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European Journal of Gynaecological Oncology  2020, Vol. 41 Issue (3): 432-438    DOI: 10.31083/j.ejgo.2020.03.5241
Original Research Previous articles | Next articles
Oleic acid promotes the development of endometrial cancer by up-regulating KLF4 expression
J. Feng1, J. Wu1, M. Zhang1, J. Wang1, K. Chen1, X. Li1, J. Xie1, J. Zhang1, C. Wang1()
1Shihezi University School of Medicine, Shihezi, Xinjiang 832000, China
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Objective: To investigate the role and possible molecular mechanism of the transcription factor Krüppel-like factor 4 (KLF4) in the biological phenotype of endometrial cancer cells following induction by oleic acid (OA). Materials and Methods: Ishikawa endometrial cancer cells were grown in vitro with different concentrations of oleic acid. Cell proliferation was measured using the Counting Kit-8 (CCK-8) assay. Cell migration and invasion were evaluated by transwell chamber assay and scratch assay, respectively. The mRNA and protein expression levels of KLF4, ERα, Bcl2, MMP1, MMP9, were evaluated by qRT-PCR and Western blot, respectively. Results: The expression levels of KLF4, ERα, Bcl2, MMP1 and MMP9 increased significantly (p < 0.05) upon stimulation of the endometrial cancer cells with oleic acid (200 μM), together with the migration and invasion abilities of the cells (p < 0.05). Up-regulation of KLF4 caused the expression of ERα, Bcl2 and MMP9 to increase significantly (p < 0.05), whereas down-regulation of KLF4 caused the expression of ERα, Bcl2, MMP1 and MMP9 to significantly decrease (p < 0.05). The increased expression levels of ERα, Bcl2, MMP1 and MMP9 upon 200 μM oleic acid stimulation were significantly blocked (p < 0.05) by down-regulation of KLF4. Conclusion: oleic acid promotes the expression of ERα, Bcl2, MMP1 and MMP9 via up-regulation of KLF4, thus resulting in increased migration and invasion abilities of endometrial cancer cells.

Key words:  OA      KLF4      Endometrial cancer      Migration      Invasion     
Submitted:  11 April 2019      Accepted:  15 July 2019      Published:  15 June 2020     
Fund: Projects of Shihezi University(GJHZ201703)
*Corresponding Author(s):  C. Wang     E-mail:

Cite this article: 

J. Feng, J. Wu, M. Zhang, J. Wang, K. Chen, X. Li, J. Xie, J. Zhang, C. Wang. Oleic acid promotes the development of endometrial cancer by up-regulating KLF4 expression. European Journal of Gynaecological Oncology, 2020, 41(3): 432-438.

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Figure 1.  — The growth rate of Ishikawa cells was evaluated under different concentrations of OA. *50 μM, 1000 μM stimulation group vs. blank control group, #1500 μM vs. blank control group, *,#p < 0.05, **,##p < 0.01.

Figure 2.  — OA stimulation of Ishikawa cells and its effects on cell migration, invasion and proliferation ability. A) 50 μM and 200 μM OA-stimulated Ishikawa cells for 0, 24, 48, 72, and 96 h. CCK-8 assay was used to assess cell proliferation. B, C) 50 μM and 200 μM OA- stimulated Ishikawa cells incubated for 0, 24, and 48 h. The area of scratch healing was assessed with a × 100 microscope. D, E) Transwell migration experiment with 50 μM and 200 μM OA-stimulated Ishikawa cells grown for 24 h, × 200 microscope; F, G) Transwell invasion test with 50 μM and 200 μM OA-stimulated Ishikawa cells grown for 48 h, × 200 microscope; *NC vs. 50 μM; #NC vs. 200 μM; *, #p < 0.05, **, ##p < 0.01.

Figure 3.  — Expression levels of ERα, Bcl2, MMP1 and MMP9 in Ishikawa cells following stimulation with 50 μM and 200 μM OA for 24 h. A, B, G, H and I show mRNA expression. C, D, E and F show protein expression. *NC vs. 200 μM; *p < 0.05.

