Upregulation of TSPAN12 is associated with the colorectal cancer growth and metastasis

Abstract

Tetraspanin 12 (TSPAN12), as an earliest member of tetraspanin family, has been recently shown to be highly expressed in several malignant tumors, such as lung cancer and breast cancer, which plays an important role in regulating cell proliferation, migration and invasion. However, the functional roles of TSPAN12 in colorectal cancer (CRC) remain largely unclear. In this study, the expression of TSPAN12 was up-regulated compared to that in paracarcinoma tissues. Higher TSPAN12 expression was significantly correlated with TNM stage, tumor size and lymph node metastasis. The vitro functional analysis, including MTT, colony formation, flow cytometry and transwell assays indicated that lentivirus-mediated TSPAN12 knockdown significantly suppressed cell proliferation, migration and invasion, induced cell apoptosis of CRC cells. In addition, knockdown of TSPAN12 remarkably decreased the growth of subcutaneously inoculated tumors in nude mice. Our findings for the first time supported that TSPAN12 might play a positive role in the regulation of CRC cell proliferation, migration and invasion. The inhibition of TSPAN12 may serve as a novel promising therapeutic strategy against human CRC.

Keywords: Colorectal cancer; TSPAN12; cell proliferation; invasion; shRNA.

Figure 1

Expression of TSPAN12 was upregulated in CRC tissues. A. The mRNA levels of TSPAN12 in 112 paired CRC tissues by real-time quantitative PCR analysis. B. Western blotting analysis of TSPAN12 protein levels in 8 matched CRC tumor (T) and non-tumor tissues (N). GAPDH was used as internal control. ***P < 0.001.

Figure 2

TSPAN12 expression was effectively silenced in CRC cells. (A) Expression analysis of TSPAN12 in five different CRC cell lines by Western blotting. The mRNA levels analysis of TSPAN12 in HT29 (B) and SW1116 (D) cells after shTSPAN12-1 or shTSPAN12-2 infection by RT-qPCR analysis. The protein levels analysis of TSPAN12 in HT29 (C) and SW1116 (E) cells after shTSPAN12-1 or shTSPAN12-2 infection by Western blot analysis. GAPDH was used as internal control. ***P < 0.001 in comparison with non-silencing scrambled control.

Figure 3

Loss-of-function analysis of TSPAN12 in CRC cells, including proliferation and invasion. The number of colonies in HT29 (A) and SW1116 (B) cells after shTSPAN12-1 infection analyzed by colony formation assay. (C) MTT assay was used to determine the proliferative rates of HT29 and SW1116 cells after shTSPAN12-1 infection. Cell migration and invasion assays were conducted on HT29 (D) and SW1116 (E) cells after shTSPAN12-1 infection, as confirmed by statistical analysis. *P < 0.05; ***P < 0.001 in comparison with non-silencing scrambled control.

Figure 4

Knockdown of TSPAN12 significantly promoted cell early and late apoptosis in CRC cells. Flow cytometer combined with Annexin V/7-AAD double staining was used to analyze the percentage of apoptotic cells in HT29 (A) and SW1116 (B) cells following TSPAN12 silencing, as confirmed by statistical analysis. *P < 0.05; ***P < 0.001 in comparison with non-silencing scrambled control.

Figure 5

TSPAN12 silencing reduced tumorigenesis of CRC in vivo. (A and D) Representative images of tumors formed in the mice in which shTSPAN12-1 or scrambled control shRNA-infected HT29 and SW1116 cells were injected. Tumor volume altered curve of nude mice injected by HT29 (B) and SW1116 (E) cells transfected with shTSPAN12-1 or scrambled shRNA. Tumor weight alteration of nude mice injected by HT29 (C) and SW1116 (F) cells transfected with shTSPAN12-1 or scrambled shRNA. Expression analysis of TSPAN12 in tumor tissues collected from nude mice injected by HT29/shTSPAN12-1 (G), SW1116/shTSPAN12-1 (H), respectively. **P < 0.01; ***P < 0.001 in comparison with non-silencing scrambled control.

Figure 6

TSPAN12 silencing regulated proteins associated with cell proliferation and metastasis. Western blot analysis of TSPAN12-related molecules, including p-Akt, p-Erk, caspase-3, E-cadherin and Vimentin in HT29 (A) and SW1116 (B) cells, respectively; GAPDH was used as internal control.