FHL1 Inhibits the Progression of Colorectal Cancer by Regulating the Wnt/β-Catenin Signaling Pathway

Abstract

Purpose: This study aims to explore the FHL1 expression level in colorectal cancer (CRC) patients, analyze its association with patient survival and investigate the role of FHL1 in CRC. Methods: We used secondary sequencing to profile mRNA expression in CRC tissue and corresponding adjacent normal tissue from four CRC patients. We focus on FHL1 and analyzed the association between its expression level and clinical indicators. Furthermore, we explored the functional role of FHL1 in colorectal cancer tumorigenesis by transfecting cells with siRNA or overexpression plasmids. Results: Hierarchical clustering revealed significantly differentially expressed mRNAs. FHL1 expression was significantly lower in CRC tissue than in adjacent normal tissue as well as in CRC cell lines relative to NCM460. Low FHL1 expression in CRC tissue correlated with poor patient survival. Our data demonstrated that overexpression of FHL1 inhibited the proliferation, colony formation potential, and expression of CdK4 and Cyclin D1, whereas ablating FHL1 promoted their proliferation and colony formation potential, suggesting that FHL1 acts as a tumor suppressor in CRC. Moreover, we showed that FHL1 inhibited the proliferation of colorectal cancer cells by negatively regulating the Wnt/β-catenin signaling pathway. Conclusion: FHL1 is a potential tumor suppressor gene in colorectal cancer, and regulation of the FHL1-Wnt/β-catenin pathway may be part of its antitumor mechanism.

Keywords: Colorectal cancer; FHL1; RNA sequencing; Wnt/β-catenin signaling pathway.

Figure 1

Downregulation of FHL1 was identified in colorectal cancer tissues. A. RNA expression profiles in 4 paired CRC tissues and the corresponding adjacent normal tissues. B,C. FHL1 mRNA expression levels in 40 paired CRC tissues and the corresponding adjacent normal tissues were measured by using qRT-PCR. D. FHL1 protein levels in 8 representative CRC tissues and the corresponding adjacent normal tissues were tested by Western blot. E. Representative IHC staining images of FHL1 in a tissue microarray that contained 177 paired CRC and adjacent normal tissues (scale bar, 100 µm). F. Comparison of the relative FHL1 expression levels in CRC tumors compared to adjacent normal tissues in the tissue microarray. G. Kaplan-Meier survival curve indicating overall survival in relation to FHL1 expression levels in CRC tumor tissues from the tissue microarray. Data represent the mean ± SD. * P < 0.05, ** P < 0.01.

Figure 2

Effects of FHL1 overexpression or knockdown on aggressive proliferation of colorectal cancer cells were studied. A,B. FHL1 mRNA and protein levels were measured in 5 CRC cell lines and normal colon epithelial cells by qRT-PCR (A) and Western blot (B). C, E. FHL1 gene expression and protein levels were evaluated in HT-29 cells after transfection with the overexpression plasmid. D, F. The silencing effect on FHL1 by three siRNAs was evaluated in HCT-116 cells using qRT-PCR (D) and Western blot (F). G. The colony formation potential of HCT-116 and HT-29 cells after FHL1 knockdown by siRNA-3 or FHL1 overexpression by Lv-GFP+FHL1 was analyzed with a colony formation assay. H-I. The proliferation of HCT-116 and HT-29 cells after FHL1 knockdown by siRNA-3 or FHL1 overexpression via Lv-GFP+FHL1 was measured using a CCK-8 cell-counting kit. Data are presented as mean ± SD and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3

FHL1 negatively regulates the Wnt/β-catenin pathway. A-B. The p-GSK, GSK and β-catenin protein levels were measured by Western blot after FHL1 knockdown with siRNA-3 or FHL1 overexpression with Lv-GFP+FHL1 in HCT-116 cells and HT-29 cells, respectively. C-D. Real-time PCR analysis of the expression levels of LEF1 and TCF4 in FHL1 knockdown or overexpression cells. E. The LEF1 and TCF4 protein levels were assessed by Western blot after using FHL1 siRNA-3 or/and PRI-724 in HCT-116 cells. Data are presented as mean ± SD and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01.

Figure 4

The effect of FHL1 overexpression or knockdown on cell cycle proteins in colorectal cancer cells was elucidated. A. The proliferation of HCT-116 cells after FHL1 knockdown by siRNA-3 or treatment with PRI-724 was measured using a CCK-8 cell-counting kit. B-C. Expression of cell cycle-related proteins was measured by Western blot after FHL1 knockdown with siRNA-3 or FHL1 overexpression with Lv-GFP+FHL1 in HCT-116 cells and HT-29 cells, respectively. Data are presented as mean ± SD and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01.

Figure 5

Schematic illustration of FHL1 inhibition of the Wnt/β-catenin pathway in colorectal cancer cells.