Effects of lncRNA AFAP1-AS1 on proliferation and invasion of thyroid cancer cells and its mechanisms

doi:10.3760/cma.j.cn371439-20191129-00030

Abstract

Abstract:

Objective To explore the effects of long noncoding RNA (lncRNA) actinfilament-associated protein 1-antisense RNA1 (AFAP1-AS1) on proliferation and invasion of thyroid cancer cells and its mechanisms. Methods Quantitative real time fluorescence PCR (qRT-PCR) was performed to assess the expression of AFAP1-AS1 in normal thyroid cells and thyroid cancer cells. The thyroid cancer cell line WRO was divided into three groups, AFAP1-AS1 silencing group (AFAP1-AS1-siRNA group), negative control group (NT-siRNA group) and blank control group (Blank group). AFAP1-AS1-siRNA group and NT-siRNA group were transfected with AFAP1-AS1 siRNA and NT-siRNA respectively using Lipofectamine^TM 3000, and Blank group was treated with PBST. The proliferation ability was measured by CCK-8. The invasion ability was measured by Transwell assay. The expression levels of Rho A, Cyclin D1 and MMP-9 protein were measured by Western blotting. Results The relative expressions of AFAP1-AS1 in normal thyroid cell line FRTL-5, thyroid cancer cell lines SW579, CAL-62, FRO and WRO were 1.03±0.04, 2.95±0.17, 5.31±0.35, 7.26±0.49 and 9.67±0.53 respectively, and the difference among the five groups was statistically significant (F=16.932, P=0.027). The expression of AFAP1-AS1 was highest in WRO cells, therefore, the WRO cells were selected for subsequent experiments. The relative expressions of AFAP1-AS1 in AFAP1-AS1-siRNA group, NT-siRNA group and Blank group were 0.23±0.02, 1.02±0.04 and 1.03±0.05 respectively, and the difference among the three groups was statistically significant (F=13.590, P=0.006). Compared with NT-siRNA group, the expression of AFAP1-AS1 in AFAP1-AS1-siRNA group was significantly lower (P<0.001). The A₄₅₀ values in the three groups were 0.68±0.06, 1.17±0.09, 1.22±0.09, and 0.96±0.08, 1.69±0.11, 1.72±0.12 respectively 3 and 4 days after transfection, and the differences were statistically significant (F=7.318, P=0.016; F=10.351, P=0.004). The differences between AFAP1-AS1-siRNA group and NT-siRNA group 3 and 4 days after transfection were statistically significant (P=0.043; P=0.013). The numbers of invasive cells in the three groups were 72.8±5.7, 145.6±8.9, 148.4±7.3, and the difference was statistically significant (F=37.273, P=0.034). The number of invasive cells in AFAP1-AS1-siRNA group was significantly less than that in NT-siRNA group (P=0.021). The expressions of Rho A protein in the three groups were 0.34±0.03, 1.02±0.04 and 1.04±0.03 respectively, and the difference was statistically significant (F=9.667, P=0.013). The expression of Rho A protein in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group (P=0.018). The expressions of Cyclin D1 protein in the three groups were 0.52±0.04, 1.03±0.02 and 1.05±0.04 respectively, with a statistically significant difference (F=15.464, P=0.010). The expression of Cyclin D1 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group (P=0.023). The expressions of MMP-9 protein in the three groups were 0.42±0.04, 1.05±0.03 and 1.02±0.04 respectively, and the difference was statistically significant (F=10.328, P=0.032). The expression of MMP-9 in AFAP1-AS1-siRNA group was significantly lower than that in NT-siRNA group (P=0.035). Conclusion The silencing of lncRNA AFAP1-AS1 can inhibit the proliferation and invasion of thyroid cancer cells, and the mechanism may be related to the down-regulation of Rho A, Cyclin D1 and MMP-9 proteins.

Key words: Thyroid neoplasms, RNA,long noncoding, Cell proliferation, Neoplasm invasiveness, Actinfilament-associated protein 1-antisense RNA1

Cheng Hu, Liu Mingkui, Chen Tianping. Effects of lncRNA AFAP1-AS1 on proliferation and invasion of thyroid cancer cells and its mechanisms[J]. Journal of International Oncology, 2020, 47(6): 327-332.

