Hsa_circ_0000190 Promotes NSCLC Cell Resistance to Cisplatin via the Modulation of the miR-1253/IL-6 Axis

Background This study explored the mechanistic basis for nonsmall cell lung cancer (NSCLC) cisplatin (DDP) treatment resistance in an effort to define effective approaches to abrogating the emergence of such chemoresistance. Methods Analyses of NSCLC expression of hsa_circ_0000190, miR-1253, and interleukin 6 (IL-6) were conducted via a quantitative real-time polymerase chain reaction (qPCR) approach, while the ability of these tumor cells to resist DDP treatment was evaluated with a CCK-8 assay. Interactions between different RNA molecules were assessed using both RNA immunoprecipitation and dual-luciferase reporter assays. Results NSCLC cell lines and tissues resistant to DDP were found to express higher levels of hsa_circ_0000190, and knocking down this circRNA in NSCLC cells was associated with greater sensitivity to DDP exposure. Further research identified miR-1253 as a hsa_circ_0000190 target, with the ability of hsa_circ_0000190 knockdown to restore DDP sensitivity being largely attributable to the ability of this circRNA to suppress miR-1253 activity. IL-6 was identified as a major miR-1253 target in this context, with miR-1253 regulating chemoresistance in NSCLC cells in part by preventing IL-6 upregulation. Conclusion Together, these data suggest that hsa_circ_0000190 can promote DDP chemoresistance in NSCLC cells through its ability to modulate miR-1253/IL-6 axis activity, highlighting a novel pathway that can be targeted in an effort to guide the more effective diagnosis and management of DDP-resistant tumors.


Introduction
Nonsmall cell lung cancer (NSCLC) cases make up an estimated 85% of all primary lung tumor diagnoses [1].NSCLC patients are often treated with a chemotherapeutic regimen that includes cisplatin (DDP) [2], but the emergence of DDP resistance ultimately constrains the efficacy of this interventional strategy and contributes to poor prognostic outcomes [3].Research focused on elucidating the mechanistic basis for the emergence of chemoresistance in NSCLC thus has the potential to provide a novel means of restoring therapeutic sensitivity to target tumors, ultimately contributing to better antitumor efficacy.
Circular RNAs (circRNAs) are a series of endogenously encoded RNA transcripts that form a closed loop as a result of the covalent linkage of the 3′ and 5′ ends of exonic sequences [4,5].These circRNAs have increasingly been codified as key regulators of a diverse array of oncogenic processes [6].Hsa_circRNA_104348, for example, is capable of targeting the miR-187-3p/rhotekin 2 (RTKN2) axis and driving the activation of Wnt/β-catenin signaling in a manner conducive to hepatocellular carcinoma progression [7].In NSCLC, circRNA_0000429 has been reported as a molecular sponge capable of sequestering miR-1197, thereby modulating the expression of MAP-kinase activating death domain (MADD) [8].Hsa_circ_0000190 can also promote NSCLC tumor growth by inducing soluble PD-L1 upregulation such that these tumors can better evade immune-mediated elimination [9].Hsa_circ_0000190 can similarly regulate epidermal growth factor receptor (EGFR)/extracellular regulated protein kinases (ERK) pathway activity in a manner beneficial to NSCLC tumor cells [10].Despite its important tumorigenic role in this form of lung cancer, however, no publications to date have documented the impact of hsa_circ_0000190 on the emergence of DDP resistance in NSCLC.
Small single-stranded noncoding transcripts known as miR-NAs are capable of regulating the vast majority of known biological processes [11], doing so by pairing with complementary target mRNA 3 ′ untranslated region (UTR) sequences and thereby reducing the translation of these transcripts [12].Many tumors reportedly exhibit the dysregulation of miR-1253, and this miRNA has been reported to be a target of several circRNAs, functioning in a tumor suppressor-like manner [13][14][15].Little research to date, however, has focused on the association between hsa_circ_0000190 and miR-1253 in NSCLC.IL-6 is a cytokine that can drive NF-κB-mediated TIM4 upregulation and consequent NSCLC cell metastatic progression [16], in addition to inducing the phosphorylation of BECN1 so as to modulate chemoresistance and autophagic activity [17].
In this study, an in-depth analysis was conducted of the potential role of the hsa_circ_0000190/miR-1253/IL-6 axis as a regulator of DDP resistance in NSCLC.Following initial analyses of hsa_circ_0000190 expression patterns in NSCLC, the mechanistic role of this circRNA as a modulator of NSCLC cell malignancy and chemoresistance was assessed.

