Downregulation of Circ_0071589 Suppresses Cisplatin Resistance in Colorectal Cancer by Regulating the MiR-526b-3p/KLF12 Axis

Abstract

Background: Chemoresistance is one key factor for the failure of cisplatin (CDDP)-based therapy in colorectal cancer (CRC). Although circular RNAs (circRNAs) are associated with chemoresistance development, the role and mechanism of hsa_circ_0071589 (circ_0071589) in the development of CDDP resistance in CRC remain unclear.

Methods: CDDP-resistant and sensitive CRC samples were collected. CDDP-resistant HCT116/CDDP and LOVO/CDDP cells were established. The levels of circ_0071589, microRNA (miR)-526b-3p and Krüppel-like factor 12 (KLF12) were detected via quantitative reverse transcription polymerase chain reaction, Western blot or immunohistochemistry. Cell viability, proliferation, cycle process, apoptosis, migration and invasion were examined via Cell Counting Kit-8, flow cytometry, transwell assay and Western blot. The association between miR-526b-3p and circ_0071589 or KLF12 was predicted by starBase, and explored via dual-luciferase reporter assay and RNA immunoprecipitation. The effect of circ_0071589 on CDDP resistance in CRC in vivo was investigated using a xenograft model.

Results: Circ_0071589 level was upregulated in CDDP-resistant CRC tissue samples and cell lines. Circ_0071589 knockdown inhibited CDDP resistance, proliferation, migration and invasion, and promoted apoptosis in CDDP-resistant CRC cells. Circ_0071589 was a sponge for miR-526b-3p. MiR-526b-3p knockdown reversed the role of circ_0071589 inhibition in CDDP resistance. MiR-526b-3p suppressed CDDP resistance by directly targeting KLF12. Circ_0071589 regulated KLF12 expression through modulating miR-526b-3p. Circ_0071589 knockdown aggravated CDDP-induced reduction of xenograft tumor growth by upregulating miR-526b-3p and decreasing KLF12.

Conclusion: Knockdown of circ_0071589 repressed CDDP resistance in CDDP-resistant CRC cells by regulating the miR-526b-3p/KLF12 axis.

Keywords: KLF12; circ_0071589; cisplatin resistance; colorectal cancer; miR-526b-3p.

Figure 1

Circ_0071589 expression in CRC tissues and cells. (A) Circ_0071589 level was detected in CRC tissues from CDDP-resistant (n=37) or sensitive patients (n=19) and control samples (n=56) via qRT-PCR. (B and C) Circ_0071589 level was measured in normal human colorectal mucosa cells FHC, CRC cell lines (HCT116 and LOVO cells) and corresponding resistant cell lines (HCT116/CDDP and LOVO/CDDP cells). (D and E) Cell viability and IC50 of CDDP were examined in HCT116 and LOVO cells and corresponding resistant HCT116/CDDP and LOVO/CDDP cells. *P<0.05, versus the indicated group.

Figure 2

The effect of circ_0071589 silence on CDDP-resistant CRC cell development. (A) Circ_0071589 expression was detected in HCT116/CDDP and LOVO/CDDP cells transfected with si-circ_0071589 or si-NC. (B and C) Cell viability and IC50 of CDDP were measured in HCT116/CDDP and LOVO/CDDP cells transfected with si-circ_0071589 or si-NC after treatment of different concentrations of CDDP for 48 h. Cell proliferation (D and E), cycle distribution (F and G), apoptosis (H), migration and invasion (I and J), and related protein levels (K and L) were examined in HCT116/CDDP and LOVO/CDDP cells transfected with si-circ_0071589 or si-NC. *P<0.05, versus the indicated group.

Figure 3

The association between circ_0071589 and miR-526b-3p. The target correlation between circ_0071589 and miR-526b-3p was explored via starBase (A), and confirmed via dual-luciferase reporter assay (B and C), and RIP (D and E). (F) MiR-526b-3p expression was measured in CDDP-resistant (n=37) or sensitive (n=19) CRC tissues and control samples (n=56). (G and H) MiR-526b-3p level was detected in CDDP-resistant or sensitive CRC cells. (I) MiR-526b-3p expression was detected in HCT116/CDDP and LOVO/CDDP cells transfected with si-circ_0071589 or si-NC. *P<0.05, versus the indicated group.

Figure 4

The effect of miR-526b-3p knockdown on silence of circ_0071589-mediated CDDP-resistant CRC cell development. MiR-526b-3p expression (A), cell viability and IC50 of CDDP (B and C), cell proliferation (D and E), cycle distribution (F and G), apoptosis (H), migration (I), invasion (J), and related protein levels (K and L) were detected in HCT116/CDDP and LOVO/CDDP cells transfected with si-NC, si-circ_0071589, si-circ_0071589 + anti-miR-NC or anti-miR-526b-3p. *P<0.05, versus the indicated group.

Figure 5

The association between miR-526b-3p and KLF12. The target correlation between miR-526b-3p and KLF12 was explored via starBase (A), and validated by dual-luciferase reporter assay (B and C). (D and E) KLF12 expression was measured in CDDP-resistant or sensitive CRC tissues and control samples. (F and G) KLF12 level was detected in CDDP-resistant or sensitive CRC cells. *P<0.05, versus the indicated group.

Figure 6

The effects of miR-526b-3p and KLF12 on CDDP-resistant CRC cell development. KLF12 expression (A and B), cell viability and IC50 of CDDP (C and D), cell proliferation (E and F), cycle distribution (G and H), apoptosis (I), migration (J), invasion (K), and related protein levels (L and M) were measured in HCT116/CDDP and LOVO/CDDP cells transfected with miR-NC, miR-526b-3p mimic, miR-526b-3p mimic + pcDNA-NC or pcDNA-KLF12. *P<0.05, versus the indicated group.

Figure 7

The correlation among circ_0071589, miR-526b-3p and KLF12. (A–C) The linear correlation among circ_0071589, miR-526b-3p and KLF12 levels in CDDP resistant CRC tissues was analyzed. (D) KLF12 expression was detected in HCT116/CDDP and LOVO/CDDP cells transfected with si-NC, si-circ_0071589, si-circ_0071589 + anti-miR-NC or anti-miR-526b-3p. *P<0.05, versus the indicated group.

Figure 8

The effects of circ_0071589 and CDDP on CRC xenograft tumor growth. (A) Tumor volume was measured every four days from CDDP treatment at 7th day (n=6). (B) The representative tumor images in each group. (C) Tumor weight was detected in each group. (D–G) circ_0071589, miR-526b-3p and KLF12 levels were measured in each group. *P<0.05, versus the indicated group.

Figure 9

The schematic diagram of this study. Circ_0071589 could target KLF12 via modulating miR-526b-3p, thus to regulate the proliferation, apoptosis, migration and invasion of CDDP-resistant CRC cells.