Downregulation of Wnt3 Suppresses Colorectal Cancer Development Through Inhibiting Cell Proliferation and Migration - PubMed (original) (raw)
doi: 10.3389/fphar.2019.01110. eCollection 2019.
Fulin Xia 1, Ying Liu 1, Yun Zhou 1, Wenling Ye 1, Panha Hean 2, Jiming Meng 2, Huiyang Liu 1, Lei Liu 1, Jianxun Wen 3, Xuequn Ren 2, Wei-Dong Chen 1 3, Yan-Dong Wang 4
Affiliations
- PMID: 31632267
- PMCID: PMC6779829
- DOI: 10.3389/fphar.2019.01110
Downregulation of Wnt3 Suppresses Colorectal Cancer Development Through Inhibiting Cell Proliferation and Migration
Xiaobo Nie et al. Front Pharmacol. 2019.
Abstract
The aberrant expression of Wnt3 has linked to several types of human malignancies. However, it is not known for its role in tumorigenesis of colorectal cancer (CRC). Herein, we show that Wnt3 is upregulated in human CRC tissues and is essential for the CRC progression. Knockdown of Wnt3 in human CRC cells delayed tumor formation in nude mouse xenografts through silencing of canonical Wnt pathway and glycolysis. We further found that silencing of Wnt3 enhanced the sensitivity of CRC cells to cisplatin through inducing apoptotic cell death. Taken together, it demonstrates that Wnt3 is a novel clinical biomarker for the detection of CRC and plays an important role in colorectal tumorigenesis. Therefore, downregulation of Wnt3 will be a valuable strategy in CRC treatment.
Keywords: HCT-116; Wnt3; apoptosis; colorectal cancer; glycolysis; proliferation.
Copyright © 2019 Nie, Xia, Liu, Zhou, Ye, Hean, Meng, Liu, Liu, Wen, Ren, Chen and Wang.
Figures
Figure 1
Expression of Wnt3 is upregulated in human colorectal cancer (CRC) tissues. (A) Wnt3 messenger RNA (mRNA) levels in CRC tumor tissues and paired normal tissues were examined by quantitative real-time PCR (qRT-PCR; N = 46, ***P < 0.001). (B) Online TCGA data analysis of Wnt3 mRNA level in colorectal normal and cancerous tissues from CRC patients (N = 279). (C) Representative immunoblot of Wnt3 protein expression in human CRC tissues and corresponding nontumor ones by Western blot analysis, normalized to β-actin protein levels. Densitometry was used to quantify relative Wnt3 protein levels normalized to β-actin (**P < 0.01). (D) Representative immunohistochemical staining of Wnt3 in human normal or CRC tissue sections (100× and 200×).
Figure 2
The association between Wnt3 messenger RNA (mRNA) levels and various clinicopathological factors of colorectal cancer (CRC) patients. (A) Wnt3 mRNA levels of CRC tumor tissues in stage II and III. (*P < 0.05). (B) Wnt3 mRNA levels in CRC patients with different age, drinking history, and fecal occult blood test (FOBT) (N = 46, *P < 0.05). (C1–C2) Wnt3 mRNA levels in CRC patients with different gender and smoking history (N = 46, *P<0.05). (C3–C5) Wnt3 mRNA levels of CRC tumor tissues in TNM stages are shown (*P < 0.05). (C6–C10) The association between Wnt3 mRNA levels and level of alanine transaminase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (CREA), and urea levels in CRC patients (N = 46). (D) The overall survival curve of CRC patients (N = 279) according to Wnt3 mRNA level were analyzed with data from the TCGA.
