 0.05). Therefore, miR-196a-5p has a key role in epithelial-mesenchymal transition and invasion by targeting Smad4 in the gastric cancer stem cells. miR-196a-5p may serve as a potential target for gastric cancer therapy.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stomach Neoplasms, Neoplasm Invasiveness, Neoplastic Stem Cells, MicroRNAs, Smad4 Protein, Tissue Engineering

CLC Number:

R394.2

Guo Rui, Dai Jian-feng, Luo Yu-ming, Zhao Hai-ming, Tang Peng. Regulation of Smad4 by miRNA-196a-5p influences gastric cancer stem cell characteristics[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3310-3315.

Figures/Tables 1

2.1 胃癌干细胞的分选采用流式细胞术分选胃癌BGC-823细胞系中的肿瘤干细胞，分选前后CD44+细胞百分比分别为(3.58±0.95)%和(94.65±6.47)%，差异有显著性意义(t=-24.121，P=0.000)，成功分选得到胃癌干细胞。 2.2 胃癌干细胞的侵袭能力增强 Transwell实验结果显示，CD44+细胞的侵袭细胞数为(169.54±17.24)个，显著高于CD44-细胞(86.49±9.37)个，差异有显著性意义(t=7.331，P=0.001)，见图1。 2.3 miRNA-196a-5p调节胃癌干细胞的侵袭能力 Real-time PCR结果显示，CD44+细胞中miRNA-196a-5p相对表达量(4.28±0.59)显著高于CD44-细胞(1.13±0.43)，差异有显著性意义(t=7.473，P=0.001)。 Transwell实验结果显示，CD44++miRNA-196a-5p INH组的侵袭细胞数(75.36±9.24)个，明显低于CD44++NC组(175.64±14.26)个，差异有显著性意义(t=11.385，P=0.000)，而CD44++miRNA-196a-5p INH组与CD44-+NC组[(71.65±6.85)个]之间的侵袭细胞数差异无显著性意义(t=0.559，P=0.303)，见图2。 Western blotting实验结果显示，CD44++miRNA- 196a-5p INH组E-cadherin蛋白相对表达量(0.41±0.05)高于CD44++NC细胞(0.11±0.02)，差异有显著性意义(t=9.649，P=0.003)，CD44++miRNA-196a-5p INH组与CD44-+NC组(0.43±0.07)之间差异无显著性意义(t=-0.643，P=0.278)，见图3；CD44++miRNA-196a-5p INH组N-cadherin蛋白相对表达量(0.09±0.02)低于CD44++NC组(0.22±0.04)，差异有显著性意义(t=-5.422，P=0.003)，CD44++miRNA-196a-5p INH组与CD44-+NC组(0.09± 0.03)之间差异无显著性意义(t=-0.480，P=0.328)，见图3；CD44++miRNA-196a-5p INH组中Vimentin蛋白相对表达量(0.13±0.04)低于CD44++NC组(0.89±0.19)，差异有显著性意义(t=-6.780，P=0.001)，CD44++miRNA-196a-5p INH组与CD44-+NC组(0.12±0.03)之间差异无显著性意义(t=0.346，P=0.373)，见图3。 2.4 Smad4是miRNA-196a-5p靶基因生物信息学软件(www.targetscan.org，www.microrna.org)分析结果显示，miRNA-196a-5p与Smad4 mRNA 3’-UTR区域具有潜在结合位点，见图4。Western blotting实验结果显示，CD44++miRNA-196a-5p INH组Smad4蛋白相对表达量(2.06±0.29)高于CD44++NC组(0.38±0.06)，差异有显著性意义(t=9.826，P=0.000)，CD44++miRNA-196a-5p INH组与CD44-+NC组(1.98±0.34)之间差异无显著性意义(t=0.310，P=0.386)，见图5。双荧光素酶实验结果显示，miRNA-196a-5p mimics+psicheck-Smad4-wt细胞的相对荧光强度(0.46±0.12)显著低于NC+psicheck-Smad4-wt细胞(1.06±0.14) (t=-5.636，P=0.002)，而miRNA-196a-5p mimics+psicheck-Smad4- mut细胞的相对荧光强度(1.02±0.13)与NC+psicheck- Smad4-wt细胞(1.01±0.15)之间差异无显著性意义(t=0.087，P=0.467)。"

References

[1] Chen W, Zheng R, Zhang S, et al. Report of incidence and mortality in China cancer registries, 2009. Chin J Cancer Res. 2013;25(1):10-21.

