Mechanisms of miR-103a-3p/CHI3L1 in proliferation and vascular mimicry of ovarian cancer cells

Abstract

Abstract:

ObjectiveTo investigate the mechanisms of microRNA (miR)-103a-3p/chitinase-3-like protein 1 (CHI3L1) in the proliferation and vascular mimicry of ovarian cancer cells and its effect on transforming growth factor-β (TGF-β) pathway.MethodsThe relationship between the expression level of miR-103a-3p and the overall survival rate of ovarian cancer patients was analyzed by bioinformatics. The human ovarian adenocarcinoma SKOV3 cells were divided into 4 groups: control group, miR-103a-3p mimic group, miR-103a-3p mimic+CHI3L1 group and CHI3L1 group. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the expression levels of miR-103a-3p, CHI3L1 mRNA and CHI3L1 protein respectively. The expression level of YKL-40 in cell culture fluid was detected by enzyme-linked immunosorbent assay. The cell viability, proliferation ability and angiogenesis ability of the 4 groups were detected by CCK-8 method, clone formation experiment and angiogenesis experiment. The dual luciferase report verified that miR-130a-3p targeted CHI3L1.ResultsThe overall survival rate of ovarian cancer patients with high expression of miR-103a-3p was higher than that of patients with low expression of miR-103a-3p (χ²=6.187,P=0.048). The differences in miR-103a-3p and CHI3L1 mRNA levels among the control group, miR-103a-3p mimic group, miR-103a-3p mimic+CHI3L1 group and CHI3L1 group were statistically significant (F=198.254,P<0.001;F=60.214,P<0.001), miR-103a-3p mimic group and miR-103a-3p mimic+CHI3L1 group had higher miR-103a-3p levels than the control group (allP<0.001), CHI3L1 group had higher CHI3L1 mRNA level than the control group (P<0.001). The expression levels of CHI3L1 protein in the 4 groups were 2.25±0.23, 1.19±0.12, 2.29±0.28 and 4.31±0.37, and the difference was statistically significant (F=18.675,P<0.001). The expression levels of YKL-40 in the cell culture fluids of the 4 groups were (1.84±0.20) ng/ml, (0.95±0.08) ng/ml, (2.64±0.25) ng/ml, (6.27±0.79) ng/ml, and the difference was statistically significant (F=35.297,P<0.001). The YKL-40 level of the CHI3L1 group was significantly higher than that of the control group (P<0.001), the miR-103a-3p mimic group was lower than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was higher than the miR-103a-3p mimic group (P<0.001). The cell viabilities of the 4 groups were 100%±2.54%, 76.23%±2.13%, 104.89%±3.56% and 137.42%±2.80%, and the difference was statistically significant (F=23.584,P<0.001). The cell viability of the miR-103a-3p mimic group was significantly lower than that of the control group (P<0.001), the CHI3L1 group was higher than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was higher than the miR-103a-3p mimic group (P<0.001). The number of clones formed in the 4 groups were 76.85±4.67, 21.56±2.85, 72.06±5.07 and 169.63±9.21, and the difference was statistically significant (F=31.541,P<0.001). The proliferation capacity of the miR-103a-3p mimic group was significantly lower than that of the control group (P<0.001), the CHI3L1 group was higher than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was significantly higher than the miR-103a-3p mimic group (P<0.001). The differences in the relative tube lengths and the tube bramches of the 4 groups were both statistically significant (F=24.254,P<0.001;F=27.564,P<0.001). The differences in TGF-β and Smad levels of the 4 groups were both statistically significant (F=30.254,P<0.001;F=34.187,P<0.001). The results of dual luciferase experiments showed that compared with the NC group, the luciferase activity in cells co-transfected of miR-103a-3p and CHI3L1-wt was significantly reduced. The difference of luciferase activity between the cells transfected with NC and co-transfected with miR-103a-3p and CHI3L1-mut was not significant.ConclusionMiR-103a-3p can directly inhibit the expression of CHI3L1 and inhibit the proliferation and angiogenesis of ovarian cancer SKOV3 cells to inhibit ovarian lymphatic metastasis and distant metastasis, which may be related to the TGF-β pathway.

