Distinct roles and differential expression levels of Wnt5a mRNA isoforms in colorectal cancer cells - PubMed (original) (raw)
Distinct roles and differential expression levels of Wnt5a mRNA isoforms in colorectal cancer cells
Tsui-Chin Huang et al. PLoS One. 2017.
Abstract
The canonical Wnt/β-catenin pathway is constitutively activated in more than 90% of colorectal cancer (CRC) cases in which β-catenin contributes to CRC cell growth and survival. In contrast to the Wnt/β-catenin pathway, the non-canonical Wnt pathway can antagonize functions of the canonical Wnt/β-catenin pathway. Wnt5a is a key factor in the non-canonical Wnt pathway, and it plays diverse roles in different types of cancers. It was shown that reintroducing Wnt5a into CRC cells resulted in inhibited cell proliferation and impaired cell motility. However, contradictory results were reported describing increased Wnt5a expression being associated with a poor prognosis of CRC patients. Recently, it was shown that the diverse roles of Wnt5a are due to two distinct roles of Wnt5a isoforms. However, the exact roles and functions of the Wnt5a isoforms in CRC remain largely unclear. The present study for the first time showed the ambiguous role of Wnt5a in CRC was due to the encoding of distinct roles of the various Wnt5a mRNA isoforms. A relatively high expression level of the Wnt5a-short (S) isoform transcript and a low expression level of the Wnt5a-long (L) isoform transcript were detected in CRC cell lines and specimens. In addition, high expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with tumor depth of CRC patients. Furthermore, knockdown of the endogenous expression of the Wnt5a-S mRNA isoform in HCT116 cells drastically inhibited their growth ability by inducing apoptosis through induction of FASLG expression and reduction of TNFRSF11B expression. Moreover, reactivation of methylation inactivation of the Wnt5a-L mRNA isoform by treatment with 5-azacytidine (5-Aza) enhanced the siWnt5a-S isoform's ability to induce apoptosis. Finally, we showed that the simultaneous reactivation of Wnt5a-L mRNA isoform and knockdown of Wnt5a-S mRNA isoform expression enhanced siWnt5a-S isoform-induced apoptosis and siWnt5a-L isoform-regulated suppression of β-catenin expression in vitro. High expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with high mRNA levels of β-catenin detection in vivo. Altogether, our study showed that, for the first time, different Wnt5a mRNA isoforms play distinct roles in CRC and can be used as novel prognostic markers for CRC in the future.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. Wnt5a mRNA isoforms expressions in colorectal cancer (CRC) cells.
(A) Expression levels of Wnt5a in nine types of NCI60 cancer cell lines which were retrieved from the CellMiner database. The Wnt5a-long (L) isoform (B) and—short (S) isoform (C) mRNA analyses were conducted on RNA isolated from nine CRC cell lines and one non-cancerous human CRL-1459 colon cell line. (D) The Wnt5a-L isoform and -S isoform mRNA expression levels were detected in CRC tissues.
Fig 2. Effects of the Wnt5a-long (L) and Wnt5a-short (S) mRNA isoforms on cell proliferation and spheroid formation of colorectal cancer (CRC) cells.
(A) Validation of expression levels of the different Wnt5a mRNA isoforms after transfection with specific siRNAs of the different Wnt5a isoforms for 48 h in the HCT116 cell line. (B) The proliferative ability of HCT116 cells after transfection with specific siRNAs of different Wnt5a isoforms. (C) HCT116 cells were transfected with specific siRNAs of different Wnt5a isoforms, and foci were visualized after crystal violet staining. (D) The combined results of all transfections, wherein total focal counts ± SD are illustrated. (E) HCT116 cells were grown in 3D NanoCulture Plates (NCPs) with transfection with specific siRNAs of different Wnt5a isoforms. In the 3D-NCP condition, siWnt5a-L isoform-transfected spheroid cells appeared to increase in spheroid size, but siWnt5a-S isoform-transfected spheroid cells appeared to spread out from the spheroids, and the spheroid sizes were reduced.
Fig 3. siWnt5a-short (S) isoform-induced apoptotic death in HCT116 cells.
