Knockdown of RhoC Inhibits Oral Squamous Cell Carcinoma Cell Invasion and Metastasis via Regulation of HMGA2

Abstract

Ras homolog family member C (RhoC) is an important component of intracellular signal transduction and its overexpression has been reported to be involved in regulating tumor proliferation, invasion, and metastasis in various malignant tumors. However, its role and underlying mechanism in oral squamous cell carcinoma (OSCC) still remain obscure. In our study, RhoC expression, its relation with clinical stages, and survival rate in OSCC were analyzed using datasets from The Cancer Genome Atlas (TCGA). Next, a RhoC knockdown cell model was established in vitro, and the effects of RhoC knockdown in OSCC cells were detected by the MTT assay, colony formation assay, transwell invasion assay, scratch assay, and F-actin phalloidin staining. An in vivo tongue-xenografted nude mouse model was established to measure the effects of knockdown of RhoC on tumor cell growth and lymph node metastasis. A mechanism study was conducted by real-time PCR and immunocytochemistry. The results of TCGA analysis showed that RhoC was overexpressed in OSCC tumor tissues. In vitro assays indicated that knockdown of RhoC did not have much effect on OSCC cell growth but significantly suppressed cell colony formation, invasion, and migration abilities, and F-actin polymerization was also reduced. The tongue-xenografted in vivo model demonstrated that knockdown of RhoC suppressed OSCC cell growth and inhibited metastasis to the superficial cervical lymph nodes. Further mechanism studies showed that knockdown of RhoC downregulated HMGA2 expression, and HMGA2 expression was highly correlated with RhoC expression in OSCC tumor tissues via the analysis of TCGA datasets. Overall, our study showed that knockdown of RhoC inhibited OSCC cells invasion and migration in vitro and OSCC cell growth and lymph node metastasis in vivo. Moreover, the potential mechanisms involved in these activities may be related to the regulation of HMGA2 expression. The RhoC gene could serve as a promising therapeutic target for OSCCs in the future.

Figure 1

Assessment of RhoC in OSCC patients analyzed in TCGA datasets. (a) The expression levels of RhoC in OSCC tumor tissues are higher than in normal human epithelial tissues analyzed in the TCGA dataset (P < 0.01). (b) Relative RhoC expression levels at each stage, the expression levels of RhoC in stages II–IV are higher than in stage I. (c) The association of RhoC expression with overall survival (OS) in OSCC patients (P=0.281).

Figure 2

RhoC gene knockdown in OSCC cells. (a) Stable transfection of lentivirus with scrambled or RhoC/shRNA in OSCC cells is indicated by red fluorescence (magnification, ×200). (b) Expression of RhoC at the protein level is significantly decreased in RhoC/shRNA cells by ICC, bar = 100 μm. (c) Expression of RhoC at the RNA level is significantly decreased in RhoC/shRNA cells compared to controls as assessed by RT-PCR. ^∗P < 0.05 versus control.

Figure 3

Knockdown of RhoC has no effect on OSCC cell proliferation but could inhibit colony formation. (a) Knockdown of RhoC has no effect on the growth of OSCC cells (CAL-27 and SCC-15 cells) as shown by the MTT assay. (b) Knockdown of RhoC inhibits the colony formation of CAL-27 compared with control group. ^∗P < 0.05 versus control. (c) Knockdown of RhoC inhibits the colony formation of SCC-15 compared with control group versus control.

Figure 4

Knockdown of RhoC decreases the invasive ability of OSCC cells ((a) CAL-27 and (b) SCC-15 cells) in the transwell invasion assay (magnification, ×200). ^∗P < 0.05 versus control. Bar = 100 μm.

Figure 5

Knockdown of RhoC suppresses the migration ability of OSCC cells ((a) CAL-27 and (b) SCC-15 cells) in the scratch migration assay. ^∗P < 0.05 versus control. Bar = 500 μm.

Figure 6

Knockdown of RhoC suppresses the F-actin polymerization of OSCC cells ((a) CAL-27 and (b) SCC-15 cells). ^∗P < 0.05 versus control. Bar = 50 μm.

Figure 7

Knockdown of RhoC suppresses CAL-27 cell growth in the tongue-xenografted nude mice model. (a) H&E staining of the tongue in nude mice, bar above = 200 μm, bar below = 2 mm. The degree of epithelial cell proliferation was lower in the RhoC/shRNA group than in controls. (b) The proportion of tumor tissue in the tongue of nude mice is lower in RhoC/shRNA group than in controls. ^∗P < 0.05. (c) Comparison of mouse body weights.

Figure 8

Knockdown of RhoC inhibits CAL-27 cell metastasis to the superficial cervical lymph nodes and reduces the metastasis rate of lymph nodes. (a) H&E staining of the tumor mass in the superficial cervical lymph nodes, bar = 500 μm. (b) The proportion of metastatic tumor tissue in the superficial cervical lymph nodes (% of control) is lower in the RhoC/shRNA group than in controls. ^∗P < 0.05 versus control. (c) The metastasis rate of lymph node tissues is lower in the RhoC/shRNA group than in controls. ^∗P < 0.05 versus controls.

Figure 9

Knockdown of RhoC regulates the expression of HMGA2. (a) Relative RNA expression level of HMGA2 is lower in the RhoC/shRNA group. ^∗P < 0.05 versus controls. (b) Protein expression of HMGA2 is lower in the RhoC/shRNA group by ICC, bar = 100 μm.

Figure 10

The analysis of HMGA2 in the TCGA datasets. (a) The expression of HMGA2 is higher in OSCC than in normal epithelial tissues analyzed in TCGA datasets. (b) RhoC is co-expressed with HMGA2 in the correlation analysis (Pearson's correlation) in OSCC tissues, P < 0.001, R = 0.39. (c) The association of HMGA2 expression with overall survival (OS) in OSCC patients P=0.267.