Inhibition of GATA5 expression in HELA cells promotes expression of sox2, c-myc and CD44 in HELA cells

doi:10.3969/j.issn.2095-4344.1566

Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (5): 710-715.doi: 10.3969/j.issn.2095-4344.1566

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Inhibition of GATA5 expression in HELA cells promotes expression of sox2, c-myc and CD44 in HELA cells

Feng Haipeng^{1, 2}, Zheng Yifei^{1, 2}, Zhou Ying^{1, 2}, Li Zhilu^{1, 2}, Sun Yan^{1, 2}, Liu Kun^{1, 2}, Zhang Ruizhu^{1, 2}, Wang Qiaoyun^{1, 2}, Meng Bo², Lin Bo^{1, 2}, Li Mengsen^{1, 2, 3}

¹Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; ²Hainan Medical University, Haikou 571199, Hainan Province, China; ³Institute of Oncology, Hainan Medical University, Haikou 571199, Hainan Province, China

Revised:2018-10-30 Online:2019-02-18 Published:2019-02-18
Contact: Li Mengsen, MD, Professor, Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; Hainan Medical University, Haikou 571199, Hainan Province, China; Institute of Oncology, Hainan Medical University, Haikou 571199, Hainan Province, China； Lin Bo, MD, Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; Hainan Medical University, Haikou 571199, Hainan Province, China
About author:Feng Haipeng, Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; Hainan Medical University, Haikou 571199, Hainan Province, China. Zheng Yifei, Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; Hainan Medical University, Haikou 571199, Hainan Province, China. Zhou Ying, Hainan Provincial Key Laboratory of Oncology Occurrence and Intervention, Haikou 571199, Hainan Province, China; Hainan Medical University, Haikou 571199, Hainan Province, China. Feng Haipeng, Zheng Yifei and Zhou Ying contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81560450 (to LMS) and 31560243 (to LB); Hainan Provincial Postgraduate Innovation Project, No. Hys2017-178 (to MB), Hys2017-180 (to FHP), Hys2018-277 (to FHP), Hys2018-278 (to WQY), and Hys2018-279 (to ZRZ)

Abstract

Abstract:

BACKGROUND: GATA5, as a tumor suppressor gene, plays a role in inhibiting the development of cancer. GATA5 can be methylated in gastrointestinal tumors and then lose its function. GATA5 can be also methylated during hepatocarcinogenesis, and then cannot promote the differentiation of stem cells and maintain the normal function. Cervical cancer HELA cells express GATA5 protein, in which whether GATA5 plays a tumor suppressor function is still unknown.
OBJECTIVE: To analyze the expression of sox2, c-myc and CD44 related to cancer stem cell formation after suppressing the expression of GATA5 in cervical cancer HELA cells.
METHODS: Transfection of CMV-siRNA-gata5 further inhibited the expression of GATA5 in cervical cancer HELA cells. The expression of c-myc mRNA and sox2 mRNA were then detected by RT-PCR. The proliferation of HELA cells was detected by MTT. The colony formation and soft agar monoclonal formation of HELA cells were observed. The expression of GATA5, c-myc and sox2 was detected by western blot. The expression and location of CD44 was detected by laser confocal microscope.
RESULTS AND CONCLUSION: After suppressing the expression of GATA5 in cervical cancer HELA cells by CMV-siRNA-gata5, the proliferation, cell monoclonal formation and colony formation of HELA cells were enhanced. The expression of c-myc, sox2 and CD44 was also strengthened. These findings indicate that the malignant activity of HELA cells is increased by inhibiting the GATA5 protein expression. The mechanism may be that inhibition of GATA5 promotes the expression of cancer stem cell factors sox2, c-myc, and CD44.

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: Uterine Cervical Neoplasms, Hela Cells, GATA5 Transcription Factor, Proto-Oncogene Proteins c-myc, SOXB2 Transcription Factors, Stem Cells

CLC Number:

Feng Haipeng, Zheng Yifei, Zhou Ying, Li Zhilu, Sun Yan, Liu Kun, Zhang Ruizhu, Wang Qiaoyun, Meng Bo, Lin Bo, Li Mengsen. Inhibition of GATA5 expression in HELA cells promotes expression of sox2, c-myc and CD44 in HELA cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 710-715.