(A) HCT116 cells were transfected with specific siRNAs of different Wnt5a isoforms for 48 h. Cells were subjected to an annexin-V assay using a Muse cell analyzer. The method led to four different populations of cells: live cells (annexin-V negative (lower right quadrant)), early apoptotic cells (positive for annexin-V (lower right corner)), late apoptotic/dead cells (annexin V positive (upper right quadrant)), and non-apoptotic dead cells (upper left quadrant). (B) Quantification of total apoptotic cells (early apoptosis + late apoptotic cells) determined by annexin-V positivity. (C) HCT116 cells transfected with specific siRNAs of different Wnt5a isoforms for 48 h, subjected to a Western blot analysis, and probed for CDK4, caspase-3, poly(ADP ribose) polymerase (PARP), and β-catenin antibodies, respectively. β-Actin was used as the loading control.
Fig 4. Wnt5a-short (S) mRNA isoform-mediated regulation of the expressions of multiple genes associated with apoptosis.
(A) Total RNA from control siRNA and siWnt5a-S isoform-transfected HCt116 cells was characterized using a Human Apoptosis PCR Array. The figure shows a scatter plot of differences in relative transcript abundances of 84 key genes that either change their expression during apoptosis or regulate those apoptosis-related gene expression changes. Three genes showed significant changes in siWnt5a-S isoform-transfected HCT116 cells compared to control siRNA-transfected cells. (B) Dysregulated genes identified by the Apoptosis PCR Array experiments and verified by an independent RT-qPCR of HCT116 cells. An RT-qPCR was performed using independent primers to those used in the array experiments. Relative mRNA abundances were calculated using GAPDH as an endogenous control. RT-qPCR values are the mean ± SD of n = 3 and normalized to the mean of control siRNA-transfected cells. * p<0.05, *** p<0.001.
Fig 5. Aberrant expressions of Wnt5a-short (S) mRNA isoform-modulated apoptosis-related genes associated with the survival rate of colorectal cancer (CRC) patients.
mRNA expressions of FASLG (A) and TNFRSF11B (B) in normal colorectal and colorectal tumor tissues were obtained from the Oncomine database (
). (C) Box plots comparing differences in FASLG and TNFRSF11B mRNA expressions between risk groups using a _t_-test. (D) Two-gene (FASLG and TNFRSF11B) combinations were used to accurately predict patient outcomes using Kaplan-Meier analyses from the CRC Survival Metabase showing stratification of FASLG and TNFRSF11B for longer-surviving (patients with high expression of FASLG and low expression of TNFRSF11B) versus shorter-surviving patients (patients with low expression of FASLG and high expression of TNFRSF11B).
Fig 6. Re-activation of Wnt5a mRNA isoforms by treatment with a demethylation agent.
Re-activation of the Wnt5a-long (L) mRNA isoform (A) or Wnt5a-short (S) mRNA isoform (B) after treatment with 5-azacytidine (5-Aza) at the indicated concentration.
Fig 7. Knockdown of the Wnt5a-short (S) mRNA isoform enhanced the sensitivity of colorectal cancer (CRC) cells to 5-azacytidine (5-Aza).
(A) The Wnt5a-S mRNA isoform was silenced for 48 h with 100 nM of specific siRNA, following which cells were treated with 5-Aza at the indicated concentrations for an additional 48 h. Cells were stained with annexin V and analyzed using a Muse Cell Analyzer. (B) The quantification result of annexin V-positive cells with knockdown of the Wnt5a-S isoform alone or in the presence of 5-Aza. (C) Western blot analysis of DNMT1, caspase-3, poly (ADP ribose) polymerase (PARP) cleavage, and β-catenin in HCT116 cells transfected with control siRNA or Wnt5a-S isoform-specific siRNA oligomers for 48 h and subsequently incubated with 5-Aza at the indicated concentrations for an additional 48 h. Arrow symbol indicated that siWnt5a-S isoform knockdown HCT116 cells in the present with 5-Aza showed more significant at inducing caspase-3 cleavage than control siRNA-transfected HCT116 cells in the presence of 5-Aza. β-actin is shown as the loading control.
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