Hypoxia Induces Drug Resistance in Colorectal Cancer through the HIF-1α/miR-338-5p/IL-6 Feedback Loop

Abstract

Hypoxia is associated with poor prognosis and therapeutic resistance in cancer patients. Accumulating evidence has shown that microRNA (miRNA) plays an important role in the acquired drug resistance in colorectal carcinoma (CRC). However, the role of miRNA in hypoxia-induced CRC drug resistance remains to be elucidated. Here, we identified a hypoxia-triggered feedback loop that involves hypoxia-inducible transcription factor 1α (HIF-1α)-mediated repression of miR-338-5p and confers drug resistance in CRC. In this study, the unbiased miRNA array screening revealed that miR-338-5p is downregulated in both hypoxic CRC cell lines tested. Repression of miR-338-5p was required for hypoxia-induced CRC drug resistance. Furthermore, we identified interleukin-6 (IL-6), which mediates STAT3/Bcl2 activation under hypoxic conditions, as a direct miR-338-5p target. The resulting HIF-1α/miR-338-5p/IL-6 feedback loop was necessary for drug resistance in colon cancer cell lines. Using CRC patient samples, we found miR-338-5p has a negative correlation with HIF-1α and IL-6. Finally, in a xenograft model, overexpressing miR-338-5p in CRC cells and HIF-1α inhibitor PX-478 were able to enhance the sensitivity of CRC to oxaliplatin (OXA) via suppressing the HIF-1α/miR-338-5p/IL-6 feedback loop in vivo. Taken together, our results uncovered an HIF-1α/miR-338-5p/IL-6 feedback circuit that is critical in hypoxia-mediated drug resistance in CRC; targeting each member of this feedback loop could potentially reverse hypoxia-induced drug resistance in CRC.

Keywords: HIF-1α; IL-6; colorectal cancer; drug resistance; miR-338-5p.

Graphical abstract

Figure 1

Hypoxia Induces Colorectal Cancer Drug Resistance by IL-6 (A and B) The IC₅₀ values of OXA, 5-Fu, DOX, and CTX in CRC (A) HCT116 and (B) HCT8 cells under hypoxic or normoxic conditions were determined using the CCK-8 assay. (C) Hypoxia decreased apoptosis level in CRC cells induced by oxaliplatin. A DNA fragmentation assay was used. (D) Culture supernatants from hypoxic or normoxic CRC cells with or without OXA were used to examine IL-6 level by ELISA. (E) Protein levels of the IL-6/STAT3/Bcl2 pathway were determined using western blot. (F) Anti-IL-6 slowed down the growth of hypoxic CRC cells under oxaliplatin treatment. A CCK-8 assay was used. (G) DNA fragmentation assays showed increased apoptosis level in hypoxic CRC cells induced by oxaliplatin (H) and decreased Bcl2 level or increased cleaved caspase-3 level by western blot analysis. *p < 0.05, **p < 0.01. Each bar represents the mean ± SD of three independent experiments.

Figure 2

Hypoxia-Decreased miR-338-5p Expression Confers the Resistance of CRC Cells to Oxaliplatin (A) The heatmap showed 22 differentially expressed miRNAs in hypoxic versus normoxic CRC cells by miRNA microarray (FC > 2-fold and p < 0.05). (B) miRNA microarray results showed miR-338-5p expression levels significantly decreased in hypoxic CRC cells. (C) qPCR validation showing miR-338-5p expression level was significantly decreased in hypoxic CRC cells. Overexpression of miR-338-5p induced by mimic transfection significantly (D) enhanced the growth-inhibitory effect of oxaliplatin in hypoxic CRC cells by CCK assay; (F) increased hypoxic CRC cell apoptosis level to oxaliplatin by DNA fragmentation assays; and (H) decreased Bcl2 level or increased cleaved caspase-3 level by western blot analysis. Downregulation of miR-338-5p induced by inhibitor transfection significantly (E) decreased normoxic CRC cell growth under oxaliplatin treatment by CCK assay; (G) decreased normoxic CRC cell apoptosis level to oxaliplatin by DNA fragmentation assays; and (I) increased Bcl2 level or decreased cleaved caspase-3 level by western blot analysis. *p < 0.05, **p < 0.01. Each bar represents the mean ± SD of three independent experiments.

Figure 3

miR-338-5p Targeting IL-6 Is Required for Hypoxia-Mediated CRC Drug Resistance (A) hsa-miR-338-5p was the intersection miRNA of miRDB, miRWalk, and TargetScan databases that targeted IL-6. (B) IL-6 protein expression level was found regulated directly by miR-338-5p, as reflected by the decreased IL-6 expression in hypoxic CRC cells after transient transfection of miR-338-5p mimic and increased IL-6 expression in normoxic CRC cells after miR-338-5p inhibitor transfection. IL-6 concentration of culture supernatants was (C) decreased by miR-338-5p mimic in hypoxic CRC cells and (D) increased by miR-338-5p inhibitor in normoxic CRC cells. Recombination human IL-6 (rhIL-6) significantly (E) decreased the growth-inhibitory effect of OXA combined with miR-338-5p in hypoxic CRC cells by CCK-8 assay; (F) decreased hypoxic CRC cell apoptosis level by DNA fragmentation assays; and (G) restored Bcl2 level or decreased cleaved caspase-3 level by western blot analysis. (H) The wild-type and mutant variant of the putative miR-338-5p target sequences of the IL-6 gene. (I) Two copies of the wild-type and mutant miR-338-5p target sequences were fused with a luciferase reporter and transfected into control oligonucleotide- and miR-338-5p mimic-infected HEK293T cells. miR-338-5p significantly suppressed the luciferase activity of the wild-type IL-6 3′ UTR. *p < 0.05, **p < 0.01. Each bar represents the mean ± SD of three independent experiments.