Figure 4.  — Expression levels of ERα, Bcl2, MMP1 and MMP9 following up-regulation of KLF4. A, B, G, H and I show mRNA expression. C, D, E and F show protein expression. *experimental group vs. control group; *p < 0.05.

Figure 5.  — Expression levels of ERα and oncogene following down-regulation of KLF4. A, B, G, H and I show mRNA expression. C, D, E and F show protein expression. *experimental group vs. control group, *p < 0.05, **p < 0.01.

Figure 6.  — ERα and oncogene expression following simultaneous OA stimulation and down-regulation of KLF4. A, B, G, H, and I show mRNA expression. D, E, and F show protein expression. *OA + NC vs. OA + KLF4; *p < 0.05, **p < 0.01.

Table 1  - Primer sequences
Primer name Sequence (5′→3′) Size (bp)
[1] Parkin D.M., Bray F., Ferlay J., Pisani P.: “Global cancer statistics, 2002” CA Cancer J. Clin., 2005, 55, 74.
doi: 10.3322/canjclin.55.2.74 pmid: 15761078
[2] Torre L.A., Bray F., Siegel R.L., Ferlay J., Lortet Tieulent J., Jemal A.: “Global cancer statistics, 2012”r. CA Cancer J. Clin., 2015, 65, 87.
doi: 10.3322/caac.21262 pmid: 25651787
[3] Greenwald Z.R., Huang L.N., Wissing M.D., Franco E.L., Gotlieb W.H.: “Does hormonal therapy for fertility preservation affect the survival of young women with early-stage endometrial cancer?” Cancer, 2017, 123, 1545.
pmid: 28026855
[4] Chathyan P., Clifford J.B., Helen R.G.: “Aging, adipose tissue, fatty acids and inflammation”. Biogerontology, 2015, 16, 235.
[5] Nomura D.K., Long J.Z., Niessen S., Hoover H.S., Ng S.W., Cravatt .: “Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis”. Cell, 2010, 140, 49.
[6] Przybytkowski E., Joly E., Nolan C.J., Hardy S., Francoeur A.M., Langelier Y., Prentki M.: “Upregulation of cellular triacylglycerol-free fatty acid cycling by oleate is associated with long-term serum-free survival of human breast cancer cells”. Biochem. Cell. Biol., 2007, 85, 301.
doi: 10.1139/o07-001 pmid: 17612624
[7] Liotti A., Cosimato V., Mirra P., Calì G., Conza D., Secondo A., et al.: “Oleic acid promotes prostate cancer malignant phenotype via the G protein-coupled receptor FFA1/GPR40”. J. Cell. Physiol., 2018, 233, 7367.
pmid: 29663374
[8] Yang P., Su C., Luo X., Zeng H., Zhao L., Wei L., et al.: “Dietary oleic acid-induced CD36 promotes cervical cancer cell growth and metastasis via up-regulation Src/ERK pathway”. Cancer Lett., 2018, 438, 76.
doi: 10.1016/j.canlet.2018.09.006
[9] Mcconnell B.B., Yang V.W.: “Mammalian Krüppel-like factors in health and diseases”. Physiol. Rev., 2010, 90, 1337.
pmid: 20959618
[10] Birsoy K., Chen Z., Friedman J.: “Transcriptional regulation of adipogenesis by KLF4”. Cell Metab., 2008, 7, 339.
doi: 10.1016/j.cmet.2008.02.001 pmid: 18396140
[11] Xu Q., Liu M., Zhang J., Xue L., Zhang G., Hu C., et al.: “Overex-pression of KLF4 promotes cell senescence through microRNA-203-survivin-p21 pathway”. Oncotarget, 2016, 7, 60290.
doi: 10.18632/oncotarget.11200 pmid: 27531889
