MiR-520b regulates Wnt/β-catenin signaling pathway to promote invasion and metastasis of tongue squamous cell carcinoma

doi:10.3877/cma.j.issn.1674-1366.2020.03.005

Abstract

Abstract:

Objective

To investigate the expression of miR-520b in tongue squamous cell carcinoma (TSCC) and explore its role and molecular mechanism in the invasion and metastasis of TSCC.

Methods

Real time quantity polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-520b in different tongue squamous cell carcinoma tissues and cell lines. After silencing or over-expressing miR-520b in tongue squamous cell carcinoma cell lines SCC-9 and UM1, RT-qPCR was used to detect the expression of EMT-related genes; transwell assay was used to verify cell invasion; dual luciferase assay, RT-qPCR, Western blot, etc. were used to detect the effect of miR-520b expression in Wnt/β-catenin signaling pathway; bioinformatics, Western blot, and dual luciferin enzyme experiments verified that miR-520b regulated the potential targeted gene Dickkopf 1 (DKK1) in Wnt/β-catenin signaling pathway. SPSS statistics 21.0 was used for analysis, and student′s t test was used for comparison between groups.

Results

Compared with normal adjacent tissues, miR-520b was significantly up-regulated in TSCC tissues (5.28±1.63 vs. 2.59±1.43, t=-16.04, P=0.0005) , and was significantly positively correlated with the more aggressive TSCC phenotype in patients (5.81±0.74 vs.3.08±0.89, t = 12.11, P = 0.0011) ; overexpression of miR-520b promoted invasion and metastasis of TSCC cells (SCC-9: 110.8±17.8 vs. 74.7±9.8, t_SCC-9 = 32.58, P_SCC-9 = 0.0011; UM1: 178.8±39.7 vs. 90.3±22.5, t_UM1 = 99.67, P_UM1 = 0.0002) , while low expression on the contrary suppressed (SCC-9: 74.7±9.8 vs. 30.9±7.8, t_SCC-9 = -31.47, P_SCC-9 = 0.0024; UM1: 90.3±22.5 vs. 35.7±10.6, t_UM1 = -37.89, P_UM1 = 0.0019) . In addition, overexpression of miR-520b inhibited DKK1 (negative regulator of Wnt/β-catenin signaling pathway) , thereby activating the Wnt/β-catenin signaling pathway and promoting epithelial-mesenchymal transition (EMT) of TSCC cells.

Conclusions

MiR-520b is highly expressed in TSCC, which may promote the invasion and metastasis of TSCC by inhibiting DKK1 and activating the Wnt/β-catenin signaling pathway.

Key words: Carcinoma, squamous cell, Tongue, Neoplasm invasiveness, MiR-520b, Wnt/β-catenin, DKK1

Junquan Weng, Haidong Fan, Liqiang Xu, Huijuan Liu. MiR-520b regulates Wnt/β-catenin signaling pathway to promote invasion and metastasis of tongue squamous cell carcinoma[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2020, 14(03): 155-163.

