Blocking hsa_circ_0074027 suppressed non-small cell lung cancer chemoresistance via the miR-379-5p/IGF1 axis

ABSTRACT Cancer cell chemoresistance is the primary reason behind cancer treatment failure. Previous reports suggest that circular RNA (circRNA) hsa_circ_0074027 (HC0074027) is a crucial modulator of non-small cell lung cancer (NSCLC) disease progression. Herein, we delineated the underlying mechanism of HC0074027-regulated chemoresistance in NSCLC. We employed quantitative real-time polymerase chain reaction (qRT-PCR) or Elisa in the detection of HC0074027, micoRNA-379-5p (miR-379-5p), and insuline-like growth factor I (IGF1) expressions. Cell survival was evaluated via the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Direct associations among HC0074027, miR-379-5p, and IGF1 were examined via dual-luciferase reporter (DLR) and RNA immunoprecipitation (RIP) assays. Lastly, HC0074027 was incorporated into nude mice to examine its biological activity in vivo. Based on our analysis, HC0074027 levels strongly correlated with NSCLC chemoresistance to docetaxel (DTX), cisplatin (DDP), and paclitaxel (PTX). Alternately, HC0074027 silencing enhanced chemosensitivity in vitro. In vivo, HC0074027 downregulation suppressed tumor expansion and increased cancer cell sensitivity to chemotherapy. We also revealed that HC0074027 physically interacts with miR-379-5p to exert its biological function in vitro. Moreover, IGF1 is a functionally crucial target of miR-379-5p in modulating NSCLC chemoresistance in vitro. Finally, we demonstrated that HC0074027 can indirectly modulate IGF1 levels via sequestering miR-379-5p. We demonstrated that HC0074027 promotes NSCLC chemoresistance via sequestering miR-379-5p activity, and modulating IGF1 expression. Our work highlights the significance of HC0074027 in NSCLC chemoresistance and suggests HC0074027 to be an excellent candidate for targeted NSCLC therapy.


Introduction
Non-small cell lung cancer (NSCLC) contributes to a large portion of cancer morbidity and mortality on the global stage [1]. Traditionally, NSCLC is treated with chemotherapeutic agents like docetaxel (DTX), cisplatin (DDP), and paclitaxel (PTX) [2][3][4][5][6]. However, chemoresistance to these drugs is a major reason of failure in the fight against NSCLC [7][8][9]. Given the prevalence and severity of NSCLC, it is both urgent and necessary to establish an underlying mechanism behind NSCLC chemoresistance to advance the development of targeted and effective drugs that can successfully combat this disease.
Herein, we are aimed at exploring the biological role of HC0074027 in NSCLC. We examined the expressions and functions of HC0074027 and miR-379-5p in chemoresistant NSCLC cells. To achieve a complete understanding of the underlying mechanism behind HC0074027 activity, we assessed chemoresistance in vitro and tumor expansion in vivo. We hypothesized direct binding between HC0074027 and miR-379-5p, which ultimately affected the expression of Insuline-like growth factor I (IGF1). Our work highlights a new pathway of HC0074027-regulated NSCLC chemoresistance and opens up new avenues for targeted anti-NSCLC treatment.

Patients and tissue samples
120 NSCLC patients, cured with DTX therapy, were selected from the second people's Hospital of Chengdu from January 2014 to November 2019. Drug effectiveness was evaluated in these patients once in 2 weeks during DTX therapy. Moreover, they were followed up once in 3 months till disease progression or death. Recurrent NSCLC was defined (SolidTumors, RECIST 1.1) as a rise in lesion diameter sum by 5 mm or 20% versus baseline [25,26]. An overall of 120 tissue samples were collected, with 60 identified as primary NSCLC (sensitive) and another 60 as recurrent NSCLC (resistant) tissues. We also acquired informed written consent from all participants and received ethical approval from our institution prior to the commencement of the study.

Dual-luciferase reporter (DLR) assay
MiR-379-5p docking sites harboring HC0074027 or IGF1 were cloned into psiCHECK2 (Promega, Fitchburg, WI) to generate the luciferase reporter plasmids WT-HC0074027 and WT-IGF1, respectively. We also generated the mutated forms of the above luciferase reporter plasmids (MUT-HC0074027 and MUT-IGF1). Next, the aforementioned vector, along with miRNA-NC or miR-379-5p-M, were co-incorporated into 293 T cells and incubated for 48 h. Finally, luciferase activity was measured with the DLR Assay Kit (Promega).

RNA immunoprecipitation (RIP) assay
The EZ-Magna RIP ™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was employed for the RIP evaluation. In short, after H1299/DTX and A549/DTX cellular lysis with RIP lysis buffer and RNase I (Millipore), 100 µL cellular lysate was exposed to RIP buffer carrying antibody-precoated magnetic beads. HC0074027 and miR-379-5p were then precipitated as described in the kit directions and their levels were assessed via RT-qPCR.

Elisa assay
An ELISA kit (R&D Systems) and its operational guidelines were employed for the quantification of IGF1 in different culture media. We performed 3 replicates of each experiment and the average values are presented in this manuscript.

Animal studies
Our animal protocols abided by the animal ethical review board of our hospital. 20 female BALB/c nude mice (ALF Biotechnology, Nanjing, China), aged 7-week-old, were recruited for this study and arbitrarily separated into 4 populations (n = 5/ group). To achieve a subcutaneous xenograft, sh-NC or sh-circ-incorporated A549/DTX cells were administered to the right flanks of nude mice. The xenograft tumors were allowed to grow and once it reached 100 mm 3 in size, DTX (5 mg/kg) or PBS (corresponding control group) was provided via tail vein once in every 3 days. Tumor volume (mm 3 ) was monitored and recorded once a week, computed as follows: length × width 2 × 0.5. After 28 days, the tumors were excised, weighed, and used for subsequent analysis.