Key words:Ovarian neoplasms,Chitinase-3-like protein 1,MicroRNAs,Transforming growth factor beta,Cell proliferation

Yang Lifen, Song Wei, Xu Dawei, Wu Jun, Gao Ran. Mechanisms of miR-103a-3p/CHI3L1 in proliferation and vascular mimicry of ovarian cancer cells[J]. Journal of International Oncology, 2020, 47(6): 333-339.

Figures/Tables7

组别	miR-103a-3p	CHI3L1
对照组	0.86±0.07	3.26±0.29
miR-103a-3p mimic组	19.83±1.72^a	1.21±0.12^a
miR-103a-3p mimic+CHI3L1组	18.72±2.04^a	3.95±0.40^b
CHI3L1组	0.85±0.09	24.95±2.17^a
F值	198.254	60.214
P值	<0.001	<0.001

组别	TGF-β	Smad
对照组	1.56±0.13	2.42±0.22
miR-103a-3p mimic组	0.92±0.08^a	1.10±0.10^a
miR-103a-3p mimic+CHI3L1组	1.60±0.15^b	2.73±0.24^b
CHI3L1组	2.48±0.21^a	4.18±0.32^a
F值	30.254	34.187
P值	<0.001	<0.001

References20

[1]	Xie X, Yang M, Ding Y, et al. Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer[J]. Oncol Rep, 2017,38(6):3297-3308. DOI: 10.3892/or.2017.6034. doi:10.3892/or.2017.6034pmid:29039544
[2]	Jefri M, Huang YN, Huang WC, et al. YKL-40 regulated epithelial-mesenchymal transition and migration/invasion enhancement in non-small cell lung cancer[J]. BMC Cancer, 2015,15:590. DOI: 10.1186/s12885-015-1592-3. doi:10.1186/s12885-015-1592-3pmid:26275425
[3]	Boisen MK, Madsen CV, Dehlendorff C, et al. The prognostic value of plasma YKL-40 in patients with chemotherapy-resistant ovarian cancer treated with bevacizumab[J]. Int J Gynecol Cancer, 2016,26(8):1390-1398. DOI: 10.1097/IGC.0000000000000798. doi:10.1097/IGC.0000000000000798pmid:27648712
[4]	Pan G, Hu T, Chen X, et al. Upregulation of circMMP9 promotes osteosarcoma progression via targeting miR-1265/CHI3L1 axis[J]. Cancer Manag Res, 2019,11:9225-9231. DOI: 10.2147/CMAR.S226264. doi:10.2147/CMAR.S226264pmid:31754311
[5]	Paraskevopoulou MD, Vlachos IS, Karagkouni D, et al. DIANA-lncBase v2: indexing microRNA targets on non-coding transcripts[J]. Nucleic Acids Res, 2016,44(D1):D231-D238. DOI: 10.1093/nar/gkv1270. doi:10.1093/nar/gkv1270pmid:26612864
[6]	杨宗元, 魏晓, 周晓水, 等. microRNA-103下调Dicer1对卵巢癌侵袭转移影响及其机制探讨[J]. 中华肿瘤防治杂志, 2014,21(22):1757-1763. DOI: 10.16073/j.cnki.cjcpt.2014.22.002.
[7]	Li W, Zhang X, Wang J, et al. TGFβ1 in fibroblasts-derived exosomes promotes epithelial-mesenchymal transition of ovarian cancer cells[J]. Oncotarget, 2017,8(56):96035-96047. DOI: 10.18632/oncotarget.21635. doi:10.18632/oncotarget.21635pmid:29221185
[8]	Rosendahl M, Høgdall CK, Mosgaard BJ. Restaging and survival analysis of 4036 ovarian cancer patients according to the 2013 FIGO classification for ovarian, fallopian tube, and primary peritoneal cancer[J]. Int J Gynecol Cancer, 2016,26(4):680-687. DOI: 10.1097/IGC.0000000000000675. doi:10.1097/IGC.0000000000000675pmid:26937751
