Sevoflurane Suppresses the Proliferation, Migration and Invasion of Colorectal Cancer Through Regulating Circ_0000423/miR-525-5p/SGPP1 Network

Abstract

Introduction: Sevoflurane (SEV) has been shown to inhibit the malignant progression in many cancers, including colorectal cancer (CRC). However, it is not clear whether SEV regulates the progression of CRC by mediating the circular RNA (circRNA) axis.

Methods: Different concentrations of SEV were used to treat CRC cells. Cell proliferation, migration and invasion were determined by cell counting kit 8 assay, colony formation assay and transwell assay. The expression of circ_0000423, microRNA (miR)-525-5p and sphingosine-1-phosphate phosphatase 1 (SGPP1) mRNA was measured by quantitative real-time PCR. Cell apoptosis was assessed using flow cytometry, and protein expression was measured by western blot analysis. Dual-luciferase reporter assay and RIP assay were performed to confirm the interactions among circ_0000423, miR-525-5p and SGPP1. Animal experiments were performed to explore the effect of SEV and circ_0000423 on CRC tumorigenesis.

Results: SEV could inhibit CRC cell proliferation, migration and invasion. Circ_0000423 was upregulated in CRC and its expression could be reduced by SEV. Overexpressed circ_0000423 reversed the inhibitory effect of SEV on CRC cell proliferation, migration and invasion and the promotion effect on cell apoptosis. MiR-525-5p could be sponged by circ_0000423, and its overexpression also abolished the regulation of circ_0000423 on the progression of SEV-treated CRC cells. In addition, SGPP1 was confirmed to be a target of miR-525-5p, and its expression was positively regulated by circ_0000423. MiR-525-5p inhibitor promoted CRC cell progression under the treatment of SEV, while these effects could be overturned by SGPP1 silencing. Furthermore, the inhibition effect of SEV on CRC tumorigenesis also could be abolished by overexpressing circ_0000423.

Conclusion: Our results showed that SEV inhibited CRC progression through the regulation of circ_0000423/miR-525-5p/SGPP1 axis.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-021-00717-5.

Keywords: Colorectal cancer; SGPP1; Sevoflurane; circ_0000423; miR-525-5p.

Figure 1

SEV inhibited the proliferation, migration and invasion of CRC cells. SW480 and HCT116 cells were treated with different concentrations (1.7, 3.4 and 5.1%) of SEV. CCK8 assay (a, b) and colony formation assay (c) were used to measure cell viability and colony formation rate. (d, e) Transwell assay was performed to detect cell migration and invasion. *p < 0.05.

Figure 2

Circ_0000423 overexpression reversed the inhibition effect of SEV on CRC progression. (a) Circ_0000423 expression in CRC tumor tissues and adjacent normal tissues was measured by qRT-PCR. (b) The expression of circ_0000423 was detected by qRT-PCR in CRC cells (SW480 and HCT116) and NCM460 cells. (c) QRT-PCR was used to test circ_0000423 expression in SW480 and HCT116 treated with different concentrations (1.7, 3.4 and 5.1%) of SEV. (d) The transfection efficiency of circ_0000423 overexpression vector was confirmed by detecting circ_0000423 expression using qRT-PCR. (E-N) SW480 and HCT116 were transfected with vector or circ_0000423 overexpression vector and then treated with 5.1% SEV. Non-transfected and non-treated cells were used as control. (e) The circ_0000423 expression was measured by qRT-PCR. CCK8 assay (f, g), colony formation assay (h), flow cytometry (i) and transwell assay (j, k) were used to determine cell proliferation, apoptosis, migration and invasion. (l, m) The protein levels of CyclinD1 and Cleaved-Caspase-3/Caspase-3 were examined by WB analysis. *p < 0.05.

