MicroRNA-433 inhibits oral squamous cell carcinoma cells by targeting FAK

Abstract

We investigated the involvement of microRNA-433 (miR-433) in the proliferation, migration, and invasiveness of oral squamous cell carcinoma (OSCC). Totally 108 OSCC tissues and adjacent normal tissues from patients with OSCC were collected. Also, transplanted tumor formation experiment in nude mice was conducted to verify the effect of miR-433 and FAK on subcutaneous transplanted tumor. The CD44⁺ stem cell from SCC-9 were collected and assigned into the blank, miR-433 mimics, mimics control, miR-433 inhibitors, inhibitors control, siFAK and miR-433 inhibitors + siFAK groups. The qRT-PCR and western blotting were used to detect miR-433, FAK, ERK, MEK, pERK and pMEK after transfection. Flow cytometry, MTT assay, scratch test and Transwell assay were performed to determine the cell proportion, growth, migration and invasion of SCC-9 cells. In cell line SCC-9, expression of CD133, Oct-4, and BIM-1 was greater in CD44⁺ cells than CD44^- cells, indicating that CD44⁺ cells had characteristics of tumor stem cells. Expression of FAK, ERK, MEK, p-ERK and p-MEK was decreased in tumor tissues from the CD44^-, miR-433, and siFAK groups. Expression of MiR-433 mRNA was elevated, while levels of FAK, ERK, MEK, p-ERK, and p-MEK mRNA were all decreased in the miR-433 mimics group. In the miR-433 mimics and siFAK groups, cell proliferation, migration, and invasion were all decreased, while the opposite trends were seen in the miR-433 inhibitor group. These results indicate that miR-433 downregulates FAK through the ERK/MAPK signaling pathway to inhibit the proliferation, migration, and invasiveness of SCC-9 OSCC cells.

Keywords: ERK; FAK; SCC-9 cell; microRNA-433; oral squamous cell carcinoma.

Figure 1. Sorting and identification of stem cells from cell line SCC-9

(A-B), CD44⁺ cells sorted by flow cytometry; (C), relative expression of miR-433 and FAK mRNA in the stem cells and non-stem cells; (D), cell cycle detected by flow cytometry; (E), specific protein expressions of stem cells detected by immunofluorescence staining; ^*, P < 0.05, compared with non-stem cells; SCC, squamous cell carcinoma; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 2. Effects of miR-433 and FAK on subcutaneous transplanted tumor in nude mice in sorted CD44 cells and unsorted SCC-9 cells

(A), transplanted tumor growth curve; (B), tumor formation results; (C), comparisons of miR-433 relative expressions; (D), histogram of protein expressions; (E), comparisons of protein expressions; ^*, P < 0.05, compared with the control group; #, P < 0.05, compared with the CD44^- group; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 3. Targeting relationships between miR-433 and FAK

(A), Combination of miR-433 and FAK 3’UTR; (B), results of luciferase assay; miR-433 mimics had no significant influence on the luciferase activity of Mut-miR-433/FAK, while it reduced luciferase activity of Wt-miR-433/FAK; ^*, P < 0.05; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 4. Expressions of miR-433, FAK and relevant proteins in the ERK/MAPK signaling pathway

(A), relative mRNA expressions of miR-433, FAK, ERK and MEK in each group; (B), histogram of transfecting FAK, ERK, MEK and phosphorylated proteins by western blotting; (C), expression analysis of FAK, ERK MEK and phosphorylated proteins; ^*, P < 0.05, compared with the blank group; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 5. Effects of miR-433 on cell proliferation ability in each group

^*, P < 0.05, compared with the blank group; miR-433, microRNA-433; OD, optical density; FAK, focal adhesion kinase.

Figure 6. Effects of miR-433 on cell migration ability in each group

(A), Scratch test results of each group; (B), comparisons of scratch width of cells in each group, ^*, P < 0.05, compared with the blank group; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 7. Effects of miR-433 on cell invasion ability in each group

(A), Transwell assay results of each group; (B), the number of cells penetrating through the bottom membrane of the chamber in each group; ^*, P < 0.05, compared with the blank group; miR-433, microRNA-433; FAK, focal adhesion kinase.

Figure 8. MiR-433 expressions in the OSCC tissues and adjacent normal tissues by qRT-PCR

^*, P < 0.05, compared with the normal adjacent tissues; miR-433, microRNA-433; OSCC, oral squamous cell carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; FAK, focal adhesion kinase.

Figure 9. FAK, ERK and MEK expressions in the OSCC tissues and the normal adjacent tissues (× 200)

FAK and MEK were mainly expressed as pervasive and brown particles in the cytoplasm, while ERK was expressed primarily in the nucleus as indicated by brown staining. The positive expression rates of these three proteins in the OSCC tissues were all significantly higher than those in the adjacent normal tissues; OSCC, oral squamous cell carcinoma; FAK, focal adhesion kinase.

Figure 10. Correlation analysis among miR-433, FAK, ERK, and MEK

(A), correlation analysis between miR-433 and FAK; (B), correlation analysis between miR-433 and ERK; (C), correlation analysis between miR-433 and MEK; miR-433, microRNA-433; FAK, focal adhesion kinase.