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References 31

[1]	Torre LA, Bray F, Siegel RL，et al. Global cancer statistics，2012[J]. CA Cancer J Clin，2015，65(2):87-108. DOI：10.3322/caac.21262.
[2]	Lian IeB, Tseng YT, Su CC，et al. Progression of precancerous lesions to oral cancer：results based on the Taiwan National Health Insurance Database[J]. Oral Oncol，2013，49(5):427-430. DOI：10.1016/j.oraloncology.2012.12.004.
[3]	Xie B, Ding Q, Han H，et al. miRCancer：a microRNA-cancer association database constructed by text mining on literature[J]. Bioinformatics，2013，29(5):638-644. DOI：10.1093/bioinformatics/btt014.
[4]	Yu X, Li Z. MicroRNA expression and its implications for diagnosis and therapy of tongue squamous cell carcinoma[J]. J Cell Mol Med，2016，20(1):10-16. DOI：10.1111/jcmm.12650.
[5]	Wong TS, Liu XB, Chung-Wai Ho A，et al. Identification of pyruvate kinase type M2 as potential oncoprotein in squamous cell carcinoma of tongue through microRNA profiling[J]. Int J Cancer，2008，123(2):251-257. DOI：10.1002/ijc.23583.
[6]	Siriwardena S, Tsunematsu T, Qi G，et al. Invasion-Related Factors as Potential Diagnostic and Therapeutic Targets in Oral Squamous Cell Carcinoma-A Review[J]. Int J Mol Sci，2018，19(5):E1462. DOI：10.3390/ijms19051462.
[7]	Škovierová H, Okajčeková T, Strnádel J，et al. Molecular regulation of epithelial-to-mesenchymal transition in tumorigenesis （Review）[J]. Int J Mol Med，2018，41(3):1187-1200. DOI：10.3892/ijmm.2017.3320.
[8]	Zhang JJ, Zhou XH, Zhou Y et al. Bufalin suppresses the migration and invasion of prostate cancer cells through HOTAIR，the sponge of miR-520b[J]. Acta Pharmacol Sin，2019，40(9):1228-1236. DOI：10.1038/s41401-019-0234-8.
[9]	Liu H, Du F, Sun L，et al. GATA6 suppresses migration and metastasis by regulating the miR-520b/CREB1 axis in gastric cancer[J]. Cell Death Dis，2019，10(2):35. DOI：10.1038/s41419-018-1270-x.
[10]	Cai Y, Dong ZY, Wang JY. MiR-520b inhibited metastasis and proliferation of non-small cell lung cancer by targeting CHAF1A[J]. Eur Rev Med Pharmacol Sci，2018，22(22):7742-7749. DOI：10.26355/eurrev_201811_16396.
[11]	Salinas-Vera YM, Marchat LA, Gallardo-Rincón D，et al. AngiomiRs：MicroRNAs driving angiogenesis in cancer（Review）[J]. Int J Mol Med，2019，43(2):657-670. DOI：10.3892/ijmm.2018.4003.
[12]	Guan R, Cai S, Sun M，et al. Upregulation of miR-520b promotes ovarian cancer growth[J]. Oncol Lett，2017，14(3):3155-3161. DOI：10.3892/ol.2017.6552.
[13]	Liu X, Liu J, Zhang X，et al. MiR-520b promotes the progression of non-small cell lung cancer through activating Hedgehog pathway[J]. J Cell Mol Med，2019，23(1):205-215. DOI：10.1111/jcmm.13909.
[14]	Hu N, Zhang J, Cui W，et al. miR-520b regulates migration of breast cancer cells by targeting hepatitis B X-interacting protein and interleukin-8[J]. J Biol Chem，2011，286(15):13714-13722. DOI：10.1074/jbc.M110.204131.
[15]	Zhang W, Kong G, Zhang J，et al. MicroRNA-520b inhibits growth of hepatoma cells by targeting MEKK2 and cyclin D1[J]. PLoS One，2012，7(2):e31450. DOI：10.1371/journal.pone.0031450.
[16]	Li M, Bu X, Cai B，et al. Biological role of metabolic reprogramming of cancer cells during epithelial-mesenchymal transition （Review）[J]. Oncol Rep，2019，41(2):727-741. DOI：10.3892/or.2018.6882.
[17]	Wang WX, Yu HL, Liu X. MiR-9-5p suppresses cell metastasis and epithelial-mesenchymal transition through targeting FOXP2 and predicts prognosis of colorectal carcinoma[J]. Eur Rev Med Pharmacol Sci，2019，23(15):6467-6477. DOI：10.26355/eurrev_201908_18530.
[18]	Kim EJ, Kim JS, Lee S，et al. QKI，a miR-200 target gene，suppresses epithelial-to-mesenchymal transition and tumor growth[J]. Int J Cancer，2019，145(6):1585-1595. DOI：10.1002/ijc.32372.
[19]	Gulei D, Magdo L, Jurj A，et al. The silent healer：miR-205-5p up-regulation inhibits epithelial to mesenchymal transition in colon cancer cells by indirectly up-regulating E-cadherin expression[J]. Cell Death Dis，2018，9(2):66. DOI：10.1038/s41419-017-0102-8.
[20]	Johansson J, Berg T, Kurzejamska E，et al. MiR-155-mediated loss of C/EBPβ shifts the TGF-β response from growth inhibition to epithelial-mesenchymal transition，invasion and metastasis in breast cancer[J]. Oncogene，2013，32(50):5614-5624. DOI：10.1038/onc.2013.322.
[21]	Luo Y, Wu J, Wu Q，et al. miR-577 Regulates TGF-β Induced Cancer Progression through a SDPR-Modulated Positive-Feedback Loop with ERK-NF-κB in Gastric Cancer[J]. Mol Ther，2019，27(6):1166-1182. DOI：10.1016/j.ymthe.2019.02.002.
[22]	Xu X, Liu M. miR-522 stimulates TGF-β/Smad signaling pathway and promotes osteosarcoma tumorigenesis by targeting PPM1A[J]. J Cell Biochem，2019，120(10):18425-18434. DOI：10.1002/jcb.29160.
[23]	Gurzu S, Kobori L, Fodor D，et al. Epithelial Mesenchymal and Endothelial Mesenchymal Transitions in Hepatocellular Carcinoma：A Review[J]. Biomed Res Int，2019(1):1-12. DOI：10.1155/2019/2962580.
[24]	Teeuwssen M, Fodde R. Wnt Signaling in Ovarian Cancer Stemness，EMT，and Therapy Resistance[J]. J Clin Med，2019，8(10):1658. DOI：10.3390/jcm8101658.
[25]	Wang C, Xu X, Jin H，et al. Nicotine may promote tongue squamous cell carcinoma progression by activating the Wnt/β-catenin and Wnt/PCP signaling pathways[J]. Oncol Lett，2017，13(5):3479-3486. DOI：10.3892/ol.2017.5899.
[26]	Jing Y, Cui D, Guo W，et al. Activated androgen receptor promotes bladder cancer metastasis via Slug mediated epithelial-mesenchymal transition[J]. Cancer Lett，2014，348(1-2):135-145. DOI：10.1016/j.canlet.2014.03.018.
[27]	Zhang X, Tian Y, Yang Y，et al. Development of anticancer agents targeting the Hedgehog signaling[J]. Cell Mol Life Sci，2017，74(15):2773-2782. DOI：10.1007/s00018-017-2497-x.
[28]	Liu P, Chen B, Gu Y，et al. PNMA1，regulated by miR-33a-5p，promotes proliferation and EMT in hepatocellular carcinoma by activating the Wnt/β-catenin pathway[J]. Biomed Pharmacother，2018，108:492-499. DOI：10.1016/j.biopha.2018.09.059.
[29]	Huang X, Zhu H, Gao Z，et al. Wnt7a activates canonical Wnt signaling，promotes bladder cancer cell invasion，and is suppressed by miR-370-3p[J]. J Biol Chem，2018，293(18):6693-6706. DOI：10.1074/jbc.RA118.001689.
[30]	Croset M, Pantano F, Kan C，et al. MicroRNA-30 family members inhibit breast cancer invasion，osteomimicry，and bone destruction by directly targeting multiple bone metastasis-associated genes[J]. Cancer Res，2018，78(18):5259-5273. DOI：10.1158/0008-5472.CAN-17-3058.
[31]	Feng ZY, Xu XH, Cen DZ，et al. miR-590-3p promotes colon cancer cell proliferation via Wnt/β-catenin signaling pathway by inhibiting WIF1 and DKK1[J]. Eur Rev Med Pharmacol Sci，2017，21(21):4844-4852.

