Has_circ_0010220 regulates the miR-574-3p/IL-6 axis to increase doxorubicin resistance in osteosarcoma

Abstract

Background

Osteosarcoma (OS) is the most common primary bone malignancy. It has an aggressive nature and produces drug resistance in diseased patients, which in turn causes obstacles in treating cancer with chemotherapy. The objective of our investigation was to analyze the function and hsa_circ_0010220 mechanism in doxorubicin (DOX) resistance to OS.

Methods

The hsa_circ_0010220, IL-6, and miR-574-3p levels in OS diseased tissues and cell resistance towards DOX drug were elucidated by qRT-PCR and Elisa assay. The DOX half-inhibitory concentration (IC₅₀) was quantified by Cell Counting Kit-8. For this study, we used RNA pull-down, RNA immunoprecipitation, and a dual-luciferase reporter experiment to identify the proteins that interacted with has_circ_0010220, IL-6, and miR-574-3p in OS cells that have developed resistance towards DOX.

Results

The results indicated upregulated Hsa_circ_0010220 and IL-6 expression, However, DOX-resistant OS tissues and cells showed a downregulation of miR-574-3p. Reducing DOX resistance in vitro was achieved by silencing Has_circ_0010220. Further, by sponging miR-574-3p, increasing has_circ_0010220 boosted DOX resistance. However, miR-574-3p bound to IL-6 and inhibited DOX resistance. Additionally, it was discovered that hsa_circ_0010220 sponged miR-574-3p for upregulating IL-6 expression.

Conclusions

Hsa_circ_0010220 encouraged OS resistance to DOX by miR-574-3p/IL-6 axis regulation, suggesting its potency as a promising biomarker for treating OS.

Keywords

Hsa_circ_0010220 doxorubicin resistance miR-574-3p osteosarcoma IL-6

Introduction

Osteosarcoma (OS) is a severe kind of primary bone tumor that most often arises in young adults.^1,2 The progress in medicinal technology has allowed the use of surgical resection and different therapies including chemotherapy, these techniques not only broaden the treatment options for OS patients but also prolonged the 5-year survival rate by 70%³. But resistance to chemotherapy has become a major concern as it acts as an obstacle during treatment, hence, the success of therapy for OS may be greatly enhanced by addressing this issue of medication resistance.

New noncoding RNAs known as circular RNAs (circRNAs) are very stable due to their covalent closed-loop structure.⁴ Previous literature supports the fact that circRNAs have a crucial part in the establishment and advancement of various types of cancers.^5–7 The accumulated data also suggest that these RNAs function aberrantly in OS, and their imbalance is associated with OS pathogenesis.⁸ For instance, via circ_001422, miR-195-5p/FGF2/PI3K/Akt axis induces OS advancement and metastasis.⁹ Circ_0000527 induces OS cell progression by regulating miR-646/ARL2 axis.¹⁰ Additionally, hsa_circ_0010220, which is a transcript of Rho guanine nucleotide exchange factor 10 like (ARHGEF10L), when upregulation causes substandard OS prognosis.¹¹ However, despite extensive research, the role of hsa circ 0010220 in OS chemoresistance is still unclear.

The objective of this investigation is to discover the influential activity of hsa_circ_0010220 on (doxorubicin) DOX resistance during OS treatment and to uncover its underlying molecular mechanism.

Materials and Methods

Specimen collection

The study included a total of 284 OS patients, of which, 142 were sensitive to treatment (i.e. chemo-sensitive) and 142 were resistant to DOX treatment (i.e. Chemo-resistant). This classification was done using the Huvos scoring system. Our investigation was done after The Affiliated Wuxi No. Two People’s Hospital of Nanjing Medical University Ethics Committee approval. Corresponding informed consent was taken from every participant.

Cell culture and treatment

American Type Culture Collection was the source for the U2OS and KHOS (OS) cell lines (ATCC, Manassas, VA). By subjecting cells to increasing doses of DOX, the DOX-resistant cell lines U2OS/DOX and HOS/DOX were formed (Solarbio, Beijing, China). Each cell line was maintained in a 37°C, 5% CO₂ incubator with DMEM (DMEM; Gibco, Carlsbad, CA) supplemented with 10% FBS (FBS; Gibco).

