Circular RNA CirCHIPK3 promotes cell proliferation and invasion of breast cancer by sponging miR-193a/HMGB1/PI3K/AKT axis

Abstract

Background: The aim of this study was to explore the potential mechanism of circular RNA (circRNA) CirCHIPK3 on the malignant proliferation and metastasis of breast cancer (BC).

Methods: Human BC samples and their matched normal adjacent tissues were obtained from 50 patients to assess the expression of CirCHIPK3 and its relationship with BC prognosis. A series of in vitro and in vivo functional experiments were carried out to elucidate the role of CirCHIPK3 in BC progression and its underlying molecular mechanisms. Moreover, the interaction of CirCHIPK3, miR-193a, and HMGB1 was examined using bioinformatics, FISH, RIP, RNA-pull down and luciferase reporter assays. Western blot analysis was performed to examine the expression of HMGB1, p-PI3K, total PI3K, p-AKT, and AKT after si-CirCHIPK3 transfection.

Results: Upregulation of CirCHIPK3 was identified in BC, which predicted a worse prognosis in BC patients. Furthermore, it was found that CirCHIPK3 facilitated cell proliferation, migration, and invasion in BC by regulating miR-193a/HMGB1/PI3K/AKT signaling. CirCHIPK3 acted as a sponge for miR-193a to facilitate HMGB1 expression. si-CirCHIPK3 also inhibited tumor growth of BC in nude mice. si-CircCHIPK3 decreased HMGB1/PI3K/AKT signal expression in MDA-MB-231 cells, whereas overexpression of CircCHIPK3 enhanced HMGB1/PI3K/AKT signal.

Conclusions: CirCHIPK3 regulated miR-193a/HMGB1/PI3K/AKT signaling to facilitate BC development and progression, providing a novel therapeutic target for BC.

Keywords: Breast cancer; CirCHIPK3; endogenous competitive RNA; high-mobility group box-1; miR-193a.

Figure 1

(a) Comparison of CirCHIPK3 expressions; (b) miR‐193a expressions; and (c) HMGB1 expressions between BC tissue and adjacent normal tissue in BC patients. (d) Correlation of miR‐193a expressions with CirCHIPK3 expressions in BC tissue. (e) Correlation of HMGB1 expressions with CirCHIPK3 expressions in BC tissue. (f) Kaplan‐Meier overall survival curves based on CirCHIPK3 expression levels. (g) HMGB1 expressions detected by IHC between normal tissue and BC tissue. (h) Quantitative analysis of HMGB1 expressions between normal tissue and BC tissue.

Figure 2

(a) The expressions of CirCHIPK3 in normal and breast cancer cells. (b) MDA‐MB‐231 cell proliferation was detected by MTT assay following si‐CirCHIPK3 and OE‐ CirCHIPK3 (*P < 0.05,**P < 0.01) () si‐CircHIPK3, () si‐CircNC, () OE‐CircNC, () OE‐CircHIPK3. (c) MCF‐7 cell proliferation was detected by MTT assay following si‐CirCHIPK3 and OE‐CirCHIPK3 (*P < 0.05,**P < 0.01) () si‐CircHIPK3, () si‐CircNC, () OE‐CircNC, () OE‐CircHIPK3. (d and f) Wound healing assay for migration rate after 0, 24 and 48 hours using MDA‐MB‐231 cells following si‐CirCHIPK3 and OE‐CirCHIPK3 (*P < 0.05,**P < 0.01) () si‐CircHIPK3, () si‐CircNC, () OE‐CircNC, () OE‐CircHIPK3. (e and g) Transwell invasion assay for tumor cell invasion after 0, 24 and 48 hours using MDA‐MB‐231 cells following si‐CirCHIPK3 and OE‐ CirCHIPK3 (*P < 0.05,**P < 0.01).