基因	引物序列
miR-520b	上游：5′-AAAGTGCTTCCTTTTAGAGGG-3′
?	下游：5′-GCGAGCACAGAATTAATACGAC-3′
U6	上游：5′-CTCGCTTCGGCAGCACA-3′
?	下游：5′-AACGCTTCACGAATTTGCGT-3′
E-cadherin	上游：5′-GACCGGTGCAATCTTCAAA-3′
?	下游：5′-TTGACGCCGAGAGCTACAC-3′
CK18	上游：5′-AAGGCCTACAAGCCCAGATT-3′
?	下游：5′-CACTGTGGTGCTCTCCTCAA-3′
N-cadherin	上游：5′-GTGCCATfAGCCAAGGGAA-3′
?	下游：5′-GCGTTCCTGTTCCACTCATAGGAG-3′
Vimentin	上游：5′-TTCTTCCCTGAACCTGAGAGA-3′
?	下游：5′-GAGTGGGTGTCAACCAGAGG-3′
C-myc	上游：5′-TCAAGAGGCGAACACACAAC-3′
?	下游：5′-GGCCTTTTCATTGTTTTCCA-3′
LEF1	上游：5′-CACTGTAAGTGATGAGGGGG-3′
?	下游：5′-TGGATCTCTTTCTCCACCCA-3′
CCND1	上游：5′-TTCTGCCTTTGATGTTAC-3′
?	下游：5′-AGGCTGAATCAATGTCTT-3′
CD44	上游：5′-AGTCCCTGGATCACCGACAG-3′
?	下游：5′-GTTTCTTGCCTCTTGGTTGCT-3′
MMP-7	上游：5′-GCATCTCCTTGAGTTTGGCT-3′
?	下游：5′-GAGCTACAGTGGGAACAGGC-3′
DKK1	上游：5′-ATAGCACCTTGGATGGGTATTCC-3′
?	下游：5′-CTGATGACCGGAGACAAACAG-5′
GAPDH	上游：5′-GACTCATGACCACAGTCCATGC-3′
?	下游：5′-AGAGGCAGGGATGATGTTCTG-3′

