Hsa_circ_0004194 suppresses colorectal cancer progression via hsa-miR-27a-3p

Abstract

Aims: To investigate the functional role and the underlying molecular mechanisms associated with hsa_circ_0004194 in the context of colorectal cancer (CRC) and to elucidate its impact on cancer progression.

Results: A notable and statistically significant decrease in the expression levels of hsa_circ_0004194 was observed specifically within CRC tissues when compared to non-tumor colorectal mucosa tissues. Functional evaluations, such as CCK8 assays, plate clone formation analysis, and transwell migration assays, our study revealed hsa_circ_0004194 significantly reduced the activity behavior of CRC cells. This overexpression of hsa_circ_0004194 effectively hindered these key cellular processes, demonstrating its role in suppressing the aggressive behaviors of CRC cells. Additionally, in vivo experiments utilizing mouse xenograft models exhibited that the upregulation of hsa_circ_0004194 significantly attenuated tumor growth, reduced tumor volume, and diminished liver metastasis. Further mechanistic investigation, through the utilization of RNA pull-down and luciferase reporter assays, uncovered that hsa_circ_0004194 sequestered hsa-miR-27a-3p, thereby enhancing retinoic acid X receptor α (RXRα)' expression which is in CRC cells. Moreover, this circular RNA also impeded the signaling pathway of Wnt/β-catenin.

Conclusion: Our study is the first to demonstrate that hsa_circ_0004194 exhibits downregulated expression in CRC and functions as a ceRNA by binding to and sequestering hsa-miR-27a-3p, thereby modulating the RXRα/β-catenin signaling pathway to inhibit CRC progression. This discovery suggests that hsa_circ_0004194 holds significant potential as a therapeutic biomarker for patients with CRC.

Keywords: Colorectal cancer; Hsa-miR-27a-3p; RXRα; hsa_circ_0004194.

Fig. 1

Hsa_circ_0004194 expression in CRC. (A) The design scheme of RNA-sequencing analyses. (B-C)Based on our analysis, we observed that SW480 cells (B) exhibited 92 circular RNAs with higher expression and 207 with lower expression compared with NCM460; HCT116 cells (C) displayed 866 circular RNAs with higher expression and 580 with lower expression compared with NCM460. (D) Schematic illustration showing the composition of hsa_circ_0004194. (E) The junction sequence was amplified by divergent primers and confirmed by sanger sequencing. (F) The transcript levels of CTNNB1 mRNA and hsa_circ_0004194 with or without RNase R digestion by real-time qRT-PCR. (G) Agarose gel electrophoresis (AGE) showed the PCR products which was amplified with divergent or convergent primers using cDNA or gDNA as the template, respectively. actin as the internal reference. (H) RT-PCR analysis of hsa_circ_0004194 abundance in the cytoplasmic and nuclear fractions of SW480, while Actin and U3 as positive controls of cytoplasm and nuclear localization respectively. (I)hsa_circ_0004194 expression level in CRC cell lines and MCM460 cells by RT-PCR. (J) hsa_circ_0004194 expression levels in fresh CRC tissues (n = 52) and paired adjacent non-tumor colorectal mucosal tissues (n = 52) by RT-PCR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001,∗∗∗∗p < 0.0001.

Fig. 2

Hsa_circ_0004194 suppresses the proliferation, migration and invasion of CRC cells. (A) SW480 and HCT116 cells were infected with hsa_circ_0004194 or Mock lentivirus and Quantitative RT-PCR used to determine hsa_circ_0004194 expression. (B-C) hsa_circ_0004194 significantly suppressed cell proliferation (B) and colony formation (C) in SW480 and HCT116 cells by CCK-8 and colony formation assays assays, respectively.(D) Hsa_circ_0004194 significantly inhibited cell migration and invasion in SW480 and HCT116 cells. The scale bar in transwell assays indicated 100 μm. Data were all represented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 3

Hsa_circ_0004194 suppresses the growth and metastasis of CRC in vivo. (A–C) SW480 and HCT116 cells were injected subcutaneously into nude mice. After about a month, the tumors were dissected and photographed. Tumour volume was measured as length X width X height X 0.52. (D) H&E and IHC staining analyses were performed to demonstrate the morphology and detect Ki-67 expression in tumor tissues extracted from xenograft mice. (E) MC38 cells were infected with circ_0004194 or Mock lentivirus and Quantitative RT-PCR used to determine hsa_circ_0004194 expression. 5X10⁵ infected MC38 cells were injected into the spleen of C57 mice. Mice were then sacrificed and the liver tissues were sectioned and stained by H&E staining after about a month. (F) The number of visible metastases nodules was calculated. (G)Representative liver specimen and HE pictures. The scale bar in 40X and 400X photos indicated 625 μm and 50 μm. Data were represented as mean ± SD (n = 3). ∗∗p < 0.01. ∗∗∗p < 0 0.001.

Fig. 4

Hsa_circ_0004194 directly binds to hsa-miR-27a-3p in CRC. (A)hsa_circ_0004194 overexpression significantly down-regulated hsa-miR-27a-3p expression in SW480 and HCT116 cells.(B) Correlation analysis revealed that the expression of hsa_circ_0004194 and hsa-miR-27a-3p was negatively correlated in CRC tissues.(C-D)The expression levels of hsa_circ_0004194 and hsa-miR-27a-3p pulled down by biotin-labeled hsa_circ_0004194 probe and control probe in SW480 lysates. (E) The rnahybrid website predicted the possible binding targets of hsa_circ_0004194 and hsa-miR-27a-3p. (F)Dual luciferase assay confirmed that hsa-miR-27a-3p could bind specifically to hsa_circ_0004194 in SW480 and HCT116 cells. Data were all represented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig. 5

Hsa_circ_0004194 suppresses CRC progression partially depending on hsa-miR-27a-3p. (A) Overexpression efficiency of hsa-miR-27a-3p was detected by qRT-PCR. (B-E) hsa-miR-27a-3p could partially restore the inhibitory effect of hsa_circ_0004194 overexpression on the proliferation(B-C)、migration(D) and invasion(E)of CRC cells. The scale bar in transwell assays indicated 50 μm. Data were all represented as mean ± SD (n = 3). ns indicated no significance, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001.

Fig. 6

Hsa_circ_0004194/miR-27a-3p/RXRα/β-catenin signaling pathway involved in CRC progression. (A) The mRNA and (B) protein expression levels of RXRα were up-regulated after overexpression of Circ_0004194 in SW480 and HCT116. (C-D)The mRNA (C) and protein (D) expression levels of β-catenin, cyclinD1 and c-Myc were down-regulated in SW480 and HCT116 with overexpression of hsa_circ_0004194. (E)hsa-miR-27a-3p could partially restore the effects of Hsa_circ_0004194 overexpression on protein expression levels of RXRα, β-catenin, cyclinD1 and c-Myc in SW480 and HCT116 cells. (F) The schematic illustration showing the underlying mechanism of hsa_circ_0004194 in the progression of CRC.