Materials and Methods
2.1.Clinical Samples.In total, 136 paired NSCLC tumor and normal tissue samples were harvested from patients at Nanjing Chest Hospital, Affiliated Nanjing Brain Hospital, Nanjing Medical University.The patients from whom these samples had been harvested were separated into two subgroups based on their DDP sensitivity, including DDP-sensitive patients (n = 63) and DDP-resistant patients (n = 73).The Ethical Committee of Nanjing Chest Hospital, Affiliated Nanjing Brain Hospital, Nanjing Medical University (No. 000128) approved all work using human samples, and all patients gave written consent.The patient characteristics of hsa_circ_0000190 were showed as Table 1.

Cell
Culture.The A549 and H460 human NSCLC cell lines, control HBE1 cells, and 293T cells were from the BeNa culture collection (Beijing, China).NSCLC cells resistant to DDP (Sigma, MO, USA) were established as reported previously to generate the A549/DDP and H460/DDP cell lines.All cells were cultured in RPMI-1640 (Hyclone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, CA, USA) and penicillin/streptomycin (Sigma) in a 5% CO 2 incubator at 37°C.
2.4.RNase R and Actinomycin D Treatment.Hsa_circ_0000190containing samples were treated using RNase R (Applied BIOLOGICAL Materials, Vancouver, Canada) for 20 min at a dose of 100 μg/mL with subsequent qPCR analysis in order to confirm the circular nature of this transcript.To confirm the stability of this circRNA transcript, NSCLC cells were treated using Actinomycin D (Sigma) at a dose of 2 mg/mL, with transcript levels subsequently being analyzed via qPCR.

Subcellular Localization.
A PARIS™ Kit Protein and RNA Isolation system (Thermo Fisher Scientific, MA, USA) was used based on provided directions to isolate RNA from the nuclear and cytosolic fractions.To validate these interactions, the wild-type (WT) hsa_ circ_0000190 or IL-6 sequences harboring miR-1253 complementarity or mutated (MUT) versions of these sequences were introduced into the pmirGLO vector (Promega, WI, USA).The resultant plasmids (hsa_circ_0000190 WT, hsa_ circ_0000190 MUT, IL-6 3 ′ UTR WT, and IL-6 3 ′ UTR MUT) were transfected into 293T cells together with miR-1253 or miR-NC constructs as appropriate.A dual-luciferase reporter assay kit (Promega) was used at 24 hr posttransfection to quantify luciferase activity in these samples.

RNA Immunoprecipitation (RIP).
To confirm the ability of miR-1253 and hsa_circ_0000190 to interact directly with one another, the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) kit was used based on provided instruction.Lysis buffer supplemented with RNase inhibitor (Millipore) was used for the initial preparation of cell lysates, which were subsequently incubated with Argonaute2 (Ago2; Millipore) or Immunoglobulin G (IgG; Millipore) antibodycoated magnetic beads.A qPCR approach was subsequently used to quantify RNA enrichment.
2.10.Statistical Analysis.GraphPad Prism 8.0 (GraphPad, CA, USA) was used to conduct all analyses, and data are presented as means AE standard deviations (SD).Results were compared with Student's t tests and one-way ANOVAs.Spearman's correlation analyses were used to assess linear relationships among variables.All experiments were conducted in triplicate, with P <0:05 as the cutoff used to define statistical significance.

