circHIPK3 promotes oxaliplatin-resistance in colorectal cancer through autophagy by sponging miR-637

Abstract

Background: Resistance to oxaliplatin-based chemotherapy is a major cause of recurrence in colorectal cancer (CRC) patients. There is increasing evidence indicating that circHIPK3 is involved in the development and progression of tumours. However, little is known about the potential role of circHIPK3 in CRC chemotherapy and its molecular mechanisms in chemoresistance also remain unclear.

Methods: Quantitative real-time PCR was performed to detect circHIPK3 expression in tissues of 2 cohorts of CRC patients who received oxaliplatin-based chemotherapy. The chemoresistant effects of circHIPK3 were assessed by cell viability, apoptosis, and autophagy assays. The relationship between circHIPK3, miR-637, and STAT3 mRNA was confirmed by biotinylated RNA pull-down, luciferase reporter, and western blot assays.

Findings: In the pilot study, increased circHIPK3 expression was observed in chemoresistant CRC patients. Functional assays showed that circHIPK3 promoted oxaliplatin resistance, which was dependent on inhibition of autophagy. Mechanistically, circHIPK3 sponged miR-637 to promote STAT3 expression, thereby activating the downstream Bcl-2/beclin1 signalling pathway. A clinical cohort study showed that circHIPK3 was upregulated in tissues from recurrent CRC patients and correlated with tumour size, regional lymph node metastasis, distant metastasis, and survival.

Interpretation: circHIPK3 functions as a chemoresistant gene in CRC cells by targeting the miR-637/STAT3/Bcl-2/beclin1 axis and might be a prognostic predictor for CRC patients who receive oxaliplatin-based chemotherapy.

Funding: National Natural Science Foundation of China (81301506), Shandong Medical and Health Technology Development Project(2018WSB20002), Shandong Key Research and Development Program (2016GSF201122), Natural Science Foundation of Shandong Province (ZR2017MH044), and Jinan Science and Technology Development Plan(201805084, 201805003).

Keywords: Autophagy; Chemoresistance; Colorectal cancer; STAT3; circHIPK3; miR-637.

Fig. 1

circHIPK3 is associated with CRC chemoresistance. (a) circHIPK3 expression was higher in tissues of non-responder group (n = 18) than in responder group (n = 31); Relative circHIPK3 expression levels were calculated using 2^−ΔCT method and is represented as the median (interquartile range); [Mann–Whitney U test]. (b) ROC curve for discriminating responders from non-responders based on circHIPK3 expression; AUC = 0•768 (95%CI = 0•625 to 0•876). (c) Expression of circHIPK3 was increased in oxaliplatin-resistant HT29oxR and HCT116oxR (P < 0•001) cell lines while not in 5-FU resistant cell lines compared to their respective parental cell lines (P > 0•05) [student's t-test]. (d) Images of tumour mass of each group (n = 5) on the 28th day. (e and f) The tumour volumes and weights from circHIPK3 overexpression cells were significantly larger than those from negative control cells. Data are presented as mean ±standard deviation. [student's t-test].

Fig. 2

circHIPK3 leads to oxaliplatin resistance in CRC cell lines. (a and b) Cell proliferative ability was assessed by CCK8 assay after circHIPK3 knockdown in HT29oxR (a) and HCT116oxR (b) cells at the indicated oxaliplatin concentration. (c and d) Cell proliferative ability was assessed by CCK8 assay after circHIPK3 overexpression, in HT29 (c) and HCT116 (d) cells at the indicated oxaliplatin concentration. (e) Oxaliplatin-induced apoptotic cell death was analysed by flow cytometry after treatment with si-circHIPK3 or overexpression vector in CRC cells. Data are presented as mean ±standard deviation from at least 3 independent experiments. *P < 0•05, ^⁎⁎P < 0•01, ^⁎⁎⁎P < 0•001 [student's t-test].