[12] Wang B., Zhao M.Z., Cui N.P., Lin D.D., Zhang A.Y., Qin Y., et al.: “Krüppel-like factor 4 induces apoptosis and inhibits tumorigenic progression in SK-BR-3 breast cancer cells”. FEBS Open Bio., 2015, 5, 147.
[13] Engin A.: “Fat Cell and Fatty Acid Turnover in Obesity”. Adv. Exp. Med. Biol., 2017, 960, 135.
doi: 10.1007/978-3-319-48382-5_6 pmid: 28585198
[14] Lv W., Yang T.: “Identification of possible biomarkers for breast cancer from free fatty acid profiles determined by GC-MS and multivariate statistical analysis”. Clin. Biochem., 2012, 45, 127.
pmid: 22061338
[15] Haggerty A.F., Sarwer D.B., Schmitz K.H., Ko E.M., Allison K.C., Chu C.S.: “Obesity and Endometrial Cancer: A Lack of Knowledge but Opportunity for Intervention”. Nutr. Cancer, 2017, 69, 990.
doi: 10.1080/01635581.2017.1359313 pmid: 28937804
[16] Zhao H., Pflug B.R., Lai X., Wang M.: “Metabolic and molecular regulation of dietary polyunsaturated fatty acids on prostate cancer”. Proteomics Clin. Appl., 2016, 10, 267.
doi: 10.1002/prca.201500066 pmid: 26929070
[17] Fazio C., Piazzi G., Vitaglione P., Fogliano V., Munarini A., Prossomariti A., et al.: “Inflammation increases NOTCH1 activity via MMP9 and is counteracted by Eicosapentaenoic Acid-free fatty acid in colon cancer cells”. Sci. Rep., 2016, 6, 20670.
pmid: 26864323
[18] Xia S.H., Wang J., Kang J.X.: “Decreased n-6 /n-3 fatty acid ratio reduces the invasive potential of human lung cancer cells by down regulation of cell adhesion /invasion-related genes”. Carcinogenesis, 2005, 26, 779.
doi: 10.1093/carcin/bgi019 pmid: 15661810
[19] Blanckaert V., Ulmann L., Mimouni V., Antol J., Brancquart L., Chénais B.: “Docosahexaenoic acid intake decreases proliferation,increases apoptosis and decreases the invasive potential of the human breast carcinoma cell line MDA-MB-231”. Int. J. Oncol., 2010, 36 737.
doi: 10.3892/ijo_00000549 pmid: 20126994
[20] Kotronen A., Seppänen Laakso T., Westerbacka J., Kiviluoto T., Arola J., Ruskeepää A.L., et al.: “Comparison of lipid and fatty acid composition of the liver, subcutaneous and intra-abdominal adipose tissue, and serum”. Obesity (Silver Spring), 2010, 18, 937.
[21] Ben Fradj M.K., Ouanes Y., Hadj Taieb S., Sallemi A., Kallel A., Jemaa R., et al.: “Decreased Oleic Acid and Marine n-3 Polyunsaturated Fatty Acids in Tunisian Patients with Urothelial Bladder Cancer”. Nutr Cancer, 2018, 70, 1043.
doi: 10.1080/01635581.2018.1497668 pmid: 30183426
[22] Li S., Zhou Q., He H., Zhao Y., Liu Z.: “Peroxisome proliferator-activated receptor γ agonists induce cell cycle arrest through transcriptional regulation of Kruppel-like factor 4 (KLF4)”. J. Biol. Chem., 2013, 288, 4076.
doi: 10.1074/jbc.M111.317487 pmid: 23275339
[23] Zhang N., Zhang J., Shuai L., Zha L., He M., Huang Z., Wang Z.: “Kruppel-like factor 4 negatively regulates beta-catenin expression and inhibits the proliferation, invasion and metastasis of gastric cancer”. Int. J. Oncol., 2012, 40, 2038.
doi: 10.3892/ijo.2012.1395 pmid: 22407433