DDP-Resistant NSCLC Tissues and Cells Lines Exhibit
Hsa_circ_0000190 Upregulation.To initially probe the potential link between hsa_circ_0000190 and the emergence of chemoresistance to DDP in NSCLC, tissue samples from DDP-sensitive and DDP-resistant patients were collected and the levels of hsa_circ_0000190 therein were quantified by qPCR.This approach revealed the significant upregulation of hsa_circ_0000190 in DDP-resistant tumor tissues as compared to DDP-sensitive samples (Figure 1(a)).Hsa_circ_0000190 levels in NSCLC cell lines were also altered relative to HBE1 control cells, and the expression of this circRNA was further enhanced in DDP-resistant sublines derived from these NSCLC cells (Figure 1(b)), supporting the observed data derived from human tissues.The DDP of IC50 was enhanced in DDP resistant cells relative to normal cells (Figure S1).Treatment of RNA extracts from these cells revealed that the exonuclease RNase R was not able to effectively digest hsa_circ_0000190, consistent with its covalent closed loop structure (Figure 1(c)).
When transcription was inhibited using actinomycin D, hsa_circ_0000190 also exhibited stability superior to that of the linear CNIH4 mRNA transcript (Figure 1(d)).Subcellular localization analyses of hsa_circ_0000190 also revealed that it was primarily localized in the cytosol of NSCLC cells (Figure 1(e)), suggesting that it may function as a sponge capable of sequestering target miRNAs, providing a possible mechanism whereby this circRNA may shape the chemoresistance of NSCLC cells.

Silencing Hsa_circ_0000190
Sensitizes Chemoresistant NSCLC Cells to DDP.Successful hsa_circ_0000190 knockdown was confirmed via qPCR in treated NSCLC cell lines (Figure 2(a)), and CNIH4 mRNA expression was no difference between hsa_circ_0000190 knockdown cell and control cells (Figure S2).The silencing of this circRNA resulted in significant decreases in DDP IC50 values for both A549/DDP and H460/DDP cells (Figure 2(b)) and overexpression of hsa_circ_0000190 increased DDP IC50 values for both A549/DDP and H460/DDP cells (Figure S3), suggesting that hsa_circ_0000190 can enhance NSCLC cell resistance to DDP treatment in a manner.
The knocking down of hsa_circ_0000190 resulted in miR-1253 upregulation (Figure 3(b)), and a miR-1253 binding site was present within hsa_circ_0000190 (Figure 3(c)).RIP and dual-luciferase reporter assays were next used to probe the ability of these two transcripts to interact with one another.Following miR-1253 mimic transfection, WT hsa_circ_0000190 luciferase reporter activity was suppressed, whereas the same was not true for the MUT reporter, confirming a direct targeting relationship between hsa_circ_ 0000190 and miR-1253 within 293T cells (Figure 3(d)).An Ago2 antibody preferentially precipitated both hsa_circ_ 0000190 and miR-1253 relative to a control IgG (Figure 3 Analytical Cellular Pathology upregulation of miR-1253 sensitized these chemoresistant cells to DDP, whereas pcDNA3.1-IL-6transfection partially restored DDP resistance to these cells as determined based on measured IC50 values (Figure 6(d)).These data suggest that miR-1253 can control NSCLC resistance to DDP in part through its ability to suppress IL-6 expression.