Statistical analysis
The presented data are mean + standard deviation from 3 replicates. Statistical assessment employed Student's t-test and one-way analysis of variance (ANOVA). Association between HC0074027, IGF1, and miR-379-5p mRNA was assessed with the Spearman rank correlation. All statistical analyses were performed using GraphPad Prism Software 7.0. P < 0.05 was the significance threshold.

Results
In this study, we studied the biological role and the molecular mechanisms of HC0074027 in NSCLC. Our result indicated HC0074027 levels strongly correlate with NSCLC DTX-resistance. HC0074027 enhanced chemosensitivity of DRNCs in vitro and in vivo. Additionally, HC0074027 sponged miR-379-5p, and miR-379-5p targeted IGF1 to prevent chemoresistance in DRNCs. Thus, we revealed that HC0074027 modulates NSCLC chemoresistance via the miR-379-5p/IGF1 pathway, implying that our work highlighted a new underlying mechanism of NSCLC chemoresistance, which could be targeted to improve chemotherapy against NSCLC.

HC0074027 levels strongly correlate with NSCLC DTX-resistance
HC0074027 levels were substantially elevated in DTX-resistant versus DTX-sensitive tissues and cells (Figure 1(a,b)). Patient clinicopathological characteristics are summarized in Table 1. The 5-year survival rate of the DTX-resistant patients, with elevated HC0074027 levels, was considerably lower, compared to the DTXsensitive patients, with reduced HC0074027 levels (Figure 1(c)). We also demonstrated that the IC50s of DTX (Figure 1(d)), DDP (Figure 1 (e)) and PTX (figure 1(f)) were high in the DDP-resistant cells verses normal cells.

HC0074027 enhanced chemosensitivity of DRNCs
To elucidate HC0074027 function in DRNCs, HC0074027 was silenced in both A-DTX and HN-DTX cells via siRNA incorporation. Based on our RT-qPCR data, si-HC0074027-incorporated cells exhibited markedly reduced HC0074027 expression, relative to control cells (Figure 2(a)). Moreover, the IC50s of DTX (Figure 2(b)), DDP (Figure 2(c)), and PTX (Figure 2(d)) displayed a significant reduction, compared to controls. Hence, there is a strong possibility that HC0074027 levels regulate NSCLC chemoresistance

HC0074027 knockdown enhanced DTX sensitivity in vivo
We, next, tested HC0074027 regulation of DTX sensitivity in nude mice. Sh-HC0074027 transduction resulted in a strong suppression of tumor expansion, in presence or absence of DTX ( Figure 5(a,b)). Surprisingly, DTX alone did not affect tumor expansion. However, a combined administration of DTX and sh-HC0074027 generated a strong suppression of tumor expansion ( Figure 5(a,b)). Additionally, HC0074027 and IGF1 levels were markedly reduced and miR-379-5p levels were remarkably elevated in the sh-HC0074027-incorporated A-DTX tumors ( Figure 5(c,d)).

Discussion
Chemoresistance is the principal obstacle in cancer treatment [27]. Delineating the mechanism-(s) behind drug resistance can help establish new targeted therapeutics that can efficiently suppress NSCLC progression. CircRNAs are well established chief modulators of NSCLC chemoresistance. Therefore, targeting relavant circRNAs may be a crucial step toward NSCLC resistance management. It is known that circ_0076305 modulates DDP NSCLC resistance via positive modulation of signal transducer and activator of transcription 3 (STAT3) via miR-296-5p sequestering [28]. Similarly, circ_0000079 serves as a decoy for the RNAbinding fragile X-related 1 (FXR1), preventing the assembly of the FXR1/ complex-partner protein kinase C, iota (PRCKI) complex, and reducing cell invasion and drug resistance in NSCLC [29]. Unfortunately, even though HC0074027 has long been established as a NSCLC oncogene [21][22][23][24], its role in NSCLC chemoresistance is unknown. We, therefore, examined the underlying mechanism affecting the HC0074027mediated NSCLC resistance. We demonstrated that high HC0074027 levels correspond to DTXresistance in NSCLC tissues and cells. Conversely, HC0074027 downregulation prevented NSCLC chemoresistance in vitro and in vivo.
IGF1 expression is predominant in cancerous tissues like glioma [34] and colorectal cancers [35]. It is also prevalent in NSCLC and in small-cell lung cancer [36,37], and is known to contribute to NSCLC chemoresistance [38]. Herein, IGF1 levels were markedly elevated in DRNC and tissues. We are the first to report the miR-379-5p-mediated regulation of IGF1 activity, particularly in terms of NSCLC chemoresistance. Moreover, we also established a novel mechanism involving the HC0074027-miR-379-5p-IGF1 pathway. Based on these evidences, we propose that HC0074027 is an excellent candidate for targeted anti-NSCLC therapy.

Conclusion
In summary, we revealed that HC0074027 modulates NSCLC chemoresistance via the miR-379-5p/IGF1 pathway. Our work highlights a new underlying mechanism of NSCLC chemoresistance, which can be targeted to improve chemotherapy against NSCLC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by the Clinical application of transnasal high-flow oxygen therapy in chronic obstructive pulmonary disease combined with hypercarbonemia [No. 18PJ408].