Figures/Tables 2

2.1 CMV-SiRNA-gata5降低GATA5蛋白表达并促进细胞生长用荧光观察转染CMV-siRNA-gata5的细胞后，细胞转染效率在70%以上，见图1A。经过G418筛选，干扰GATA5表达后，通过Western blotting实验发现GATA5的表达量降低，见图1B。同时通过激光共聚焦实验发现GATA5主要分布细胞核中，胞浆中也有分布，干扰GATA5表达后，细胞核中GATA5含量明显减少，见图1C。 MTT实验发现干扰GATA5表达后，HELA细胞增长速度加快，见图1D。以上结果表明CMV-siRNA-gata5可以有效干扰GATA5的表达，干扰GATA5表达后，HELA细胞增殖加快。 2.2 抑制GATA5表达后促进细胞单克隆形成软琼脂单克隆实验中，通过CMV-siRNA-gata5降低GATA5表达后的HELA细胞形成的克隆球体大于正常对照组，且CMV-SiRNA-gata5干扰GATA5表达后，克隆球周围有明显分散细胞，见图2A。细胞集落形成实验发现，降低GATA5表达后的HELA细胞与正常组相比形成的细胞集落更大，形成的个数也多，见图2B。提示GATA5的表达与细胞单克隆及集落形成相关，降低GATA5表达后，有利于细胞单克隆及集落形成。 2.3 抑制GATA5表达后促进c-myc，sox2 mRNA及蛋白表达见图3。实验发现，在干扰GATA5表达后，通过RT-PCR定量检测发现干细胞重编程因子c-myc，sox2 mRNA在细胞中表达升高，见图3A；通过Western blotting 实验发现细胞中的c-myc，sox2蛋白表达量同样升高，见图3B；激光共聚焦实验也发现c-myc蛋白及sox2蛋白的表达增强，见图3C。降低GATA5表达后，c-myc mRNA及sox2 mRNA在细胞中含量升高，表明GATA5从基因转录水平抑制c-myc，sox2蛋白的表达；抑制GATA5表达后，Western blot和激光共聚焦证实了c-myc，sox2蛋白表达升高并入核。 2.4 抑制GATA5表达后促进干细胞因子CD44表达激光共聚焦发现，抑制GATA5表达后，经过G418筛选后，HELA细胞表达的CD44增强，见图4。提示GATA5的表达降低可能促进CD44的表达，而CD44与成瘤性、侵袭性及淋巴转移性等细胞恶性行为相关。进一步提示抑制GATA5表达，促使HELA细胞的恶行性为增加。"

References

[1] Li B, Yang XX, Wang D, et al. MicroRNA-138 inhibits proliferation of cervical cancer cells by targeting c-Met. Eur Rev Med Pharmacol Sci. 2016;20(6):1109-1114.

[2] Feng D, Peng C, Li C, et al. Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri. Oncology Reports.2009;22(5):1129-1134.

[3] Todaro M, Lombardo Y, Stassi G. Evidences of cervical cancer stem cells derived from established cell lines. Cell Cycle. 2010; 9(7):1231-1240.

[4] Shu J, Zhang K, Zhang M, et al. GATA family members as inducers for cellular reprogramming to pluripotency. Cell Res. 2015;25(2):169-180.

[5] Molkentin JD, Lin Q, Duncan SA, et al. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 1997;11(8):1061-1072.

[6] Laverriere AC, Macneill C, Mueller C, et al. GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J Biol Chem. 1994;269(37): 23177-23184.

[7] Akiyama Y, Watkins N, Suzuki H, et al. GATA-4 and GATA-5 transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer.Mol Cell Biol. 2003;23(23): 8429-8439.