Quantitative real-time polymerase chain reaction

After RNA was isolated using TRIzol (Invitrogen), it was reverse transcribed using the HiScript II One-Step RT-PCR Kit (Vazyme, Nanjing, China) and the miRNA 1^st Strand cDNA Synthesis Kit (Vazyme), and then qRT-PCR analysis was performed using AceQ qRT-PCR SYBR Green Master Mix (Vazyme). Internal controls included glyceraldehyde 3-phosphate dehydrogenase and U6.

Cell counting kit-8 assay

The optimal time for incubation with escalating DOX concentration was determined by first cultivating about 3 × 10³ cells in 96-well plates. Post incubation cells were treated for 2 h with a 10 μL reagent from the CCK-8 kit (Solarbio). The optical absorbance was assessed by Multi-Mode Reader (BioTek, Burlington, VT) at 450 nm for evaluating the viability cell. DOX half-maximal inhibitory concentration (IC₅₀) was calculated.

Dual-luciferase reporter assay

The pmirGLO vector (Promega, Madison, WI) was modified to include the has_circ_0010220-wt, IL-6 3′UTR-wt, IL-6 3^′UTR-mut, and hsa circ 0010220-mut reporter sequences, all of which include the miR-574-3p binding site and mutations. Then, 293T cells were co-transfected with miR-574-3p, miR-NC, and a luciferase reporter. Luciferase activity was measured using a Dual-Luciferase Reporter Assay Kit (Promega).

RNA immunoprecipitation assay

The EZ-Magna RIP kit (Millipore) was utilized to perform RIP. The DOX-resistant cell lines were initially lysed using RIPA buffer to obtain cell lysates, which were then treated with Ago2 and IgG antibody conjugated magnetic beads. The hsa_circ_0010220, IL-6, and miR-574-3p expressions were assessed via qRT-PCR.

RNA pull-down assay

GenePharma supplied both the biotin-labeled has_circ_0010220 probe (Bio-has_circ_0010220) and the negative control probe (Bio-NC). For 2 h at 37° C, biotinylated probes were incubated with M-280 streptavidin Dynabeads (Invitrogen) to create probe-laminated beads. then the cells were lysed and the beads were incubated with the probes at 4°C for 3 hours. In the end, qRT-PCR was used to examine miR-574-3p concentrations.

Statistical analysis

The full dataset is shown as mean ± standard deviation. GraphPad Prism 7.0 was used for the statistical analysis of the data. Student’s t-test and one-way analysis of variance were used to determine the differences. A statistically significant difference was considered when p < .05.

Results

DOX-resistant OS tissues and cells increased Has_circ_0010220

The hsa_circ_0010220 expressions were markedly enhanced in chemo-resistant than in chemo-sensitive OS subjects (Figure 1(a)). Moreover, DOX IC₅₀ in DOX-resistant cell lines was significantly higher than their corresponding normal cell line, suggesting effective DOX-resistant cell lines establishment (Figure 1(b)). Moreover, hsa_circ_0010220 expression in normal OS cells was interestingly more than in hFOB1.19 cells and noticeably reduced than OS cells which were resistant towards DOX (Figure 1(c)). In light of the aforementioned, it appears that has_circ_0010220 may be a sustenance element linked to DOX resistance in OS treatment.

Figure 1.

Hsa_circ_0010220 was upregulated in DOX-resistant Osteosarcoma (OS) tissues and cells. (a). qRT-PCR detection of hsa_circ_0010220 expression in Chemosensitive and Chemoresistant OS tissues. (b). IC₅₀ values were determined by CCK-8 analysis. (c). Hsa_circ_0010220 expression was detected in hFOB1.19 cells, parental OS cells (U2OS and HOS), and DOX-resistant OS cells (U2OS/DOX and HOS/DOX) using qRT-PCR. *p < .05.

