Citation
- Authors: Zhang L. et al.
- Year: 2022
- Journal: J Transl Med 20 327
- Applications: in vitro / siRNA / INTERFERin
- Cell types:
- Name: LoVo
Description: Human colorectal adenocarcinoma cells - Name: SW620
Description: Human colon adenocarcinoma cells
- Name: LoVo
Method
To transiently knockdown the expression of FOXS1, FOXS1-siRNAs (siRNA#1, 5′-CAGGAAUGUUCUUUGATT-3′, 5′-UCAAAGAAGAACAUUCCUGTT-3′; siRNA#2, 5′-CACUCAACGAGUGCUUUGUTT-3′, 5′-ACAAAGCACUCGUUGAGUGTT-3′; siRNA#3, 5′-GGCCAAUAAAGCCAUGUGATT-3′, 5′-UCACAUGGCUUUAUUGGCCTT-3′) or control-siRNA were transfected into SW620 and LoVo cells. Shanghai GenePharma Company (Shanghai, China) provided these siRNAs. 2 × 105 CRC cells per well plated into a six-well plate were treated with siRNA (1–2 µg) encapsulated by the interferin reagent (Polyplus, New York, NY, USA) based on the protocol. The knockdown efficiency of FOXS1 siRNA was examined by western blot. FOXS1 shRNA (Shanghai Gene Chem Co, Ltd., China) was used to establish stable cell lines to knock down the expression of FOXS1.
Abstract
Background: Recent studies have shown that the fox family plays a vital role in tumorigenesis and progression. Forkhead Box S1 (FOXS1), as a newly identified subfamily of the FOX family, is overexpressed in certain types of malignant tumors and closely associated with patient's prognosis. However, the role and mechanism of the FOXS1 in colorectal cancer (CRC) remain unclear.
Method: FOXS1 level in CRC tissues and cell lines was analyzed by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry (IHC) was used to detect the relationship between FOXS1 expression and clinicopathological features in 136 patients in our unit. The expression of FOXS1 was knocked down in CRC cells using small interfering RNA (siRNA) technology. Cell proliferation was assessed by CCK8 assay, colony formation, and 5-Ethynyl-20-deoxyuridine (EdU) incorporation assay. Flow cytometry detected apoptosis and wound healing, and Transwell assays determined cell migration and invasion. Western blotting was used to detect the levels of proteins associated with the Wnt/β-catenin signaling pathway. Then, we used short hairpin RNA (shRNA) to knock down FOXS1 to see the effect of FOXS1 on the proliferation, migration, invasion, and metastasis of CRC cells in vivo. Finally, we investigated the impact of Wnt activator LiCl on the proliferation, migration, invasion, and metastasis of CRC cells after FOXS1 knockdown.
Result: Compared to those in normal groups, FOXS1 overexpressed in CRC tissues and CRC cells (P < 0.05). Upregulation of FOXS1 association with poor prognosis of CRC patients. si-FOXS1 induced apoptosis and inhibited proliferation, migration, invasion, the epithelial-mesenchymal transition (EMT), and the Wnt/β-catenin signaling pathway in vitro; sh-FOXS1 inhibited the volume and weight of subcutaneous xenografts and the number of lung metastases in vivo. LiCl, an activator of Wnt signaling, partially reversed the effect of FOXS1 overexpression on CRC cells.
Conclusion: FOXS1 could function as an oncogene and promote CRC cell proliferation, migration, invasion and metastasis through the Wnt/βcatenin signaling pathway, FOXS1 may be a potential target for CRC treatment.