[8] Wen X, Akiyama Y, Zhou J, et al. Loss of transcription factor GATA-4/-5 by hypermethylation correlated with progression of gastric carcinomas. Cancer Res. 2007;67:2873-2873.

[9] Lei X, Yan G, Zhang A, et al. Loss of GATA5 expression due to gene promoter methylation induces growth and colony formation of hepatocellular carcinoma cells. Oncol Lett. 2016; 11(1):861-869.

[10] Liu P, Zhou TF, Qiu BA, et al. Methylation-Mediated Silencing of GATA5 Gene Suppresses Cholangiocarcinoma Cell Proliferation and Metastasis. Transl Oncol. 2018;11(3): 585-592.

[11] Pei Y, Qin Y, Yuan S, et al. GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3. Oncotarget. 2016;7(47):77890-77901.

[12] Lin B, Liu K, Wang W, et al.Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system.Biosci Rep. 2017;37(1). pii: BSR20160161.

[13] Sun Y, Huang YH, Huang FY, et al. 3′-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells. Theranostics. 2018; 8(7):2044-2060.

[14] Li M, Li H, Li C, et al. Cytoplasmic alpha-fetoprotein functions as a co-repressor in RA-RAR signaling to promote the growth of human hepatoma Bel 7402 cells. Cancer Lett. 2009;285(2): 190-199.

[15] Li M, Zhu M, Li W, et al. Alpha-fetoprotein receptor as an early indicator of HBx-driven hepatocarcinogenesis and its applications in tracing cancer cell metastasis. Cancer Lett. 2013; 330(2):170-180.

[16] Gandarillas A, Watt FM. c-Myc promotes differentiation of human epidermal stem cells. Genes Dev. 1997;11(21): 2869-2882.

[17] Satoh Y, Matsumura I, Tanaka H, et al.Roles for c-Myc in self-renewal of hematopoietic stem cells.J Biol Chem. 2004; 279(24):24986-24993.

[18] Martinez-Fernandez A, Nelson TJ, Ikeda Y, et al.c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells.J Cardiovasc Transl Res. 2010;3(1):13-23.

[19] Ellis P, Fagan BM, Magness ST, et al. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult.Dev Neurosci. 2004;26(2-4):148-165.

[20] Giorgetti A, Montserrat N, Aasen T, et al. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell. 2009;5(4):353-357.

[21] Park SB, Seo KW, So AY, et al. SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC. Cell Death Differ. 2012; 19(3):534-545.

[22] Xiang R, Liao D, Cheng T, et al. Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer. Br J Cancer. 2011;104(9): 1410-1417.

[23] Leis O, Eguiara A, Lopezarribillaga E, et al. Sox2 expression in breast tumours and activation in breast cancer stem cells. Oncogene. 2012;31(11):1354-1365.

[24] Bourguignon LY, Peyrollier K, Xia W, et al.Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells.J Biol Chem. 2008;283(25): 17635-17651.

[25] Zhu H, Mitsuhashi N, Klein A, et al. The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem Cells.2010;24(4):928-935.

[26] Yang X, Sarvestani SK, Moeinzadeh S, et al. Effect of CD44 binding peptide conjugated to an engineered inert matrix on maintenance of breast cancer stem cells and tumorsphere formation. Plos One. 2013;8(3):e59147.

[27] Hong I, Hong SW, Chang YG, et al.Expression of the Cancer Stem Cell Markers CD44 and CD133 in Colorectal Cancer: An Immunohistochemical Staining Analysis.Ann Coloproctol. 2015; 31(3):84-91.

[28] Moghbeli M, Moghbeli F, Forghanifard MM, et al. Cancer stem cell detection and isolation. Med Oncol. 2014;31(9):69.

[29] Maddox J, Shakya A, South S, et al. Transcription factor Oct1 is a somatic and cancer stem cell determinant. Plos Genetics. 2012; 8(11):e1003048.

Inhibition of GATA5 expression in HELA cells promotes expression of sox2, c-myc and CD44 in HELA cells

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