Figure 3

Circ_0000423 sponged miR-525-5p in CRC. (a) The binding sites and mutate sites between circ_0000423 and miR-525-5p were shown. Dual-luciferase reporter assay (b, c) and RIP assay (d, e) were performed to assess the interaction between circ_0000423 and miR-525-5p. (f) The miR-525-5p expression in CRC tumor tissues and adjacent normal tissues was detected by qRT-PCR. (g) QRT-PCR was used to detect the miR-525-5p expression in CRC cells (SW480 and HCT116) and NCM460 cells. (h) The correlation between miR-525-5p and circ_0000423 was analyzed by Pearson correlation analysis. (i) The miR-525-5p expression was measured by qRT-PCR in SW480 and HCT116 treated with different concentrations (1.7, 3.4 and 5.1%) of SEV. (j) QRT-PCR was utilized to examine miR-525-5p expression in SW480 and HCT116 co-transfected with circ_0000423 overexpression vector and miR-525-5p mimic under the treatment of SEV. *p < 0.05.

Figure 4

MiR-525-5p reversed the regulation of circ_0000423 on the progression of SEV-treated CRC cells. SW480 and HCT116 were transfected with vector, circ_0000423, circ_0000423 + miR-NC or circ_0000423 + miR-525-5p, and then treated with 5.1% SEV. Cell proliferation, apoptosis, migration and invasion were measured using CCK8 assay (a, b), colony formation assay (c), flow cytometry (d) and transwell assay (e, f). (g, h) The CyclinD1 and Cleaved-Caspase-3/Caspase-3 protein levels were detected by WB analysis. *p < 0.05.

Figure 5

SGPP1 was a target of miR-525-5p. (a) The binding sites and mutate sites between SGPP1 3′UTR and miR-525-5p were shown. The interaction between SGPP1 and miR-525-5p was confirmed by dual-luciferase reporter assay (b, c) and RIP assay (d, e). (f, g) The mRNA and protein expression of SGPP1 in CRC tumor tissues and adjacent normal tissues was detected by qRT-PCR and WB analysis. (h) WB analysis was used to evaluate the protein expression of SGPP1 in CRC cells (SW480 and HCT116) and NCM460 cells. (i, j) The correlation between SGPP1 and miR-525-5p or circ_0000423 was analyzed by Pearson correlation analysis. (k) The protein expression of SGPP1 was measured by WB analysis in SW480 and HCT116 treated with different concentrations (1.7, 3.4 and 5.1%) of SEV. (l) The SGPP1 protein expression in SW480 and HCT116 co-transfected with circ_0000423 overexpression vector and miR-525-5p mimic was measured by WB analysis under the treatment of SEV. (m) The transfection efficiency of anti-miR-525-5p was confirmed by detecting miR-525-5p expression using qRT-PCR. (n) Under the treatment of SEV, SGPP1 protein expression was measured by WB analysis in SW480 and HCT116 co-transfected with anti-miR-525-5p and si-SGPP1. *p < 0.05.

Figure 6

MiR-525-5p targeted SGPP1 to regulate the progression of CRC mediated by SEV. SW480 and HCT116 were transfected with anti-miR-NC, anti-miR-525-5p, anti-miR-525-5p + si-NC or anti-miR-525-5p + si-SGPP1, and then treated with SEV. Cell proliferation, apoptosis, migration and invasion were determined by CCK8 assay (a, b), colony formation assay (c), flow cytometry (d) and transwell assay (e, f). (g, h) WB analysis was performed to measure the CyclinD1 and Cleaved-Caspase-3/Caspase-3 protein levels. *p < 0.05.

Figure 7

SEV reduced CRC tumorigenesis by regulating circ_0000423/miR-525-5p/SGPP1 axis. SW480 cells transfected with circ_0000423 overexpression vector or vector or treated with or without SEV were injected into nude mice. (a) Tumor volume of each group was measured every week. (b) Tumor weight was detected after 35 days. (c, d) The expression of circ_0000423 and miR-525-5p in the tumor tissues of each group was measured by qRT-PCR. (e) The protein expression of SGPP1 in the tumor tissues of each group was assessed using WB analysis. (f) IHC staining was performed to assess the Ki67 positive cells in the tumor tissues of each group. *p < 0.05.