Hsa_circ_0010220 knockdown impeded DOX resistance in DOX-resistant OS cells

Resistant cell lines were treated with si-NC and si-has_circ_0010220 to evaluate the effect of has_circ_0010220 on DOX resistance. As a first step, qRT-PCR was used to evaluate the success of the transfection, and it was determined that si-circ#2 had the highest knockdown potency, hence this was the construct chosen for future study (Figure 2(a)). It was revealed from the CCK-8 assay that hsa_circ_0010220 suppression reduced DOX-resistant cell lines IC₅₀ (Figure 2(b)). These results unveiled that hsa_circ_0010220 depletion, suppressed DOX-resistance.

Figure 2.

Hsa_circ_0010220 knockdown impeded DOX resistance in DOX-resistant OS cells. (a). U2OS/DOX and HOS/DOX cells were introduced with si-NC, si-circ#1, si-circ#2, or si-circ#3, and the knockdown efficiency was examined by qRT-PCR. (b). IC₅₀ value was evaluated using a CCK-8 assay. *p < .05.

Hsa_circ_0010220 was a miR-574-3p sponge

For the subsequent exploration of the potential hsa_circ_0010220 mechanism associated with OS, we first, predicted the potential hsa_circ_0010220 targets using e-databases. Putative binding sites for hsa_circ_0010220 and miR-574-3p were discovered in the Circular RNA Interactome database (Figure 3(a)). Subsequently, we determined that miR-574-3p mimics, significantly decreased luciferase function of has_circ_0010220 WT reporter by using dual-luciferase reporter analysis (Figure 3(b)). Has_circ_0010220 binding to miR758 was also confirmed using RNA pull-down and RIP experiments. RIP analysis indicated that hsa_circ_0010220 was enhanced in DOX-resistant miR-574-3p transfected cell lines by Ago2 antibody (Figure 3(c)). RNA pull-down analysis revealed that Bio-hsa_circ_0010220 WT can pull down miR-574-3p, however, not by Bio-hsa_circ_0010220 MUT (Figure 3(d)). Furthermore, miR-574-3p levels in chemo-resistant subjects was markedly decreased than in chemo-sensitive subjects (Figure 3(e)), whereas, hsa_circ_0010220 expression was inversely related to miR-574-3p levels of expression (Figure 3(f)). DOX-resistant cell lines showed significantly lower miR-574-3p concentrations compared to their non-resistant OS cell line. (Figure 3(g)). Lastly, hsa_circ_0010220 transfection suppressed miR-574-3p levels in OS, DOX-resistant cells (Figure 3(h)). Overall, the results indicated a direct target of hsa_circ_0010220 towards miR-574-3p.

Figure 3.

Hsa_circ_0010220 was a sponge of miR-574-3p. (a). The putative binding sites of hsa_circ_0010220 and miR-574-3p were exhibited. (b). Luciferase activity was detected by dual-luciferase reporter assay in U2OS/DOX and HOS/DOX cells co-transfected with hsa_circ_0010220 WT or hsa_circ_0010220 MUT and miR-574-3p or miR-NC. (c). RIP assay was performed to confirm whether hsa_circ_0010220 bound to miR-574-3p. (d). RNA pull-down assay was used to verify the relationship between hsa_circ_0010220 and miR-574-3p. (e). qRT-PCR detection of miR-574-3p expression in Chemosensitive and Chemoresistant OS patients. (f). Hsa_circ_0010220 expression was inversely correlated with miR-574-3p expression. (g). The expression of miR-574-3p was measured using qRT-PCR in U2OS, HOS, U2OS/DOX, and HOS/DOX cells. (h) The expression level of miR-574-3p was detected in U2OS/DOX and HOS/DOX cells transfected with pcDNA or hsa_circ_0010220. *p < .05.

miR-574-3p Inhibition abrogated hsa_circ_0010220 knockdown effect on DOX resistance in OS DOX-resistant cells

Since hsa_circ_0010220 acted as a miR-574-3p sponge, it was speculated that hsa_circ_0010220 can regulate resistance to DOX and OS advancement by controlling miR-574-3p. To provide a verification for this assumption, recovery experiments in si-NC, si-circ#2+in-miR-NC, si-circ#2, and si-circ#2+in-miR-574-3p transfected DOX-resistant OS cells were carried out. The miR-574-3p transfection reversed its enhanced expression, which was stimulated by silencing of hsa_circ_0010220 (Figure 4(a)). The results revealed from the CCK-8 assay indicated that a decrease in DOX-resistance produced by knockdown of hsa_circ_0010220 was terminated by down-regulation of miR-574-3p (Figure 4(b)). These data suggested that hsa_circ_0010220 modulates DOX resistance by miR-574-3p sponging.

