High expression of COL10A1 is associated with poor prognosis in colorectal cancer

Abstract

Background: High expression of collagen type X alpha 1 chain (COL10A1), a member of the collagen family, had been observed in various human cancers, but the detailed function and molecular mechanism of COL10A1 were largely unclear.

Aim: The aim of this study was to investigate the expression of COL10A1 in colorectal cancer (CRC) tissues and cells and to reveal its biological function and mechanism in CRC.

Materials and methods: Immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (QPCR) and Western blot experiments were used to determine the clinical relevance between expression levels of COL10A1 and CRC.

Results: Compared with normal tissues, COL10A1 expression was significantly higher in CRC tissues. Biological functional experiments showed that overexpression of COL10A1 enhanced proliferation, migration, and invasion of CRC cells, and knockdown of COL10A1 inhibited tumorigenesis in vivo. Western blot assays showed that COL10A1 promoted the process of epithelial-mesenchymal transition (EMT). The overexpression of COL10A1 was associated with adverse prognosis in CRC by tissue microarray (TMA) analysis.

Conclusion: Our findings had provided evidences to support the fact that COL10A1 was abnormally up-expressed in CRC and involved in the progression of CRC and the process of EMT. Furthermore, we demonstrated that the high-level expression of COL10A1 was an independent risk factor of prognosis and overall survival in CRC patients. These suggested that COL10A1 might be a new potential target for cancer therapy in the future.

Keywords: collagen type X alpha 1 chain; colorectal cancer; epithelial-mesenchymal transition; proliferation and invasion.

Figure 1

COL10A1 is overexpressed in CRC tissues. (A) Expression of COL10A1 in CRC patients was detected by Western blot assays, followed by gray value analysis. (B) COL10A1 expression levels of CRC and corresponding normal tissues detected by QPCR were shown using a histogram and pie chart. The fold change of COL10A1 expression (T/N).2 was defined as overexpressed. (C) IHC staining of COL10A1 protein in 30 pairs of CRC and nontumorous tissues and followed scores pie chart and average score analysis. ***P<0.001. Abbreviations: COL10A1, collagen type X alpha 1 chain; CRC, colorectal cancer; QPCR, quantitative polymerase chain reaction; T, tumor tissues; N, normal tissues; IHC, immunohistochemistry.

Figure 2

COL10A1 promotes the migration and invasion of CRC cells in vitro. (A) The expression of COL10A1 in six cell lines was detected by QPCR and Western blot assays. (B, C) Transfection efficiency of LoVo, HCT116, SW480, and SW620 was detected by QPCR and Western blot assays. (D, E) Cell migration was tested using wound healing assay. (F, G) The cells’ migration and invasion changes after transfection were tested by Transwell chamber migration assay and invasion assay. **P<0.01, ***P<0.001. Abbreviations: COL10A1, collagen type X alpha 1 chain; CRC, colorectal cancer; QPCR, quantitative polymerase chain reaction; NC, normal control.

Figure 2

COL10A1 promotes the migration and invasion of CRC cells in vitro. (A) The expression of COL10A1 in six cell lines was detected by QPCR and Western blot assays. (B, C) Transfection efficiency of LoVo, HCT116, SW480, and SW620 was detected by QPCR and Western blot assays. (D, E) Cell migration was tested using wound healing assay. (F, G) The cells’ migration and invasion changes after transfection were tested by Transwell chamber migration assay and invasion assay. **P<0.01, ***P<0.001. Abbreviations: COL10A1, collagen type X alpha 1 chain; CRC, colorectal cancer; QPCR, quantitative polymerase chain reaction; NC, normal control.

Figure 3

Knockdown of COL10A1 inhibits cells proliferation in vitro and tumorigenesis in vivo. (A, B) Knockdown of COL10A1 repressed SW480 cells proliferation by CCK-8 assay and colony formation assay. (C) Cells were subcutaneously injected into the right flank of nude mice. Tumor volumes were repeatedly measured on the indicated days to evaluate the effects of COL10A1 on tumor growth. Data points are showed as the mean ± SD tumor volume. **P<0.01, ***P<0.001. Abbreviations: COL10A1, collagen type X alpha 1 chain; CCK-8, Cell counting kit 8; SD, standard deviation; NC, normal control.

Figure 4

COL10A1 promotes the EMT process. The effect of COL10A1 in the EMT was detected by Western blot assay. E-cadherin, N-cadherin, Beta-catenin, Slug, and Snail were detected after successful transfection. Abbreviations: COL10A1, collagen type X alpha 1 chain; EMT, epithelial–mesenchymal transition; NC, normal control.

Figure 5

COL10A1 overexpression is correlated with adverse prognosis in CRC. (A) Tissue microarray analysis of COL10A1 in human CRC tissues. Original magnification, 100× and 200×. (B) Kaplan–Meier survival analysis of COL10A1 expression in patients with CRC (log-rank test). Abbreviations: COL10A1, collagen type X alpha 1 chain; CRC, colorectal cancer.