Figure 4

HIF-1α/miR-338-5p/IL-6 Feedback Loop May Be Responsible for Hypoxia-Mediated CRC Drug Resistance (A) qPCR analysis of miR-338-5p expression in hypoxic CRC cells treated with HIF-1α inhibitor PX-478 (25 μM) or HIF-1α KD plasmid. (B) IL-6 expression level of culture supernatants from hypoxic CRC cells treated with HIF-1α inhibitor PX-478 or HIF-1α KD plasmid. (C) qPCR analysis of miR-338-5p expression in hypoxic CRC cells treated with PX-478, with or without HIF-1α OE plasmid. (D) IL-6 expression level of culture supernatants from hypoxic CRC cells treated with PX-478 or HIF-1α KD plasmid, with or without miR-338-5p inhibitor. (E) Western blot analysis of the indicated proteins from hypoxic CRC cells treated with PX-478, with or without miR-338-5p inhibitor. (F) Western blot analysis of the indicated proteins from hypoxic CRC cells treated with miR-338-5p mimics and normoxic CRC cells treated with miR-338-5p inhibitor. (G) qPCR analysis of HIF-1α expression in hypoxic CRC cells with miR-338-5p mimics. (H) qPCR analysis of HIF-1α expression in normoxic CRC cells treated with miR-338-5p inhibitor. (I) Western blot analysis of HIF-1α expression from hypoxic CRC cells treated with miR-338-5p mimics, with or without rhIL-6/STAT3 OE plasmid. qPCR analysis of HIF-1α expression from hypoxic CRC (J) HCT116 and (K) HCT8 cells treated with miR-338-5p mimics, with or without rhIL-6 or STAT3 OE plasmid. *p < 0.05, **p < 0.01. Each bar represents the mean ± SD of three independent experiments.

Figure 5

Expression of HIF-1α/miR-338-5p/IL-6 Feedback Loop in Human CRC Specimens (A) Kaplan-Meier analysis of overall survival in patients with variable miR-338-5p expression, according to the data from the selected CRC tissues samples (p = 0.0237). (B) Relative expression levels of miR-338-5p were detected in adjacent and tumor tissues (n = 38) via qRT-PCR. Abundance of miR-338-5p was normalized to U6 RNA. Expression levels of miR-338-5p and (C) HIF-1α mRNA, (D) IL-6 mRNA, and (E) IL-6 of serum were inversely correlated among all CRC specimens (n = 38), as indicated by two-tailed Pearson’s correlation analysis, respectively. Expression levels of HIF-1α mRNA and (F) IL-6 mRNA and (G) IL-6 of serum were positively correlated among all CRC specimens (n = 38), respectively. (H) Expression levels of IL-6 mRNA and IL-6 of serum were positively correlated among all CRC specimens (n = 38). miR-338-5p expression levels were inversely correlated with (I) HIF-1α mRNA, (J) IL-6 mRNA, and (K) IL-6 of serum level expression in CRC specimens. Results are representative of three experiments.

Figure 6

Modulation of HIF-1α/miR-338-5p/IL-6 Feedback Loop Expression Alters the Sensitivity of CRC Cells to Oxaliplatin in a CRC Xenograft Model Overexpression of miR-338-5p increased the effectiveness of OXA in the inhibition of tumor growth in vivo. (A) Xenograft tumor growth curves. (B) Representative tumor pictures and tumor weights. (C) qPCR analysis of miR-338-5p expression in vivo. Overexpression of miR-338-5p reduced (D) HIF-1α mRNA expression and (E) serum level of IL-6 in the CRC xenograft model. (F) Immunohistochemical analysis of Ki67, HIF-1α, p-STAT3, Bcl2, and TUNEL in tumors. Inhibition of HIF-1α increased the effectiveness of OXA in the inhibition of tumor growth in vivo. (G) Xenograft tumor growth curves. (H) Pictures and weights of tumors. (J) qPCR analysis of HIF-1α mRNA expression in vivo. Inhibition of HIF-1α (I) increased miR-338-5p expression and (K) reduced serum level of IL-6 in the CRC xenograft model. (L) Immunohistochemical analysis of Ki67, HIF-1α, p-STAT3, Bcl2, and TUNEL in tumors. *p < 0.05, **p < 0.01. Each bar represents the mean ± SD of three independent experiments.