Figure 4.

Inhibition of miR-574-3p abrogated the effect of hsa_circ_0010220 knockdown on DOX resistance in DOX-resistant OS cells. (a) The level of miR-574-3p was examined by qRT-PCR. B IC₅₀ of DOX was determined by the CCK-8 assay. *p < .05.

miR-574-3p targets IL-6

An e-database called TargetScan provided information that miR-574-3p and IL-6 3^′UTR contain putative binding locations (Figure 5(a)). According to results from an experiment using a dual-luciferase reporter, mature miR-574-3p inhibited the luciferase activity of the IL-6 3^′UTR-wt reporter (Figure 5(b)). The IL-6 mRNA level was greatly elevated in chemoresistant subjects, relative to chemosensitive subjects (Figure 5(c)). Additionally, miR-574-3p and IL-6 expression was negatively associated with OS tissues (Figure 5(d)). The enrichment of IL-6 in the Bio-miR-574-3p probe was higher than the control probe (Figure 5(e)). In comparison with normal OS cell lines, IL-6 mRNA and protein concentration in DOX-resistant cell lines were highly upregulated (Figures 5(f) and (g)). MiR-574-3p mimics suppressed IL-6 mRNA and protein expression while miR-574-3p knockdown has the opposite effect, demonstrating that miR-574-3p specifically targets IL-6 (Figures 5(h) and (i)).

Figure 5.

IL-6 was a target of miR-574-3p. (a). The predicted binding sites of miR-574-3p and IL-6 3’UTR were displayed. (b). Dual-luciferase reporter assay was performed to validate the relationship between miR-574-3p and IL-6. (c). The expression of IL-6 was tested in Chemosensitive and Chemoresistant OS patients by qRT-PCR. (d). miR-574-3p and IL-6 levels were negatively correlated in OS tissues. (e). RNA pull down assay was used to confirm whether miR-574-3p and IL-6. (f) and (g). The mRNA and protein level of IL-6 was detected in U2OS, HOS, U2OS/DOX, and HOS/DOX cells. (h) and (i). U2OS/DOX and HOS/DOX cells were introduced with miR-NC, miR-574-3p, pcDNA, or hsa_circ_0010220, and IL-6 expression was measured using qRT-PCR and Elisa. *p < .05.

MiR-574-3p reduced DOX resistance by targeting IL-6

miR-574-3p and IL-6 association in DOX-resistance was determined by transfecting, DOX-resistant cell line with miR-574-3p, NC, miR-574-3p+pcDNA, and miR-574-3p+IL-6., The transfection with IL-6 altered the reduction in IL-6 expression produced by miR-574-3p overexpression (Figures 6(a) and (b)). Furthermore, miR-574-3p upregulation reduced DOX IC₅₀ value, whereas the effect vanished post-IL-6 transfection (Figure 6(c)).

Figure 6.

miR-574-3p reduced DOX resistance by targeting IL-6. (a) and (b). mRNA and protein levels of IL-6 were examined by qRT-PCR and Elisa. (c) and (d). CCK-8 assay was used to assess cell sensitivity to DOX. *p < .05.

Has_circ_0010220 regulated miR-574-3p and IL-6 expression

Spearman’s analysis showed a positive relationship between has_circ_0010220 and IL-6 expressions in OS tissue samples (Figure 7(a)). To determine hsa_circ_0010220, miR-574-3p, and IL-6 modulating mechanisms, DOX-resistant cell lines were transduced with si-NC, si-circ#2+in-miR-NC, si-circ#2, and si-circ#2+in-miR-574-3p. As Figure 7(b) and (c) displays, miR-574-3p inhibition altered the reduction of IL-6 expression, which was produced by hsa_circ_0010220 knockdown. According to the results presented above, miR-574-3p sponging may have reduced IL-6 expression as a result of has_circ_0010220 silencing.

