Upregulated expression of ACTL8 contributes to invasion and metastasis and indicates poor prognosis in colorectal cancer

Abstract

Background: ACTL8 is a member of the CT antigens. There are only few studies on the role of ACTL8 in malignant tumors. The aim of this study is to investigate the expression and clinical significance of ACTL8 protein in colorectal cancer (CRC).

Materials and methods: Human CRC tissues and cell lines, and paired adjacent non-tumor tissues and human intestinal epithelial cell lines were obtained to evaluate the expression of ACTL8. The association between protein expression of ACTL8 and clinicopathological parameters and prognosis of CRC patients was examined. The biological functions of ACTL8 in the invasion and metastasis of CRC were determined by wound healing and transwell invasion assays after silencing of ACTL8 in CRC cell lines. The potential target genes of ACTL8 were also identified by quantitative reverse transcription PCR and Western blotting after silencing of ACTL8 in CRC cell lines.

Results: It was found that ACTL8 was upregulated in human CRC tissues and cell lines. The expression of ACTL8 was positively associated with poor differentiation, invasion and metastasis, postoperative infection, and poor prognosis, but negatively associated with proximal margin length. In addition, silencing of ACTL8 significantly decreased the capacity of invasion and migration in HT29 and SW620 CRC cell lines. Moreover, silencing of ACTL8 significantly decreased the expression of TRIM29 in HT29 and SW620 CRC cell lines.

Conclusion: These results suggest that ACTL8 plays a key role in the invasion and metastasis of CRC, and TRIM29 may be involved in the ACTL8-mediated poor prognosis of CRC.

Keywords: ACTL8; CRC; TRIM29; invasion and metastasis; prognosis.

Figure 1

ACTL8 protein was upregulated in CRC. Notes: (A) Representative micrographs showing immunohistochemical staining of ACTL8 in normal colorectal tissue, tumor-adjacent tissue, and CRC tissue. Magnification: ×40. Arrows indicate the magnified regions in the insert (×400). Scale bar: 500 µm. (B) IRS of ACTL8 expression in cancer tissues was significantly higher than normal colorectal tissues and tumor-adjacent tissues. Red line denotes the median value. (C) IRS of ACTL8 expression in CRC samples was significantly higher than paired normal tissues. (D) IRS of ACTL8 expression in CRC samples was significantly higher than paired tumor-adjacent tissues. (E) Western blotting analysis of ACTL8 expression in human CRC cell lines and human intestinal epithelial cell line. **P<0.01, and ***P<0.001. P-value of (B–D) was obtained by Wilcoxon rank-sum test. P-value of (E) was obtained by one-way ANOVA and Student–Newman–Keuls test. Abbreviations: CRC, colorectal cancer; HIEC, human intestinal epithelial cells; IRS, immunoreactive score.

Figure 2

The correlation between ACTL8 protein expression and PML and DML in CRC. Notes: (A) The correlation between ACTL8 protein expression and PML. (B) The correlation between ACTL8 protein expression and DML. The red line represents the linear fit line. The r- and P-values were obtained by Pearson correlation analysis. Abbreviations: CRC, colorectal cancer; DML, distal margin length; IRS, immunoreactive score; PML, proximal margin length.

Figure 3

Kaplan–Meier curves showing the relationship of ACTL8 expression and OS of CRC patients. Notes: The curves show that overexpression of ACTL8 was significantly associated with shorter OS of CRC patients (P=0.003). The log-rank test was performed to test the statistical significance. Abbreviations: CRC, colorectal cancer; OS, overall survival.

Figure 4

Effect of ACTL8 silencing on invasion and migration of CRC cells. Notes: (A) Silencing efficiency of ACTL8 by three siRNAs in HT29 and SW620 cell lines was detected by Western blotting. (B) The effect of ACTL8 silencing on the migration ability of HT29 and SW620 cells was analyzed by wound healing migration assay (100×). (C) The effect of ACTL8 silencing on the invasion ability of HT29 and SW620 cells was detected by transwell invasion assay (200×). *P<0.05, **P<0.01, and ***P<0.001. P-value was obtained by one-way ANOVA and Student–Newman–Keuls test. Abbreviation: CRC, colorectal cancer.

Figure 5

Effect of ACTL8 silencing on the expression of TRIM29 and CFL1 in CRC cell lines. Notes: (A) The effect of ACTL8 silencing on the mRNA expression of TRIM29 in CRC cell lines was analyzed by qRT-PCR. (B) The effect of ACTL8 silencing on the mRNA expression of CFL1 in CRC cell lines was analyzed by qRT-PCR. (C) The effect of ACTL8 silencing on the protein expression of TRIM29 in CRC cell lines. *P<0.05 and **P<0.01. P-value was obtained by one-way ANOVA and Student–Newman–Keuls test. Abbreviations: CRC, colorectal cancer; qRT-PCR, quantitative reverse transcription PCR.