miR182 activates the Ras–MEK–ERK pathway in human oral cavity squamous cell carcinoma by suppressing RASA1 and SPRED1

Figures & data

Table 1 Clinical and pathological data of patients in this study

Patient	Site	Sex	Age (years)	Pathologic stage
1	Tongue	Male	65	T2N2bM0
2	Tongue	Male	76	T3N2cM0
3	Floor of mouth	Female	48	T2N1M0
4	Tongue	Male	67	T1N2bM0
5	Gum	Female	57	T2N1M0
6	Gum	Female	59	T2N2bM0
7	Floor of mouth	Male	55	T1N1M0
8	Tongue	Male	64	T3N2cM0
9	Tongue	Male	38	T3N2bM0
10	Tongue	Female	72	T3N0M0

Figure 1 Shared putative microRNAs potentially regulating both RASA1 and SPRED1 are identified by the prediction algorithm of TargetScan.

Notes: Based on the algorithm of TargetScan, there were 80 miRNAs that were predicted to only regulate SPRED1, 14 miRNAs to only regulate RASA1, and overlapped 24 miRNAs to regulate both SPRED1 and RASA1.

Figure 2 Expression differentiation of predicted miRNAs between malignant tissues and adjacent nonmalignant tissues from ten OSCC patients.

Notes: (A) Heat map generated by cluster analysis of the predicted miRNAs potentially regulating RASA1 and SPRED1 in OSCC patients based on the data of TaqMan qRT-PCR; (B) expression of miR182 matched between ten paired OSCC tissues (**P<0.01).
Abbreviations: T, malignant tumor tissues; N, adjacent nonmalignant tissues; miRNAs, microRNAs; OSCC, oral cavity squamous cell carcinoma; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

Table 2 Deregulated miRNAs potentially regulating RASA1 and SPRED1 between ten paired OSCC malignant tissues and adjacent noncancerous tissues

miRNA	Fold-change	P-value
miR31	4.27	0.0018
miR182	2.87	0.0009
miR200a	2.4	0.0039
miR141	1.7	0.0246
miR1	−4.46	0.0020
miR206	−2.73	0.0092
miR212	−1.96	0.0237

Notes: Fold-change values represented by ratio of mean values of malignant groups and normal groups by the method of 2^−ΔΔCt. Negative fold-change values indicate overexpression in adjacent normal tissues. P-values indicate the significance level for each miRNA analyzed using paired t-test.

Abbreviations: miRNAs, microRNAs; OSCC, oral cavity squamous cell carcinoma.

Figure 3 The reverse correlation of miR182 and RASA1 or SPRED1 protein levels.

Notes: (A) Expression of RASA1 and SPRED1 between paired OSCC tissues was examined by Western blot and was normalized by β-actin; (B) correlation between miR182 and RASA1 or SPRED1 represented by relative expression.
Abbreviations: T, malignant tumor tissues; N, adjacent nonmalignant tissues; OSCC, oral cavity squamous cell carcinoma.

Figure 4 miR182 promotes OSCC cell proliferation and cell-cycle progression.

Notes: Tca8113 cells were transfected with miR-NC, miR182 mimics, anti-miR-NC, and anti-miR182 oligonucleotides, and divided into four groups. (A) Effects of miR182 on the proliferation of Tca8113 cells were examined at 24, 36, 48, 60, and 72 hours after transfection using a CCK-8 assay kit. (B) Representative micrograph of each group’s Tca8113 cell colonies determined by colony-formation assay. (C) Effects of miR182 on cell-cycle progression analyzed by flow cytometry and quantification of cell percentages in G₀–G₁, S, and G₂–M phases. Different colors represented the cells in different phases of cell cycles: green, G₁ phase; yellow, S phase; blue, G₂ phase. *P<0.05 vs miR-NC; **P<0.01 vs miR-NC; ^##P<0.01 vs anti-miR-NC.
Abbreviations: Oligos, oligonucleotides; OSCC, oral cavity squamous cell carcinoma; NC, negative control; OD, optical density.

Figure 5 miR182 inhibits the apoptosis of OSCC cells.

Notes: Tca8113 cells were transfected with miR182 mimics, miR-NC, anti-miR182 oligonucleotides, or anti-miR-NC for 48 hours. Cell apoptosis was detected by flow cytometry by annexin V–FITC/PI staining. The experiment was performed in triplicate. **P<0.01 vs miR-NC; ^#P<0.05 vs anti-miR-NC.
Abbreviations: Oligos, oligonucleotides; OSCC, oral cavity squamous cell carcinoma; NC, negative control; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 6 miR182 promotes the invasion of OSCC cells.

Notes: Invasion assays were performed using Tca8113 cells transfected with miR182 mimics, anti-miR182 oligonucleotides, or controls. Representative images of Tca8113 cells for the four groups were captured. Magnification ×200. **P<0.01 vs miR-NC; ^##P<0.01 vs anti-miR-NC.
Abbreviations: Oligos, oligonucleotides; OSCC, oral cavity squamous cell carcinoma; NC, negative control.

Figure 7 miR182 promotes the activation of the Ras–MEK–ERK signaling pathway by suppressing the expression of RASA1 and SPRED1.

Notes: Western blot was performed to examine the expression differentiations of RASA1, SPRED1, Ras-GTP, and phospho-ERK1/2 in Tca8113 cells after transfection with miR182 mimics or anti-miR182 oligonucleotides. Relative band intensities were analyzed by Image-Pro Plus 6.0, Ras was used for normalization of Ras-GTP, and β-actin was used for others. **P<0.01 vs miR-NC; ^#P<0.05 vs anti-miR-NC; ^##P<0.01 vs anti-miR-NC.
Abbreviations: NC, negative control; p, phospho.

Figure 8 miR182 regulates RASA1 and SPRED1 by directly targeting their 3′UTRs.

Notes: (A) Schematic representation of mature human miR182 sequence and miR182 target-binding sites in the 3′UTRs of RASA1 and SPRED1 mRNAs. (B) For luciferase-activity assays, 3′UTR of RASA1 or SPRED1 was cloned into the pMirGlo reporter plasmid separately, and the constructed plasmid was cotransfected with miR182 mimics, miR-NC, anti-miR182 oligonucleotides, or anti-miR-NC individually. The normalized activity of the reporters relative to Renilla luciferase was analyzed 48 hours after transfection. **P<0.01 vs miR-NC; ^#P<0.05 vs anti-miR-NC.
Abbreviations: UTRs, untranslated regions; mRNAs, messenger RNAs; NC, negative control.