[24] Li Q., Gao Y., Jia Z., Mishra L., Guo K., Li Z., et al.: “Dysregulated Krüppel-like factor 4 and vitamin D receptor signaling contribute to progression of hepatocellular carcinoma”. Gastroenterology, 2012, 143, 799.
doi: 10.1053/j.gastro.2012.05.043
[25] Ohnishi S., Ohnami S., Laub F., Aoki K., Suzuki K., Kanai Y., et al.: “Downregulation and growth inhibitory effect of epithelialtype Kruppellike transcription factor KLF4, but not KLF5, in bladder cancer”. Biochem. Biophys. Res. Commun., 2003, 308, 251.
pmid: 12901861
[26] Hu W., Hofstetter W.L.: “Putative tumor-suppressive function of kruppel-like factor 4 in primary lung carcinoma”. Clin. Cancer Res., 2009, 15, 5688.
[27] Le Magnen C., Bubendorf L., Ruiz C., Zlobec I., Bachmann A., Heberer M., et al.: “Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells”. Eur. J. Cancer, 2013, 49, 955.
doi: 10.1016/j.ejca.2012.09.023 pmid: 23089465
[28] Nagata T., Shimada Y., Sekine S., Hori R., Matsui K., Okumura T., et al.: “Prognostic significance of NANOG and KLF4 for breast cancer”. Breast Cancer, 2014, 21, 96.
[29] Sunaga H., Matsui H., Anjo S., Syamsunarno M.R., Koitabashi N., Iso T., et al.: “Elongation of Long-Chain Fatty Acid Family Member 6 (Elovl6)-Driven Fatty Acid Metabolism Regulates Vascular Smooth Muscle Cell Phenotype Through AMP-Activated Protein Kinase/Krüppel-Like Factor 4 (AMPK/KLF4) Signaling”. J. Am. Heart Assoc., 2016, 5, 4014.
[30] Hara T., Kashihara D., Ichimura A., Kimura I., Tsujimoto G., Hirasawa A: “Role of free fatty acid receptors in the regulation of energy metabolism”. Biochim. Biophys. Acta, 2014, 1841, 1292.
pmid: 24923869
[31] Hirasawa A., Hara T., Katsuma S., Adachi T., Tsujimoto G.: “Free fatty acid receptors and drug discovery”. Biol. Pharm. Bull., 2008, 31, 1847.
doi: 10.1248/bpb.31.1847 pmid: 18827341
[32] Hardy S., St Onge G.G., Joly E., Langelier Y., Prentki M.: “Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40”. J. Biol. Chem., 2005, 280, 13285.
doi: 10.1074/jbc.M410922200 pmid: 15695516
[33] Liu Z., Hopkins M.M., Zhang Z., Quisenberry C.B., Fix L.C., Galvan B.M., Meier K.E.: “Omega-3 fatty acids and other FFA4 agonists inhibit growth factor signaling in human prostate cancer cells”. J. Pharmacol. Exp. Ther., 2015, 352, 380.
pmid: 25491146
[34] Nehra D., Pan A.H., Le H.D., Fallon E.M., Carlson S.J., Kalish B.T., Puder M.: “Docosahexaenoic acid, G protein-coupled receptors, and melanoma: is G protein-coupled receptor 40 a potential therapeutic target?”. J. Surg. Res., 2014, 188, 451.
pmid: 24576779
[35] Ishii S., Hirane M., Kato S., Fukushima N., Tsujiuchi T.: “Opposite effects of GPR120 and GPR40 on cell motile activity induced by ethionine in liver epithelial cells”. Biochem. Biophys. Res. Commun., 2015, 456, 135.
doi: 10.1016/j.bbrc.2014.11.047 pmid: 25446111
[36] Wu Q., Wang H., Zhao X., Shi Y., Jin M., Wan B., et al.: “Identification of G-protein-coupled receptor 120 as a tumor-promoting receptor that induces angiogenesis and migration in human colorectal carcinoma”. Oncogene, 2013, 32, 5541.
doi: 10.1038/onc.2013.264
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