基因	引物序列
miR-520b	上游：5′-AAAGTGCTTCCTTTTAGAGGG-3′
?	下游：5′-GCGAGCACAGAATTAATACGAC-3′
U6	上游：5′-CTCGCTTCGGCAGCACA-3′
?	下游：5′-AACGCTTCACGAATTTGCGT-3′
E-cadherin	上游：5′-GACCGGTGCAATCTTCAAA-3′
?	下游：5′-TTGACGCCGAGAGCTACAC-3′
CK18	上游：5′-AAGGCCTACAAGCCCAGATT-3′
?	下游：5′-CACTGTGGTGCTCTCCTCAA-3′
N-cadherin	上游：5′-GTGCCATfAGCCAAGGGAA-3′
?	下游：5′-GCGTTCCTGTTCCACTCATAGGAG-3′
Vimentin	上游：5′-TTCTTCCCTGAACCTGAGAGA-3′
?	下游：5′-GAGTGGGTGTCAACCAGAGG-3′
C-myc	上游：5′-TCAAGAGGCGAACACACAAC-3′
?	下游：5′-GGCCTTTTCATTGTTTTCCA-3′
LEF1	上游：5′-CACTGTAAGTGATGAGGGGG-3′
?	下游：5′-TGGATCTCTTTCTCCACCCA-3′
CCND1	上游：5′-TTCTGCCTTTGATGTTAC-3′
?	下游：5′-AGGCTGAATCAATGTCTT-3′
CD44	上游：5′-AGTCCCTGGATCACCGACAG-3′
?	下游：5′-GTTTCTTGCCTCTTGGTTGCT-3′
MMP-7	上游：5′-GCATCTCCTTGAGTTTGGCT-3′
?	下游：5′-GAGCTACAGTGGGAACAGGC-3′
DKK1	上游：5′-ATAGCACCTTGGATGGGTATTCC-3′
?	下游：5′-CTGATGACCGGAGACAAACAG-5′
GAPDH	上游：5′-GACTCATGACCACAGTCCATGC-3′
?	下游：5′-AGAGGCAGGGATGATGTTCTG-3′

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