[2] Kim TH, Shivdasani RA. Stomach development, stem cells and disease. Development. 2016;143(4):554-565.

[3] Miller KD, Siegel RL, Lin CC, et al. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016;66(4): 271-289.

[4] Zhang X, Hua R, Wang X, et al. Identification of stem-like cells and clinical significance of candidate stem cell markers in gastric cancer. Oncotarget. 2016;7(9):9815-9831.

[5] Zhang WJ, Zhou ZH, Guo M, et al. High Infiltration of Polarized CD163+ Tumor-Associated Macrophages Correlates with Aberrant Expressions of CSCs Markers, and Predicts Prognosis in Patients with Recurrent Gastric Cancer. J Cancer. 2017;8(3):363-370.

[6] Liu S, Clouthier SG, Wicha MS. Role of microRNAs in the regulation of breast cancer stem cells. J Mammary Gland Biol Neoplasia. 2012;17(1):15-21.

[7] Liu N, Zhong L, Zeng J, et al. Upregulation of microRNA-200a associates with tumor proliferation, CSCs phenotype and chemosensitivity in ovarian cancer. Neoplasma. 2015;62(4): 550-559.

[8] Lee SJ, Seo JW, Chae YS, et al. Genetic polymorphism of miR-196a as a prognostic biomarker for early breast cancer. Anticancer Res. 2014;34(6):2943-2949.

[9] Gocze K, Gombos K, Juhasz K, et al. Unique microRNA expression profiles in cervical cancer. Anticancer Res. 2013; 33(6):2561-2567.

[10] Sun M, Liu XH, Li JH, et al. MiR-196a is upregulated in gastric cancer and promotes cell proliferation by downregulating p27(kip1). Mol Cancer Ther. 2012;11(4):842-852.

[11] Tsai MM, Wang CS, Tsai CY, et al. MicroRNA-196a/-196b promote cell metastasis via negative regulation of radixin in human gastric cancer. Cancer Lett. 2014;351(2):222-231.

[12] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30.

[13] Zhang J, Gan L, Xu MD, et al. The prognostic value of age in non-metastatic gastric cancer after gastrectomy: a retrospective study in the U.S. and China. J Cancer. 2018; 9(7):1188-1199.

[14] Yin J, Song JN, Bai ZG, et al. Gastric Cancer Mortality Trends in China (2006-2013) Reveal Increasing Mortality in Young Subjects. Anticancer Res. 2017;37(8):4671-4679.

[15] 邹文斌,杨帆,李兆申.中国胃癌诊治关键在于提高早期诊断率[J].浙江大学学报:医学版,2015,44(1):9-14,53.

[16] Mitra A, Mishra L, Li S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget. 2015;6(13): 10697-10711.

[17] Jeong YJ, Oh HK, Park SH, et al. Association between inflammation and cancer stem cell phenotype in breast cancer. Oncol Lett. 2018;15(2):2380-2386.

[18] Carmon KS, Gong X, Yi J, et al. LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway. J Biol Chem. 2017;292(36): 14989-15001.

[19] Wang M, Yang F, Qiu R, et al. The role of mmu-miR-155-5p-NF-κB signaling in the education of bone marrow-derived mesenchymal stem cells by gastric cancer cells. Cancer Med. 2018;7(3):856-868.

[20] Takaishi S, Okumura T, Tu S, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells. 2009;27(5):1006-1020.

[21] Han S, Guo J, Liu Y, et al. Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation. Oncotarget. 2015;6(42): 44452-44465.

[22] Senel F, Kökenek Unal TD, Karaman H, et al. Prognostic Value of Cancer Stem Cell Markers CD44 and ALDH1/2 in Gastric Cancer Cases. Asian Pac J Cancer Prev. 2017;18(9): 2527-2531.

