circ_0038718 promotes colon cancer cell malignant progression via the miR-195-5p/Axin2 signaling axis and also effect Wnt/β-catenin signal pathway

Abstract

Objective: Colon cancer (CC) is one of the most common cancers whose progression is regulated by a number of factors, including circular RNAs (circRNAs). Nonetheless, circ_0038718 is a novel circRNA, and its regulatory mechanism in CC remains unclear.

Methods: Real-time quantitative PCR (qRT-PCR) was performed to detect the expression of circ_0038718, miR-195-5p and Axin2. Western blot was conducted to determine the protein expression of Axin2 and the key proteins on Wnt/β-catenin signaling pathway. Oligo (dT) ₁₈ primers and RNase R were employed to identify the circular features of circ_0038718, and the location of circ_0038718 in cells was detected via nucleocytoplasmic separation. Dual-luciferase reporter assay and RNA binding protein immunoprecipitation experiment were carried out to investigate the molecular mechanism of circ_0038718/miR-195-5p/Axin2. Additionally, MTT assay was conducted to assess cell proliferation; Transwell assay was performed to evaluate cell migration and invasion, respectively. The effect of circ_0038718 on CC tumor growth was tested through tumor formation in nude mice.

Results: circ_0038718 was highly expressed in CC and could sponge miR-195-5p in cytoplasm. Silencing circ_0038718 suppressed the proliferative, migratory and invasive abilities of CC cells, while the promoting effect of high circ_0038718 expression on CC cells was reversed upon miR-195-5p over-expression. Axin2 was a downstream target of miR-195-5p and could regulate the Wnt/β-catenin signaling pathway. Axin2 expression was modulated by circ_0038718/miR-195-5p. Knockdown of Axin2 could also attenuate the promoting effect of high circ_0038718 expression on CC cell malignant progression, thus inhibiting tumor growth.

Conclusion: circ_0038718 is able to facilitate CC cell malignant progression via the miR-195-5p/Axin2 axis, which will provide a new idea for finding a novel targeted treatment of CC.

Keywords: Invasion; Migration; Proliferation; Wnt/β-catenin signaling pathway; circ_0038718; colon cancer.

Fig. 1

circ_0038718 is up-regulated in CC tissue and cells. (A) 59 DE_circRNAs obtained from GSE126094 (circRNA) Series Matrix File; (B) Relative expression of circ_0038718 in the GEO database (Blue and red boxes represent normal and tumor group, respectively); (C) Expression of circ_0038718 in adjacent tissue and colorectal cancer tissue, respectively; (D) qRT-PCR was performed to detect circ_0038718 expression in normal human colon cell line CCD-18Co and CC cell lines LoVo, HT-29, HCT116 and SW480; (E) Relative expression of circ_0038718 and mIL4R in HT-29 and SW480 cells were analyzed by qRT-PCR after normalized with random primers and oligo (dT) ₁₈ primers; (F) Relative expression of circ_0038718 and mIL4R in HT-29 and SW480 cells were assessed via qRT-PCR after treatment with RNase R. * p < 0.05

Fig. 2

circ_0038718 promotes CC cell proliferation, migration and invasion. (A) qRT-PCR was performed to detect circ_0038718 expression in all groups; (B) MTT assay was conducted to assess the viability and proliferative ability of CC cells in all groups; (C-D) Transwell assay was carried out to evaluate CC cell migratory and invasive capacities in all groups (100×). * p < 0.05

Fig. 3

miR-195-5p is a direct target of and negatively regulated by circ_0038718. (A) 21 DE_miRNAs obtained from the GSE126093 Series Matrix File; (B) 12 down-regulated DE_miRNAs were intersected with 115 predicted target genes of circ_0038718; (C) Relative expression of miR-195-5p in GSE126093 Series Matrix File. Blue and red boxes stand for the normal and tumor groups, respectively; (D) Predicted binding site between miR-195-5p and circ_0038718; (E) qRT-PCR was conducted to test the expression of circ_0038718, U6 and GAPDH in cytoplasm and nucleus after nucleocytoplasmic separation of HT-29 and SW480 cells; (F-G) Dual-luciferase reporter assay and RIP experiment were performed to verify the targeting relationship between miR-195-5p and circ_0038718; (H) qRT-PCR was used to evaluate the effect of circ_0038718 over-expression on miR-195-5p; (I) The expression of miR-195-5p in colorectal cancer tissue and adjacent tissue, respectively; (J) The correlation between circ_0038718 and miR-195-5p expression level. * p < 0.05

Fig. 4

Up-regulation of miR-195-5p can reduce the promoting effect of circ_0038718 on CC cell proliferation, migration and invasion. (A) qRT-PCR was performed to detect miR-195-5p expression in all groups; CC cell viability and proliferative ability were assessed via (B) MTT assay; (C-D) Transwell assay was conducted to evaluate CC cell migratory and invasive capacities (100×). * p < 0.05

Fig. 5

miR-195-5p regulates Wnt/β-catenin signaling pathway by targeting Axin2. (A) Volcano plot of 415 DE_mRNAs in the GEO database (Red and green dots represent up-regulated and down-regulated genes, respectively); (B) Venn diagram of up-regulated DE_mRNAs in the GEO database and the predicted target mRNAs of miR-195-5p; (C) Relative expression of Axin2 in the GEO database (Blue and red boxes represent normal and tumor group, respectively); (D) The expression level of Axin2 in colorectal cancer tissue and adjaceent tissue; (E) The correlation between Axin2 and miR-195-5p expression level; (F) The correlation between Axin2 and circ_0038718 expression level; (G) Putative binding sites between miR-195-5p and Axin2; (H) Dual-luciferase reporter assay was performed to detect the targeting relationship between miR-195-5p and Axin2; (I) qRT-PCR was performed to assess the effect of over-expressing miR-195-5p on Axin2 mRNA; (J) Western blot was conducted to evaluate the effect of miR-195-5p over-expression on the protein expression of Axin, β-catenin, c-Myc and cyclin D1; (K) qRT-PCR was performed to assess the effect of sh-Axin2 knock down. * p < 0.05

Fig. 6

circ_0038718 activates Wnt/β-catenin signaling pathway via the circ_0038718/miR-195-5p/Axin2 axis. (A) qRT-PCR was conducted to detect the expression of circ_0038718, miR-195-5p and Axin2 in each group; (B) Western blot was performed to determine the protein expression of Axin2 and the key proteins of the Wnt/β-catenin signaling pathway; CC cell viability and proliferative ability were assessed via (C) MTT assay; (D-E) Transwell assay was conducted to evaluate CC cell migratory and invasive capacities (100×). * p < 0.05

Fig. 7

circ_0038718 silencing reduces CC cell tumorigenesis in vivo. (A) Tumor cell tumorigenesis of nude mice in all groups was identified; (B) Average tumor weight of nude mice after 5 weeks; (C) Tumor volume of nude mice in all groups for 5 weeks; (D) qRT-PCR was performed to detect the expression of circ_0038718, miR-195-5p and Axin2 in tumor tissue of nude mice; (E) Immunohistochemistry was conducted to assess the expression of Axin2, β-catenin, c-Myc and cyclin D1 in tumor tissue (400×). * p < 0.05