Discussion
High-throughput sequencing efforts have enabled the identification and characterization of a growing number of cir-cRNAs [18,19].As they are rich in binding sites for miRNAs, circRNAs are often studied as miRNA sponges [20], providing a mechanism through which they control the onset and development of particular cancers [21].In the present report, the ability of hsa_circ_0000190 to shape NSCLC resistance to DDP treatment was assessed in depth.Several prior publications have documented the importance of circRNAs as regulators of chemoresistant phenotypes.The circ-CPA4/let-7 miRNA/PD-L1 axis, for example, is reportedly capable of supporting NSCLC cell growth, chemoresistance, stemness, and the ability of these cells to evade immune-mediated elimination [22].Exosomal circVMP1 can also facilitate NSCLC progression and resistance to DDP owing to its ability to modulate the miR-524-5 p-methyltransferase like 3 (METTL3)/sex determining region Y box 2 (SOX2) axis [23].There is prior evidence for the ability of hsa_ circ_0000190 to control NSCLC tumor progression [9].In this study, the importance of hsa_circ_0000190 as a regulator of NSCLC cell DDP resistance was assessed.DDP-resistant NSCLC cell sublines exhibited increased hsa_circ_0000190 expression.When this circRNA was silenced in these DDPresistant cells, this partially restored their chemosensitivity, emphasizing the status of hsa_circ_0000190 as a promoter of the ability of NSCLC cells to resist DDP treatment.The most widely studied process through which cir-cRNAs exert their biological functions is the so-called miRNA sponge mechanism [24].Through, their ability to effectively sequester specific miRNAs, circRNAs can regulate the onset and progression of a range of cancer types [25].The * * * * * *  Analytical Cellular Pathology fact that hsa_circ_0000190 expression in NSCLC cells was primarily restricted to the cytosol was consistent with its ability to serve as a sponge for specific miRNAs.Consistently, predictive analyses identified miR-1253 as a candidate hsa_circ_0000190 binding target, and this was subsequently validated through RIP and luciferase reporter assays.DDPresistant NSCLC cells and tissues exhibited pronounced miR-1253 downregulation.Moreover, there have been several prior studies documenting varied roles for miR-1253 in particular cancers.In NSCLC, for example, this miRNA has been reported to suppress proliferative activity and stem-like phenotypes [13].Through its ability to target WNT5A, miR-1253 has also been reported to inhibit NSCLC invasivity and proliferation [15].In this study, rescue experiments demonstrated that silencing hsa_circ_0000190 suppressed the resistance of NSCLC cells to DDP in part owing to the ability of this circRNA to act as a miR-1253 sponge.
A growing wealth of evidence has clearly demonstrated the ability of miRNAs to facilitate posttranslational target mRNA degradation [26].The TargetScan database identified IL-6 as a putative miR-1253 target, as subsequently validated through a dual-luciferase reporter assay.IL-6 has been documented to enhance the chemoresistant properties of several types of cancers, for example through its ability to induce the phosphorylation of BECN1 and to control autophagic activity [17].In colorectal cancer, HIF-1α/miR-338-5p/IL-6 axis activation in response to hypoxic conditions is also reportedly conducive to tumor growth [27].The ability of cancerassociated fibroblasts to secrete IL-6 has also been mechanistically linked to the emergence of chemoresistant disease in individuals with NSCLC [28].DDP-resistant tissue samples and cell lines exhibited high levels of IL-6 expression in the present analyses, and further research demonstrated that hsa_circ_0000190 was able to promote IL-6 upregulation  Analytical Cellular Pathology through its ability to function as a sponge capable of sequestering miR-1253.Strikingly, interference with miR-1253 in NSCLC resistant cell lines was sufficient to enhance their resistance to DDP, at least in part owing to the consequent restoration of IL-6 expression.In summary, DDP-resistant NSCLC cells and tissues exhibit pronounced hsa_circ_0000190 and IL-6 upregulation together with the downregulation of miR-1253.Functional assays demonstrated that hsa_circ_0000190 can target this miR-1253/IL-6 regulatory axis in a manner that promotes the emergence of DDP chemoresistance in NSCLC.Overall, these data highlight novel targets for efforts aimed at more effectively treating patients with NSCLC.

TABLE 1 :
Relationship between hsa_circ_0000190 and clinicopathological data of NSCLC patients.