[9]	Sun C, Li J, Wang B, et al. Tumor angiogenesis and bone metastasis-correlation in invasive breast carcinoma[J]. J Immunol Methods, 2018,452:46-52. DOI: 10.1016/j.jim.2017.10.006. doi:10.1016/j.jim.2017.10.006pmid:29066178
[10]	Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases[J]. Nat Rev Drug Discov, 2017,16(3):203-222. DOI: 10.1038/nrd.2016.246. doi:10.1038/nrd.2016.246pmid:28209991
[11]	Liang J, Liu X, Xue H, et al. MicroRNA-103a inhibits gastric cancer cell proliferation, migration and invasion by targeting c-Myb[J]. Cell Prolif, 2015,48(1):78-85. DOI: 10.1111/cpr.12159. doi:10.1111/cpr.12159pmid:25530421
[12]	Fasihi A, M Soltani B, Atashi A, et al. Introduction of hsa-miR-103a and hsa-miR-1827 and hsa-miR-137 as new regulators of Wnt signaling pathway and their relation to colorectal carcinoma[J]. J Cell Biochem, 2018,119(7):5104-5117. DOI: 10.1002/jcb.26357. doi:10.1002/jcb.26357pmid:28817181
[13]	He Q, Zhao L, Liu X, et al. MOV10 binding circ-DICER1 regulates the angiogenesis of glioma via miR-103a-3p/miR-382-5p mediated ZIC4 expression change[J]. J Exp Clin Cancer Res, 2019,38(1):9. DOI: 10.1186/s13046-018-0990-1. doi:10.1186/s13046-018-0990-1pmid:30621721
[14]	Usemann J, Frey U, Mack I, et al. CHI3L1 polymorphisms, cord blood YKL-40 levels and later asthma development[J]. BMC Pulm Med, 2016,16(1):81. DOI: 10.1186/s12890-016-0239-8. doi:10.1186/s12890-016-0239-8pmid:27193312
[15]	Wan G, Xiang L, Sun X, et al. Elevated YKL-40 expression is associated with a poor prognosis in breast cancer patients[J]. Oncotarget, 2017,8(3):5382-5391. DOI: 10.18632/oncotarget.14280. doi:10.18632/oncotarget.14280pmid:28036271
[16]	Vom Dorp F, Tschirdewahn S, Niedworok C, et al. Circulating and tissue expression levels of YKL-40 in renal cell cancer[J]. J Urol, 2016,195(4 Pt 1):1120-1125. DOI: 10.1016/j.juro.2015.09.084. doi:10.1016/j.juro.2015.09.084pmid:26454102
[17]	Jensen BV, Spindler KLG, Christensen IJ, et al. Plasma YKL-40 as a biomarker for poor prognosis in patients with metastatic colorectal cancer treated with 3. line cetuximab and irinotecan[J]. Ann Oncol, 2016,27(suppl_6):142-145. DOI: 10.1093/annonc/mdw363.43.
[18]	刘炎, 范江涛, 李大海, 等. YKL-40蛋白检测在卵巢癌诊断中的应用[J]. 实用临床医药杂志, 2015,19(3):88-91. DOI: 10.7619/jcmp.201503026.
[19]	Low D, Subramaniam R, Lin G, et al. Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis-associated cancer by regulating S100A9[J]. Oncotarget, 2015,6(34):36535-36550. DOI: 10.18632/oncotarget.5440. doi:10.18632/oncotarget.5440pmid:26431492
[20]	Lin HW, Chiang YC, Sun NY, et al. CHI3L1 results in poor outcome of ovarian cancer by promoting properties of stem-like cells[J]. Endocr Relat Cancer, 2019,26(1):73-88. DOI: 10.1530/ERC-18-0300. doi:10.1530/ERC-18-0300pmid:30121622