MicroRNA MiR-490-5p suppresses pancreatic cancer through regulating epithelial-mesenchymal transition via targeting MAGI2 antisense RNA 3

Abstract

Pancreatic cancer with about 5% five-year overall survival rate remains a challenge. Invasion and migration of pancreatic cancer cells are the main factors leading to poor prognosis. MicroRNA-490-5p (miR-490-5p) has anti-cancer effects in a variety of tumors, but its role in pancreatic cancer has not been reported. The mRNA expressions of miR-490-5p, MAGI2 antisense RNA 3 (MAGI2-AS3), Matrix metalloproteinase (MMP)2, MMP9, N-cadherin, and E-cadherin were detected by quantitative real-time PCR, while the protein expressions of these genes except miR-490-5p were measured by Western blot analysis. The cell viability, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8), apoptosis and transwell assays. MiR-490-5p was abnormally low-expressed in pancreatic cancer, whose down-regulation generated enhanced effects on viability, migration and invasion in pancreatic cancer cells, as well as MAGI2-AS3 expression. MiR-490-5p mimic exerted the opposite effect on cells, which also down-regulated MMP2, MMP9, and N-cadherin protein expressions, while up-regulating E-cadherin protein expression. MAGI2-AS3, which was the targeted binding site of miR-490-5p, promoted viability, migration and invasion, and inhibited apoptosis of cancer cells. More importantly, miR-490-5p played an anti-cancer role in pancreatic cancer by targeting MAGI2-AS3 and regulating epithelial-mesenchymal transition (EMT), which was partially offset by MAGI2-AS3.

Keywords: Epithelial–mesenchymal transition; MAGI2-AS3; MicroRNA-490-5p.

Figure 1.

Expression of miR-490-5p in pancreatic cancer and its effect on the cell viability. (a) Expression of miR-490-5p in pancreatic cancer tissues was found to be lower than that in adjacent tissues, which was detected by quantitative real-time PCR (qRT-PCR). (b) The expression level of miR-490-5p in normal pancreatic duct epithelial cells HPDE6-C7 was significantly higher than that in pancreatic cancer cell lines PANC-1, SW1990, AsPC-1, and BxPC-3, which was detected by qRT-PCR. (c-d) The qRT-PCR was used to detect the level of miR-490-5p in cells to verify successful transfection. (e-f) Cell counting kit-8 (CCK-8) assay was used to detect cell viability. As the cell viability increased over time, the silencing of miR-490-5p inhibited cell viability, but the overexpression of miR-490-5p showed the opposite effect. *p < 0.05, **p < 0.001, vs. HPDE6-C7 or control; ^{^}p < 0.05, ^{^^}p < 0.001 vs. inhibitor control or mimic control.

Figure 2.

Effect of miR-490-5p on migration, invasion and apoptosis of cancer cells. (a-d) Detection of apoptotic rate by flow cytometry revealed that silencing of miR-490-5p reduced the apoptotic rates of cells, while overexpression of miR-490-5p showed the opposite effect. (e-f) Silencing of miR-490-5p in cells promoted migration, while overexpression of miR-490-5p did the opposite, which was detected by transwell experiment. (g-h) Silencing of miR-490-5p increased invasion, while overexpression of miR-490-5p did the opposite, which was detected by transwell experiment. **p < 0.001, vs. control; ^{^^}p < 0.001 vs. inhibitor control or mimic control.

Figure 3.

The targeting relation between MAGI2-AS3 and miR-490-5p. (a) The Venn graph achieved the intersection of MAGI2-AS3 and miR-490-5p, with the differentially expressed genes in TCGA-PAAD analyzed by the GEPIA website (http://gepia.cancer-pku.cn/) and the miR-490-5p targeted lncRNA predicted by the LncBase database (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex). (b) Prediction of target relation between MAGI2-AS3 and miR-490-5p using GEPIA website. (c) The targeting relation between MAGI2-AS3 and miR-490-5p was identified by dual-luciferase reporter gene analysis. (d) The differential expression of MAGI2-AS3 in PAAD was analyzed by GEPIA. (e) Relative expression of MAGI2-AS3 in pancreatic cancer tissues was found to be lower than that in adjacent tissues, which was detected by qRT-PCR. (f) Pearson assay manifested that MAGI2-AS3 was negatively associated with miR-490-5p (figure 3f, p = 0.020, r = −0.516). **p < 0.001, vs. control+MAGI2-AS3-3ʹ-UTR or control; ^{^^}p < 0.001, vs. miR-490-5p+MAGI2-AS3-3ʹ-UTR-mut.

Figure 4.

MiR-490-5p targeting MAGI2-AS3 to regulate cell proliferation, migration, apoptosis and invasion in rescue experiments. (a) MAGI2-AS3 partially reversed the inhibitory effect of miR-490-5p overexpression on cell proliferation, which was detected by CCK-8. (b, e) MAGI2-AS3 partially reversed the enhanced effect of miR-490-5p overexpression on cell apoptosis, which was detected by flow cytometry. (c, f) Overexpression of miR-490-5p inhibited cell migration and MAGI2-AS3 partially reversed this effect, which was detected by transwell experiment. (d, g) MAGI2-AS3 partially reversed the inhibitory effect of miR-490-5p overexpression on cell invasion, which was detected by transwell experiment. **p < 0.001, vs. control; ^{^^}p < 0.001 vs. NC; ^##P < 0.001, vs. MAGI2-AS3; ^ΔΔp<0.001, vs MAGI2-AS3+ mimic.

Figure 5.

MiR-490-5p targeting MAGI2-AS3 to regulate epithelial–mesenchymal transition (EMT) process. (a) Overexpression of miR-490-5p in cells caused by transfection of miR-490-5p mimic. (b) Up-regulation of miR-490-5p down-regulated the expression of MAGI2-AS3, which was detected by qRT-PCR. (c-d) The mimic group had the lowest protein levels of Matrix metalloproteinase (MMP)2, MMP9 and N-cadherin, and the highest protein level of E-cadherin, while the opposite condition appeared in MAGI2-AS3 group, which were measured by Western blot. **p < 0.001, vs. control; ^{^^}p < 0.001 vs. NC; ^##P < 0.001, vs. MAGI2-AS3; ^ΔΔp<0.001, vs MAGI2-AS3+ mimic.