Figures/Tables 4

References 25

[1]	Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018,68(6):394-424. DOI: 10.3322/caac.21492. doi: 10.3322/caac.21492 pmid: 30207593
[2]	Tuttle RM, Haddad RI, Ball DW, et al. Thyroid carcinoma, version 2.2014[J]. J Natl Compr Canc Netw, 2014,12(12):1671-1680. DOI: 10.6004/jnccn.2014.0169. doi: 10.6004/jnccn.2014.0169 pmid: 25505208
[3]	韩婧, 康骅. 甲状腺癌的发病现状及影响因素[J]. 实用预防医学, 2018,25(7):894-897. DOI: 10.3969/j.issn.1006-3110.2018.07.037
[4]	Wang X, Li M, Wang Z, et al. Silencing of long noncoding RNA MALAT1 by miR-101 and miR-217 inhibits proliferation, migration, and invasion of esophageal squamous cell carcinoma cells[J]. J Biol Chem, 2015,290(7):3925-3935. DOI: 10.1074/jbc.M114.596866. doi: 10.1074/jbc.M114.596866 pmid: 25538231
[5]	Deng J, Liang Y, Liu C, et al. The up-regulation of long non-coding RNA AFAP1-AS1 is associated with the poor prognosis of NSCLC patients[J]. Biomed Pharmacother, 2015,75:8-11. DOI: 10.1016/j.biopha.2015.07.003. doi: 10.1016/j.biopha.2015.07.003 pmid: 26463625
[6]	Bo H, Gong Z, Zhang W, et al. Upregulated long non-coding RNA AFAP1-AS1 expression is associated with progression and poor prognosis of nasopharyngeal carcinoma[J]. Oncotarget, 2015,6(24):20404-20418. DOI: 10.18632/oncotarget.4057. doi: 10.18632/oncotarget.4057 pmid: 26246469
[7]	Wang F, Ni H, Sun F, et al. Overexpression of lncRNA AFAP1-AS1 correlates with poor prognosis and promotes tumorigenesis in colorectal cancer[J]. Biomed Pharmacother, 2016,81:152-159. DOI: 10.1016/j.biopha.2016.04.009. doi: 10.1016/j.biopha.2016.04.009 pmid: 27261589
[8]	Ye Y, Chen J, Zhou Y, et al. High expression of AFAP1-AS1 is associated with poor survival and short-term recurrence in pancreatic ductal adenocarcinoma[J]. J Transl Med, 2015,13:137. DOI: 10.1186/s12967-015-0490-4. doi: 10.1186/s12967-015-0490-4 pmid: 25925763
[9]	Ma F, Wang SH, Cai Q, et al. Overexpression of lncRNA AFAP1-AS1 predicts poor prognosis and promotes cells proliferation and invasion in gallbladder cancer[J]. Biomed Pharmacother, 2016,84:1249-1255. DOI: 10.1016/j.biopha.2016.10.064. doi: 10.1016/j.biopha.2016.10.064 pmid: 27810781
[10]	Zhang JY, Weng MZ, Song FB, et al. Long noncoding RNA AFAP1-AS1 indicates a poor prognosis of hepatocellular carcinoma and promotes cell proliferation and invasion via upregulation of the RhoA/Rac2 signaling[J]. Int J Oncol, 2016,48(4):1590-1598. DOI: 10.3892/ijo.2016.3385. doi: 10.3892/ijo.2016.3385 pmid: 26892468
[11]	Zeng Z, Bo H, Gong Z, et al. AFAP1-AS1, a long noncoding RNA upregulated in lung cancer and promotes invasion and metastasis[J]. Tumour Biol, 2016,37(1):729-737. DOI: 10.1007/s13277-015-3860-x. doi: 10.1007/s13277-015-3860-x pmid: 26245991
[12]	Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms[J]. Nat Rev Cancer, 2003,3(5):362-374. DOI: 10.1038/nrc1075. doi: 10.1038/nrc1075 pmid: 12724734
[13]	Ridley AJ. RhoA, RhoB and RhoC have different roles in cancer cell migration[J]. J Mocrosc, 2013,251(3):242-249. DOI: 10.1111/jmi.12025.