Fig. 3

Silencing of circHIPK3 induces autophagy and sensitises CRC cells to oxaliplatin. (a) Transfection of mRFP-GFP-LC3B lentiviral vector into HCT116oxR cell line. Red puncta represent autolysosomes, and yellow puncta represent autophagosomes as visualised by confocal microscopy. Scare bar = 20 μm (b) Quantification of autophagic flux. Puncta were counted in 100 cells; ^⁎⁎⁎P < 0•001 [student's t-test]. (c) Western blot analysis to determine LC3B- II/I ratio, p62 and, beclin1 expression in HCT116oxR cells.

Fig. 4

circHIPK3 functions as an efficient miR-637 sponge. (a) The putative miR-637 binding site in circHIPK3 and the corresponding mutant motif. (b) The pull-down efficiency of biotinylated-circHIPK3 probe tested by RT-qPCR in HT29 and HCT116 cells; ^⁎⁎⁎P < 0•001 [student's t-test]. (c) miR-637 pull-down by biotinylated-circHIPK3 probe was tested by RT-qPCR in HT29 and HCT116 cells transfected with circHIPK3 overexpression vector; Oligo probe was used as a control; *P<0•05, ^⁎⁎P < 0•01 [student's t-test]. (d) circHIPK3 pull -down by biotinylated wild-type/mutant miR-637 was tested by RT-qPCR in CRC cells with circHIPK3 overexpression. Relative levels of circHIPK3 were normalised to input; GAPDH was used as an internal control; ^⁎⁎⁎P < 0•001 [student's t-test]. (e) Relative luciferase activity of wild type or mutant circHIPK3 in miR-637 mimics or controls; ^⁎⁎P < 0•01 [student's t-test]. (f) Correlation between circHIPK3 and miR-637 expression in CRC tissues; r = −0•626, P < 0•001[Spearman test].

Fig. 5

circHIPK3 reverses miR-637-induced elevation of oxaliplatin chemosensitivity. (a and b) Cell proliferative ability was assessed by CCK8 assay in HT29 (a) and HCT116 (b) cells after transfection with mimic NC, miR-637 mimics, mimic NC plus (+) circHIPK3 overexpression vector, and miR-637 mimics+ circHIPK3 overexpression vector at the indicated OXA concentration. (c and d) Oxaliplatin-induced apoptotic cell death was analysed by flow cytometry in the above mentioned four groups. ^⁎⁎⁎P < 0•001 [student's t-test].

Fig. 6

Identification and verification of circHIPK3/miR-637/STAT3/Bcl-2/beclin1 axis. (a) The putative miR-637 binding site in Stat3, and the corresponding mutant motif. (b) Relative luciferase activity of wild type or mutant Stat3 in miR-637 mimics or controls; ^⁎⁎P < 0•01 [student's t-test]. (c) Levels of STAT3 mRNA were significantly down-regulated in HCT116 cells transfected with miR-637 mimics, compared to cells transfected with mimic NC; ^⁎⁎⁎P < 0•001 [student's t-test]. (d) Western blot analysis of STAT3, p-STAT3, LC3BII/I, p62, beclin1, Bcl-2 protein expression in HCT116 cells transfected with mimic NC, miR-637 mimics, mimic NC+ circHIPK3 overexpression vector, and miR-637 mimics plus (+) circHIPK3 overexpression vector. (e) Correlation between STAT3 mRNA and miR-637 expression in CRC tissues; n = 49, r = −0•834, P < 0•001 [Spearman test]. (f) Correlation between circHIPK3 and STAT3 mRNA expression in CRC tissues; n = 49, r = 0•577, P < 0•001 [Spearman test]. (g) The proposed working model.

Fig. 7

Expression of circHIPK3 in CRC patients who received oxaliplatin-based chemotherapy after surgery. (a) circHIPK3 expression was higher in tissues of CRC recurrence group than in non-recurrence group; Data represents the median (interquartile range); P < 0•001 [Mann–Whitney U test]. (b) ROC curve for discriminating CRC patients with recurrence from those without recurrence based on circHIPK3 expression; AUC = 0•758 (95%CI = 0•689–0•819). (c and d) Kaplan-Meier curves for DFS (c) and OS (d) based on circHIPK3 expression; CRC patients were classified as high and low circHIPK3 expression according to the optimal cut off value (0•040); P < 0•001 [log-rank test]. (e and f), Multivariate Cox analysis for DFS (e) and OS (f) of CRC patients.