[23] Zhang C, Li C, He F, et al. Identification of CD44+CD24+ gastric cancer stem cells. J Cancer Res Clin Oncol. 2011; 137(11):1679-1686.

[24] Song Z, Yue W, Wei B, et al. Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer. PLoS One. 2011;6(3):e17687.

[25] Shitara K, Doi T, Nagano O, et al. Dose-escalation study for the targeting of CD44v+ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205). Gastric Cancer. 2017;20(2):341-349.

[26] Kulkarni M, Tan TZ, Syed Sulaiman NB, et al. RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer. Oncotarget. 2018; 9(18):14175-14192.

[27] Brabletz T, Kalluri R, Nieto MA, et al. EMT in cancer. Nat Rev Cancer. 2018;18(2):128-134.

[28] Suarez-Carmona M, Lesage J, Cataldo D, et al. EMT and inflammation: inseparable actors of cancer progression. Mol Oncol. 2017;11(7):805-823.

[29] Wang J, Wang X, Liu F, et al. microRNA-335 inhibits colorectal cancer HCT116 cells growth and epithelial- mesenchymal transition (EMT) process by targeting Twist1. Pharmazie. 2017;72(8):475-481.

[30] Tang CP, Zhou HJ, Qin J, et al. MicroRNA-520c-3p negatively regulates EMT by targeting IL-8 to suppress the invasion and migration of breast cancer. Oncol Rep. 2017;38(5): 3144-3152.

[31] Lu Z, Nian Z, Jingjing Z, et al. MicroRNA-424/E2F6 feedback loop modulates cell invasion, migration and EMT in endometrial carcinoma. Oncotarget. 2017;8(69): 114281-114291.

[32] Pereira-da-Silva T, Coutinho Cruz M, Carrusca C, et al. Circulating microRNA profiles in different arterial territories of stable atherosclerotic disease: a systematic review. Am J Cardiovasc Dis. 2018;8(1):1-13.

[33] Deng Z, He Y, Yang X, et al. MicroRNA-29: A Crucial Player in Fibrotic Disease. Mol Diagn Ther. 2017;21(3):285-294.

[34] Ma C, Huang T, Ding YC, et al. MicroRNA-200c overexpression inhibits chemoresistance, invasion and colony formation of human pancreatic cancer stem cells. Int J Clin Exp Pathol. 2015;8(6):6533-6539.

[35] Yu D, Shin HS, Lee YS, et al. miR-106b modulates cancer stem cell characteristics through TGF-β/Smad signaling in CD44-positive gastric cancer cells. Lab Invest. 2014;94(12): 1370-1381.

[36] Yang JP, Yang JK, Li C, et al. Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM. Biochem Biophys Res Commun. 2017;494 (3-4):674-680.

[37] Maruyama T, Nishihara K, Umikawa M, et al. MicroRNA-196a-5p is a potential prognostic marker of delayed lymph node metastasis in early-stage tongue squamous cell carcinoma. Oncol Lett. 2018;15(2):2349-2363.

[38] Zhang W, Li Y. miR-148a downregulates the expression of transforming growth factor-β2 and SMAD2 in gastric cancer. Int J Oncol. 2016;48(5):1877-1885.

[39] Wang XH, Liu MN, Sun X, et al. TGF-β1 pathway affects the protein expression of many signaling pathways, markers of liver cancer stem cells, cytokeratins, and TERT in liver cancer HepG2 cells. Tumour Biol. 2016;37(3):3675-3681.

[40] Liu TJ, Guo JL, Wang HK, et al. Semaphorin-7A contributes to growth, migration and invasion of oral tongue squamous cell carcinoma through TGF-β-mediated EMT signaling pathway.Eur Rev Med Pharmacol Sci. 2018;22(4):1035-1043.

[41] Goto M, Osada S, Imagawa M, et al. FAD104, a regulator of adipogenesis, is a novel suppressor of TGF-β-mediated EMT in cervical cancer cells. Sci Rep. 2017;7(1):16365.

[42] Yang H, Zhan L Yang T, et al. Ski prevents TGF-β-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer. Oncol Rep. 2015; 34(1):87-94.