Figure 3

si‐CirCHIPK3 inhibited tumor growth of BC. (a) Representative photographs of BC tumor tissue in si‐CirCHIPK3 and si‐CircNC groups. (b) Tumor weight in si‐CirCHIPK3 and si‐CircNC groups at four weeks. **P < 0.01. (n = 6 for each group). (c) Comparison of si‐CircHIPK3 group and si‐CircNC group tumor volume from day 1 to day 24 (**P < 0.01,*P < 0.05) (n = 6 for each group) () si‐CircHIPK3, () si‐CircNC. (d) Expressions of CircHIPK3 during the tumor growth between si‐CirCHIPK3 and si‐CircNC groups () si‐CircHIPK3, () si‐CircNC.

Figure 4

(a) Bioinformatics analysis of matching sequence of miR‐193a within 3′‐UTR of CircCHIPK3. MuT CircCPK3 3′‐UTR is the mutation of the match sequence of 3′‐UTR of CircCPK3with miR‐193a. (b) The predicted binding sites between HMGB1 and miR‐193a. MuT HMGB1 3′‐UTR is the mutation of the match sequence of 3′‐UTR of HMGB1 with miR‐193a. (c) Luciferase reporter assay revealed that miR‐193a binds to the 3′‐UTR of WT CircCHIPK3, not MuT CircCHIPK3 () miR‐NC, () miR‐193a mimic. (d) Luciferase activity was measured in MDA‐MB‐231 cells cotransfected with mimic NC or miR‐193a mimic and HMGB1‐wt or HMGB11‐mut reporter at 48 hours after transfection () miR‐NC, () miR‐193a mimic. HMGB1 was directly targeted by miR‐193a. Relative luciferase activity was quantified and the data were presented as mean ± SD (* P<0.05, ** P<0.01). (e) Representative images of western blot analysis of CircHIPK3 and miR‐193a on HMGB1 expressions. (f) Quantitative analysis of HMGB1 expressions influenced by CircHIPK3 and miR‐193a mimics (* P<0.05).

Figure 5

(a) Relative CircCHIPK3 and miR‐193a expression presented as fold enrichment in Ago2 relative to normal IgG immunoprecipitates. RIP assays disclosed that CircCHIPK3 and miR‐193a expressions were substantially enriched by Ago2 antibody compared with control IgG antibody () Anti‐IgG, () Anti‐AGOC2, () Input. (b) The biotin‐coupled probe pull‐down assay was performed and the results showed miR‐193a was detected in the CircCHIPK3 pulled‐down pellet compared with the control group. (c) CircCHIPK3 was detected in the biotin‐miR‐193a vector compared with the control group. (d) CIRCHIPK3 and miR‐193a were colocalized MCF‐7 and MDA‐MB‐231 cells by FISH using confocal microscopy. CircCHIPK3 was stained green, miR‐193a was stained red, nuclei were stained blue (DAPI) and overlapped expression was mixed (Scale bar, 20 μm). *P < 0.05,** P < 0.01.

Figure 6

si‐CircCHIPK3 attenuated the HMGB1/PI3K/AKT signaling axis in MDA‐MB‐231 cells. (a–f) Representative immunoblots (a); quantitative evaluation of HMGB1 (b); PI3K phosphorylation (c); total p‐PI3K (d); AKT phosphorylation (e); and total AKT (f) expression in MDA‐MB‐231 cells subsequent to transient transfection with si‐CircCHIPK3 or CircNC. Results are represented as mean ± SD. *P < 0.05,** P < 0.01 (compared to vector group).

Figure 7

OE‐CircCHIPK3 enhanced the HMGB1/PI3K/AKT signaling axis in MDA‐MB‐231 cells. (a–f) Representative immunoblots (a); quantitative evaluation of HMGB1 (b); PI3K phosphorylation (c); total p‐PI3K (d); AKT phosphorylation (e); and total AKT (f) expression in MDA‐MB‐231 cells subsequent to transient transfection with OE‐CircCHIPK3 or CircNC. Results are represented as mean ± SD. *P < 0.05,** P < 0.01 (compared to vector group).