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. 2020 Oct;11(5):918-931.
doi: 10.21037/jgo-20-395.

CircTBL1XR1/miR-424 axis regulates Smad7 to promote the proliferation and metastasis of colorectal cancer

Na Li  1
Affiliations

CircTBL1XR1/miR-424 axis regulates Smad7 to promote the proliferation and metastasis of colorectal cancer

Na Li. J Gastrointest Oncol. 2020 Oct.

Abstract

Background: This study analyzed the effect of circular RNA (circRNA), TBL1XR1, on the malignant progression of colon cancer cells and explored its possible molecular mechanism.

Methods: The expression levels of circTBL1XR1 in colorectal cancer and paracancerous tissues were detected by quantitative real-time polymerase chain (qRT-PCR). Changes in circTBL1XR1 expression in normal intestinal epithelial cells and colon cancer cell lines were detected at the cellular level. LoVo and SW620 cells with the highest circTBL1XR1 expression were transfected with circTBL1XR1, and the transfection efficiency was verified by qRT-PCR. The effects of circTBL1XR1 on the proliferation, migration, and invasion of colon cancer cells were detected by MTT, Transwell migration, and invasion assay, and a subcutaneous tumor model. Target genes of circTBL1XR1 were verified by luciferase reporter assay. Expression levels of circTBL1XR1 target genes were detected by qRT-PCR and Western blotting.

Results: circTBL1XR1 was highly expressed in colon cancer patients. circTBL1XR1 expression in colon cancer cells was also higher than that in normal intestinal cells. In vivo and in vitro experimental results showed that overexpression of circTBL1XR1 enhanced the proliferation, migration, and invasion of colon cancer cells. After lowering circTBL1XR1, the ability of migration and invasion of colon cancer cells was significantly reduced. Bioinformatics retrieval and luciferase reporter gene assay confirmed that circTBL1XR1 can bind to microRNA-424 (miR-424) and that the Smad7 gene is the target gene of miR-424.

Conclusions: circTBL1XR1 was highly expressed in colon cancer, and miR-424 was poorly expressed in colon cancer cells. circTBL1XR1 regulates the expression of Smad7 through miR-424, thereby affecting the malignant progression of colorectal cancer.

Keywords: Smad7; circTBL1XR1; colorectal cancer; epithelial-mesenchymal transition (EMT); miR-424.

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Conflict of interest statement

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jgo-20-395). The author has no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Expression of circTBL1XR1 in colorectal cancer and adjacent tissues and an analysis of patients’ clinical information. (A) CircTBL1XR1 levels in adjacent tissues and colorectal cancer tissues; (B) CircTBL1XR1 expression levels in normal intestinal epithelial cells and colorectal cancer cells; (C) the level of circTBL1XR1 in patients with colorectal cancer in metastatic and non-metastatic patients. **, P<0.01; ***, P<0.001.
Figure 2
Figure 2
CircTBL1XR1 promoted colorectal cancer proliferation and metastasis. (A) Detection of circTBL1XR1 transfection efficiency in LoVo cells; NC: negative control; (B) detection of circTBL1XR1 transfection efficiency in SW620 cells; (C) overexpression of circTBL1XR1 promoted the cell proliferation ability of LoVo cells; (D) overexpression of circTBL1XR1 promoted the cell proliferation ability of in SW620 cells; (E) overexpression of circTBL1XR1 promoted colon cancer cell migration ability in LoVo and SW620 cells; (F) overexpression of circTBL1XR1 promoted colon cancer cell invasion in LoVo and SW620 cells; Crystal violet staining for tumor cells: 0.1% crystal violet solution (Beyotime, Shanghai, China) was used for staining for 20 min. Next, gently wipe off the upper unmigrated cells with a cotton swab. Wash it with PBS three times. (G) overexpression of circTBL1XR1 inhibited the expression of E-cadherin; (H) overexpression of circTBL1XR1 promoted Vimentin expression; (I) overexpression of circTBL1XR1 promoted tumor proliferation; (J) the tumor volume test results indicated that circTBL1XR1 promoted tumor proliferation; (K) tumor weight test results after overexpression of circTBL1XR1; (L) Ki-67 immunohistochemical staining (magnification ×200). *, P<0.01; **, P<0.01; ***, P<0.001.
Figure 3
Figure 3
CircTBL1XR1 regulated the detection of miR-424. (A) Information maps of the binding sites of circTBL1XR1 and miR-424; (B) dual luciferase report assay detected the binding of circTBL1XR1 to miR-424; NC: negative control, WT: wild type, MT: mutant; (C) detection of transfection efficacy of circTBL1XR1 overexpression and knockdown; (D) CircTBL1XR1 inhibited miR-424 expression; (E) correlation analysis of circTBL1XR1 and miR-424 co-expression. **, P<0.01.
Figure 4
Figure 4
CircTBL1XR1 adsorbed miR-424 to increase the expression of Smad7 through sponge action to promote the malignant evolution of colorectal cancer cells. (A,B) Cell proliferation detection of LoVo and SW620 cells; (C) cell migration detection; (D) detection of cell invasion. Crystal violet staining for tumor cells: 0.1% crystal violet solution (Beyotime, Shanghai, China) was used for staining for 20 min. Next, gently wipe off the upper unmigrated cells with a cotton swab. Wash it with PBS three times. Magnification ×200. *, P<0.01; **, P<0.01; ***, P<0.001.
Figure 5
Figure 5
CircTBL1XR1 promoted EMT of colorectal cancer cells. (A) Western blot detection of E-cadherin and Vimentin expression; (B) detection of E-cadherin expression; (C) detection of Vimentin expression. ***, P<0.001.
Figure 6
Figure 6
MiR-424 inhibited Smad7 expression. (A) Information maps of Smad7 and miR-424 binding sites; (B) dual luciferase report detected the binding of Smad7 to miR-424; NC: negative control, WT: wild type, MT: mutant; (C) detection of transfection efficacy of miR-424 overexpression and knockdown; (D) the expression of Smad7 decreased with the addition of miR-424 mimics to colorectal cancer cell lines. Adding miR-424 inhibitor increased the expression of Smad7; (E) correlation analysis of Smad7 and miR-424 co-expression; (F) correlation analysis of circTBL1XR1 and Smad7 co-expression; (G) Smad7 immunohistochemical staining. **, P<0.01; ***, P<0.001.
Figure 7
Figure 7
Overexpression of Smad7 reversed the antitumor effects of miR-424. (A) Detection of Smad7 expression in LoVo cells; (B) detection of Smad7 expression in SW620 cells; (C,D) LoVo and SW620 cell proliferation detection; (E) detection of cell migration ability; (F) detection of cell invasion ability. *, P<0.01; **, P<0.01; ***, P<0.001.
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