<em>miR-19a</em> targeting <em>CLCA4</em> to regulate the proliferation, migration, and invasion of colorectal cancer cells

Abstract

The role of miR-19a in colorectal cancer (CRC), a devastating disease with high mortality and morbidity, remains controversial. In the present study, we show that the level of miR-19a is significantly higher in clinical CRC tissue samples than in paracancerous tissue samples, and significantly higher in CRC cells lines HT29, SW480, and CaCO2 than in the normal human colon mucosal epithelial cell line NCM460. miR-19a mimics and inhibitors were synthesized and validated. Overexpression of miR-19a mimics significantly promoted, while miR-19a inhibitors inhibited, the proliferation, survival, migration, and invasion of SW480 and CaCO2 CRC cells. Furthermore, mRNA and protein levels of chloride channel accessory 4 (CLCA4) were lower in CRC cells and tissues. Bioinformatics and a luciferase reporter assay confirmed that CLCA4 was a miR-19a target. Further, miR-19a inhibition increased CLCA4 expression. The inhibitory effect of miR-19a on cell growth, survival, migration, and invasion was reversed by knockdown of CLCA4 expression. The data demonstrated that the miR-19a/CLCA4 axis modulates phospho-activation of the PI3K/AKT pathway in CRC cells. In conclusion, our results revealed that miR-19a overexpression decreases CLCA4 levels to promote CRC oncogenesis, suggesting that miR-19a inhibitors have potential applications for future therapeutic of CRC.

Figure 1.

Expression levels of miR-19a are upregulated in CRC tissues and cell lines. A) RT-qPCR was used to measure expression levels of miR-19a in 19 pairs of CRC tumor tissues and adjacent paracancerous control tissues. B) The normal human colon mucosal epithelial cell line NCM460 and the CRC cell lines HT29, SW480, and CaCO were subjected to RT-qPCR to measure miR-19a levels. *p<0.05.

Figure 2.

Regulatory role of miR-19a in growth and apoptosis of CRC cells. A,B) Synthesized miR-19a mimics or inhibitors or controls (miR-NC or inhibitor-NC, respectively) were transfected into SW480 and CaCO cells, and miR-19a was measured by RT-qPCR. C,D) CRC cells transfected with miR-19a mimics or inhibitors were subjected to a CCK-8 assay. The growth rate, indicated by the OD at 450 nm, is shown. E) Cells were treated as in (C) and were subjected to flow cytometry to detect apoptosis. *p<0.05.; n=3.

Figure 3.

Effect of miR-19a on CRC cell migration and invasion. Cells were treated as in Figure 2, and were then subjected to migration and invasion assays. Representative images of migrated and invaded cells are shown (A,C), and cells were counted for quantitative analyses as shown in (B,D). **p<0.01; ***p<0.001; n=3; scale bars: 20 μm.

Figure 4.

Expression of CLCA4 in CRC. A) CLCA4 mRNA levels were detected in CRC and control cell lines. B) Protein levels of CLCA4 were detected in CRC and control cell lines, with quantified data shown in (C). D) The distribution of CLCA4 in CRC tissues was assessed by immunohistochemistry; scale bar: 50 μm. The relative expression level of CLCA4 in IHC images was measured as shown in (E). (F) Sequence of the CLCA4 3’UTR with the miR-19a target sequence is shown, and the mutant (Mut) sequence miR-19a target sequence is shown in red. Cells were transfected with luciferase reporter genes expressing WT or Mut CLCA4 3’ UTRs, with or without miR-19a mimics. Relative luciferase activity is shown. *p<0.05; **p<0.01.

Figure 5.

The miR-19a/CLCA4 axis regulates cell development of CRC cells. A,B) Cells were transfected with miR-19a inhibitor or NC, with or without an shCLCA4-encoding plasmid; cells were then subjected to Western blotting with CLCA4 antibody. The images and relative expression levels of CLCA4 are shown. C,D) CCK-8 assay to detect the growth of CRC cells treated as in (A). E,F) Flow cytometry assay to detect cell apoptosis in CRC cells treated as in (A). Transwell assay to detect cell migration (G-H) and invasion (I-J) in CRC cells treated as in (A). **p<0.01; ***p<0.001; n=3; scale bars: 20 μm.

Figure 6.

The miR-19a/CLCA4 axis regulates activation of the PI3K/AKT pathway. A) CaCO and SW480 CRC cells were transfected with miR-19a mimics fragments. NC, negative control; 48 h later, cells were subjected to Western blotting with anti-CLCA4, anti-PI3K, anti-phosphorylated PI3K (p-PI3K), anti-AKT, anti-phosphorylated AKT (p-AKT), and anti-GAPDH (loading control) antibodies. B) Quantified data of relative levels of CLCA4, p-PI3K/PI3K and p-AKT/AKT are shown. n=3; *p<0.05.