Elevated microRNA-145 inhibits the development of oral squamous cell carcinoma through inactivating ERK/MAPK signaling pathway by down-regulating HOXA1

Abstract

Background: Oral cancer is one of the most frequent solid cancers worldwide, and oral squamous cell carcinoma (OSCC) constitutes approximately 90% of oral cancers. The discovery of reliable prognostic indicators would be a potential strategy for OSCC treatment. In the present study, we aim to explore the underlying mechanism by which microRNA-145 (miR-145) affected OSCC. Methods: Forty-eight patients diagnosed with OSCC were enrolled to obtain the OSCC tissues and adjacent normal tissues. The targeting relationship between miR-145 and Homeobox A1 (HOXA1) was verified. In order to assess the effects of miR-145 in OSCC and the detailed regulatory mechanism, the SCC-9 cell line was adopted, in which expression of miR-145 and HOXA1 were altered by transfection. Then, a series of in vitro and in vivo experiments were performed to evaluate the cell viability, migration, invasion, and tumor growth. Results: miR-145 was poorly expressed and HOXA1 was highly expressed in OSCC. HOXA1 was verified as a target of miR-145 to mediate the activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway. In the circumstance of miR-145 elevation or HOXA1 depletion, the SCC-9 cell line manifested with inhibited cell viability, invasion, and migration in vitro, coupled with reduced tumor growth in vivo, with a decreased expression of ERK/MAPK signaling pathway-related genes/proteins. Conclusion: These findings suggested that miR-145 can inhibit HOXA1 to inactivate the ERK/MAPK signaling pathway, thereby suppressing OSCC cell proliferation, migration, and invasion to further inhibit the development of OSCC, highlighting a novel therapeutic target for the OSCC treatment.

Keywords: Extracellular-signal-regulated kinase/mitogen activated protein kinase; Homeobox A1; MicroRNA-145; Oral squamous cell carcinoma.

Figure 1. Poorly expressed miR-145 was found in OSCC tissues relative to adjacent normal tissues

(A) Relative expression of miR-145 in OSCC tissues and adjacent normal tissues detected by RT-qPCR. (B) Relative expression of miR-145 in OSCC patients with different ages detected by RT-qPCR (≥60, n=23; >60, n=25). (C) Relative expression of miR-145 in male and female OSCC patients determined by RT-qPCR (male, n=26; female, n=22). (D) Relative expression of miR-145 in OSCC patients with or without LNM detected by RT-qPCR (patients with LNM, n=15; patients without LNM, n=33). (E) Relative expression of miR-145 in OSCC patients at IIa/IIb and Ia/Ib stages measured using RT-qPCR (patients at Ia/Ib stage, n=27; patients at IIa/IIb stage, n=21). (F) Relative expression of miR-145 in moderately and highly differentiated and poorly differentiated and undifferentiated OSCC patients detected by RT-qPCR (moderately and highly differentiated OSCC patients, n=41; poorly differentiated and undifferentiated OSCC patients, n=7). The measurement data were expressed as mean ± standard deviation. The differences between OSCC tissues and adjacent normal tissues were analyzed by paired t test; n=48; comparisons between two groups were analyzed using independent sample t test. The experiment was repeated three times.

Figure 2. HOXA1 is a target gene of miR-145

(A) Binding site of miR-145 and HOXA1; (B) miR-145 binds to the 3′UTR of HOXA1, decreasing the luciferase activity; the measurement data were expressed as mean ± standard deviation; the data between two groups were compared by independent sample t test; the experiment was repeated three times; mirSVR refers to thermodynamic stability value (≤ −0.1): lower value suggests stronger binding stability of miRNA–mRNA, and thereby miRNA is more likely to down-regulate the expression of the gene; PhastCons refers to the evolutionary conservation of gene UTR in all species (≥0): the more conservative, the better. *, P<0.05 compared with the HOXA1-mut group.

Figure 3. miR-145 overexpression or HOXA1 depletion attenuates OSCC cell proliferation

The measurement data were expressed as mean ± standard deviation and analyzed by repeated-measurement ANOVA followed by Dunnett’s post hoc test; the experiment was repeated three times. Abbreviation: siHOXA1, siRNA against HOXA1. * P<0.05 compared with the mimic NC group or the inhibitor NC group.

Figure 4. Enforced miR-145 overexpression or reduced HOXA1 inhibits OSCC cell invasion and migration

(A) Representative images of cell width at 0 and 48 h in each group; (B) diagram of width of injury line in each group; (C) representative images of cell invasion in each group; (D) the number of invasive cells in each group; the measurement data were expressed as mean ± standard deviation and analyzed by one-way ANOVA, followed by Tukey’s post hoc test; the experiment was repeated three times. *, P<0.05 compared with the mimic NC group or the inhibitor NC group. Abbreviation: siHOXA1, siRNA against HOXA1.

Figure 5. Flow cytometry analysis indicated that miR-145 reduced yet HOXA1 facilitated the growth of CD44⁺ tumor OSCC cells by statistical analysis of ANOVA

The measurement data were expressed as mean ± standard deviation and analyzed by one-way ANOVA, followed by Tukey’s post hoc test; the experiment was repeated three times. % refers to the proportion of CD44⁺ cells in total cells. *, P<0.05 compared with the mimic NC group or the inhibitor NC group.

Figure 6. miR-145 decreased HOXA1 expression and inhibited the activation of the ERK/MAPK signaling pathway in vitro

(A) miR-145 expression and the mRNA expression of HOXA1 in each group; (B) protein bands in each group; (C) protein levels of HOXA1, ERK, MEK, and the extents of MEK and ERK phosphorylation in each group; the measurement data were expressed as mean ± standard deviation and analyzed by one-way ANOVA, followed by Tukey’s post hoc test; the experiment was repeated three times. *, P<0.05 compared with the mimic NC or inhibitor NC group. Abbreviations: p-ERK, phosphorylated-ERK; p-MEK, phosphorylated-MEK; siHOXA1, siRNA against HOXA1.

Figure 7. miR-145 up-regulation and HOXA1 knockdown suppressed the growth of xenograft tumors in nude mice

(A) Changes in tumor volume at 0, 9, 18, and 27 days; (B) xenograft tumors in each group; (C) relative expression of miR-145 in each group; (D) protein bands in each group; (E) relative protein levels of HOXA1, ERK, and MEK in each group; the measurement data were expressed as mean ± standard deviation and analyzed by one-way ANOVA; the data among multiple groups at different time points were analyzed using repeated-measurement ANOVA, followed by Dunnett’s post hoc test. Data in different groups were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. n=6; the experiment was repeated three times. *, P<0.05 compared with the blank group (the nude mice only injected with SCC-9 cell line). Abbreviation: siHOXA1, siRNA against HOXA1.