Figure 3
Overexpression of Wnt3 accelerates colorectal cancer (CRC) and tumor growth and inhibits cellular apoptosis induced by cisplatin. (A) Representative qRT-PCR analysis of Wnt3 messenger RNA (mRNA) level in selected HCT-116 clone cells that stably overexpress Wnt3-pEX4 plasmid (Wnt3-pEX4) or control one (NC-pEX4). (B) Representative immunoblot showing Wnt3 and cyclin D1 protein levels extracts from Wnt3-pEX4 or NC-pEX4 HCT-116 cells. (C and D) Real-time cell analysis (RTCA) showed the cell proliferation ability was obviously accelerated in Wnt3-pEX4 cells, and the proliferation rate of Wnt3-pEX4 cells at the 36 and 48 h were analyzed. The proliferation index was recorded every 15 min to 54 h postseeding using RTCA software. (E) Three representative images of colony formation assay of NC-pEX4 or Wnt3-pEX4 cells, and colony number was automatically counted by IPP 6.0 software, the colony formation assays were performed in triplicate. (F) The real-time tumor size, tumor weight after killing, representative nude mice and dissected tumors, and Wnt3 mRNA level (N = 6) in tumor tissue lysates are shown. (G) NC-pEX4 and Wnt3-pEX4 cells were treated with 8 µg/ml of cisplatin for 36 h after seeding; cell apoptosis was analyzed by flow cytometry analysis. The cellular apoptotic rate was a sum of early and late apoptotic rates. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC-pEX4 group.
Figure 4
Knockdown of Wnt3 inhibits colorectal cancer (CRC) proliferation, migration, and tumor growth. (A) Representative quantitative real-time PCR (qRT-PCR) analysis of Wnt3 mRNA level in selected HCT-116 clone cells that stably overexpress Wnt3-shRNA plasmid (Wnt3-shRNA) and control one (NC-shRNA). (B) Representative immunoblot showing Wnt3 and cyclin D1 protein levels extracts from Wnt3-shRNA and NC-shRNA cells. (C and D) Real-time cell analysis (RTCA) showed the cell proliferation ability was obviously inhibited in Wnt3-shRNA cells, and the proliferation rate of Wnt3-shRNA cells at the 36 and 48 h were analyzed. The proliferation index was recorded every 15 min to 54 h postseeding using RTCA software. (E) Representative photographs of Wnt3-shRNA or NC-shRNA cells taken at 0, 48, and 72 h postwound (×40). (F) Three representative images of colony formation assay of NC-shRNA or Wnt3-shRNA cells, and colony number was automatically counted by IPP 6.0 software; the colony formation assays were performed in triplicate. (G) The real-time tumor size, tumor weight after killing, representative nude mice and dissected tumors, and Wnt3 mRNA level (N = 6) in tumor tissue lysates are shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC-shRNA group.
Figure 5
Downregulation of Wnt3 arrests the cell cycle, induces the apoptosis of CRC cells, inhibits the expression level of genes involved in canonical Wnt pathway and glycolysis. (A) Flow cytometry analysis of cell cycle distribution in Wnt3-shRNA and NC-shRNA cells. Cell cycle arrest was analyzed with a NovoCyteTM flow cytometer and the results are representative of three independent experiments. (B) Wnt3-shRNA or NC-shRNA cells were treated with 8 µg/ml of cisplatin for 36 h after seeding, and the cell apoptosis was analyzed by flow cytometry analysis. The cellular apoptotic rate was a sum of early and late apoptotic rates. (C) Representative quantitative real-time PCR (qRT-PCR) analysis of mRNA levels of crucial genes involved in canonical Wnt pathway in NC-shRNA and Wnt3-shRNA cells. (D) Wnt3-shRNA and NC-shRNA cells were treated with the indicated concentration of cisplatin for 24 h, and the cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The data are presented as mean ± SD (N = 6). (E) Western blot analysis of PARP (t-PARP), cleaved PARP (c-PARP), caspase 3 (t-caspase 3), and cleaved caspase 3 (c-caspase 3) protein levels in lysates extracts from Wnt3-shRNA or NC-shRNA cells, normalized to β-actin protein levels. (F) Representative qRT-PCR analysis of mRNA levels of crucial genes involved in glycolysis in NC-shRNA and Wnt3-shRNA cells. *P < 0.05, **P < 0.01 compared with the NC-shRNA group
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