Figure 7.

Hsa_circ_0010220 modulated IL-6 expression by regulating miR-574-3p. (a). Spearman’s correlation analysis was used to detect the relationship between hsa_circ_0010220 and Il-6 in OS tissues. (b) and (c). U2OS/DOX and HOS/DOX cells were transfected with si-NC, si-circ#2, si-circ#2+in-miR-NC, or si-circ#2+in-miR-574-3p, and IL-6 expression was examined using qRT-PCR and Elisa. *p < .05.

Discussion

Variations in chemotherapy efficacy and toxicity is a major factor that limits the treatment options for OS.¹² The potency of DOX during OS treatment is often compromised because of acquired resistance.¹³ In recent years, non-coding RNAs became the topic of interest for their potential role against complex drug resistance mechanisms.¹⁴ Our investigation clarified the impact that hsa_circ_0010220 has on OS patients who develop resistance towards DOX.

The cytoplasmic circRNAs have been shown to act as competitive endogenous RNAs (ceRNAs) and miRNA sponges, hence regulating the expression levels of their target mRNAs.¹⁵ For instance, By targeting the miR-379-5p/MAP3K9 axis, Circ SIPA1L1 promotes OS development,¹⁶ and hsa_circ_0003732 causes cell proliferation in OS through the miR-545/CCNA2 axis.¹⁷ Circ_0001721 stimulates the OS progression via miR-372-3p/M/]APK7 axis.¹⁸ Previous studies have shown that has_circ_0010220 may boost OS development by regulating the miR-198/Syntaxin 6 axis.¹¹ Our data correlated with these studies as hsa_circ_0010220 was greatly enhanced in OS patients and cells that were resistant to DOX. With reference to functional experiments, hsa_circ_0010220 reduction caused inhibition of resistance caused by DOX and repressed tumor advancement in resistant OS cells.

Bioinformatics tools were applied for evaluating the molecular foundation of hsa_circ_0010220 in OS, and to suggest miRNAs that might interact for binding with hsa_circ_0010220. After the evaluation miR-574-3p was selected. Many studies have certified that miR-574-3p can suppress many tumors. For instance, miR-574-3p represses esophageal cancer progression and invasion by targeting FAM3C and MAPK1.¹⁹ It causes tumor suppression in ovarian cancer by obstructing the expression of MMP3.²⁰ Through the miR-574-3p/HMGA2 axis, the LncRNA ZEB2-AS1 promotes the esophageal squamous cell carcinoma migration, proliferation, and cellular invasion.²¹ According to our findings, miR-574-3p was significantly down-regulated in DOX-resistant tissues and cells. In addition, the impact of has_circ_0010220 silencing on DOX-resistance and tumor progression in OS DOX-resistant cells were modified by suppression of miR-574-3p. IL-6 has also been indicated to induce prostate cancer pathogenesis and processes by autocrinal cross-activation of type 1 insulin-like growth criterion receptor²² and to induce prostate tumor cell proliferation.²³ IL-6 also increased OPN-STAT3 pathway activity in MG-63 and U2OS osteosarcoma cells to promote cancer stemness and oncogenicity.²⁴ Our investigation suggests remarkably elevated levels of IL-6 in DOX-resistant tissues and cells. It was also worth noting that IL-6 altered the suppressing effect of miR-574-3p on DOX resistance.

Conclusion

This investigation concludes that hsa_circ_0010220 increased DOX-resistance in OS by miR-574-3p/IL-6 axis regulation. The findings provide a potent and novel therapeutic marker that would improve chemotherapy for OS patients. The study lacks in-vivo experiments. Additionally, the exact hsa_circ_0010220 mechanism regulating osteosarcoma still needs further exploration.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was supported by Young talent’s subsidy project in science and education of department of public health of Jiangsu Province (QNRC2016150).

ORCID iD

Baocai Ran

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