[14]	Karlsson R, Pedersen ED, Wang Z, et al. Rho GTPase function in tumorigenesis[J]. Biochim Biophys Acta, 2009,1796(2):91-98. DOI: 10.1016/j.bbcan.2009.03.003. doi: 10.1016/j.bbcan.2009.03.003 pmid: 19327386
[15]	Vega FM, Ridley AJ. Rho GTPases in cancer cell biology[J]. FEBS Lett, 2008,582(14):2093-2101. DOI: 10.1016/j.febslet.2008.04.039. doi: 10.1016/j.febslet.2008.04.039 pmid: 18460342
[16]	Vega FM, Fruhwirth G, Ng T, et al. RhoA and RhoC have distinct roles in migration and invasion by acting through different targets[J]. J Cell Biol, 2011,193(4):655-665. DOI: 10.1083/jcb.201011038. doi: 10.1083/jcb.201011038 pmid: 21576392
[17]	Zhou X, Zhan W, Bian W, et al. GOLPH3 regulates the migration and invasion of glioma cells though RhoA[J]. Biochem Biophys Res Commun, 2013,433(3):338-344. DOI: 10.1016/j.bbrc.2013.03.003. doi: 10.1016/j.bbrc.2013.03.003 pmid: 23500462
[18]	Rosman DS, Phukan S, Huang CC, et al. TGFBR1*6A enhances the migration and invasion of MCF-7 breast cancer cells through RhoA activation[J]. Cancer Res, 2008,68(5):1319-1328. DOI: 10.1158/0008-5472.CAN-07-5424. doi: 10.1158/0008-5472.CAN-07-5424 pmid: 18316594
[19]	Klein G, Vellenga E, Fraaije MW, et al. The possible role of matrix metalloproteinase (MMP)-2 and MMP-9 in cancer, eg acute leukemia[J]. Crit Rev Oncol Hematol, 2004,50(2):87-100. DOI: 10.1016/j.critrevonc.2003.09.001. doi: 10.1016/j.critrevonc.2003.09.001 pmid: 15157658
[20]	Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression[J]. Nat Rev Cancer, 2002,2(3):161-174. DOI: 10.1038/nrc745. doi: 10.1038/nrc745 pmid: 11990853
[21]	Yeh MW, Rougier JP, Park JW, et al. Differentiated thyroid cancer cell invasion is regulated through epidermal growth factor receptor-dependent activation of matrix metalloproteinase (MMP)-2/gelatinase A[J]. Endocr Relat Cancer, 2006,13(4):1173-1183. DOI: 10.1677/erc.1.01226. doi: 10.1677/erc.1.01226 pmid: 17158762
[22]	Jia W, Gao XJ, Zhang ZD, et al. S100A4 silencing suppresses proliferation, angiogenesis and invasion of thyroid cancer cells through downregulation of MMP-9 and VEGF[J]. Eur Rev Med Pharmacol Sci, 2013,17(11):1495-1508. pmid: 23771538
[23]	Fu M, Wang C, Li Z, et al. Minireview: Cyclin D1: normal and abnormal functions[J]. Endocrinology, 2004,145(12):5439-5447. DOI: 10.1210/en.2004-0959. doi: 10.1210/en.2004-0959 pmid: 15331580
[24]	Yang K, Hitomi M, Stacey DW. Variations in cyclin D1 levels through the cell cycle determine the proliferative fate of a cell[J]. Cell Div, 2006,1:32. DOI: 10.1186/1747-1028-1-32. doi: 10.1186/1747-1028-1-32 pmid: 17176475
[25]	Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells[J]. Curr Opin Cell Biol, 2003,15(2):158-163. DOI: 10.1016/s0955-0674(03)00008-5. doi: 10.1016/S0955-0674(03)00008-5

组别	A₄₅₀值
组别	0 d	1 d	2 d	3 d	4 d
AFAP1-AS1-siRNA组	0.26±0.02	0.37±0.04	0.51±0.05	0.68±0.06^a	0.96±0.08^b
NT-siRNA组	0.25±0.02	0.43±0.05	0.75±0.07	1.17±0.09	1.69±0.11
Blank组	0.26±0.02	0.44±0.04	0.75±0.07	1.22±0.09	1.72±0.12
F值	5.476	3.654	2.478	7.318	10.351
P值	0.076	0.082	0.276	0.016	0.004