LncRNA HOXA-AS2 Promotes Temozolomide Resistance in Glioblastoma by Regulated miR-302a-3p/IGF1 Axis

Background Glioblastoma (GBM) is a highly prevalent brain tumor characterized by high rates of morbidity, recurrence, and mortality. While temozolomide (TMZ) is commonly used as a first-line treatment for this cancer, the emergence of TMZ resistance limits its utility. The long noncoding RNA HOXA-AS2 reportedly drives GBM progression, but whether it can influence therapeutic resistance to TMZ has yet to be established. Methods HOXA-AS2 expression was analyzed in TMZ-resistant and sensitive GBM tissue samples and cell lines by qPCR. A siRNA-based approach was used to knock down HOXA-AS2 in GBM cells, after which TMZ resistance was tested. Bioinformatics approaches were used to predict miRNA binding targets of HOXA-AS2, after which a series of luciferase reporter assay and rescue experiments with appropriate miRNA inhibitor/mimic constructs were performed to validate these predictions and to clarify the ability of HOXA-AS2 to regulate chemoresistant activity. Results TMZ-resistant GBM patients and cell lines exhibited increased HOXA-AS2 expression that was correlated with worse overall survival. Knocking down HOXA-AS2 increased the sensitivity of resistant GBM cells to TMZ. miR-302a-3p was identified as a HOXA-AS2 target confirmed through luciferase reporter assays and rescue experiments, and IGF1 was further identified as a confirmed miR-302a-3p target. In addition, HOXA-AS2 knockdown resulted in a corresponding drop in IGF1 expression consistent with indirect regulation mediated by miR-302a-3p. Conclusion In summary, these results highlight the role of HOXA-AS2 as a driver of TMZ resistance in GBM through its ability to regulate the miR-302a-3p/IGF1 signaling axis, highlighting this pathway as a promising target for the diagnosis, therapeutic sensitization, and/or treatment of affected patients.


Introduction
Glioblastoma (GBM) is the most common form of primary intracranial malignancy, accounting for 40-50% of all brain tumor cases [1]. GBM patients exhibit symptoms associated with intracranial compression and pressure and exhibit low cure rates together with high rates of morbidity, recurrence, and death [2][3][4]. Te oral alkylating agent temozolomide (TMZ) exhibits 100% bioavailability as is able to readily transit across the blood-brain barrier without causing signifcant treatment-related side efects, leading to its frequent utilization as a frst-line chemotherapeutic drug in GBM patients following surgical tumor removal [5]. However, these patients often develop resistance to TMZ during the later stages of treatment, markedly limiting its utility and contributing to high rates of failed treatment [6]. Te etiology of TMZ resistance is complex and multifactorial, highlighting the need for further studies of these chemoresistant processes in an efort to better provide GBM patients with efective pharmacological interventions.
Long noncoding RNAs (lncRNAs) lack coding potential despite being >200 nucleotides in length, yet nonetheless serve as important regulators of diverse biological processes at the transcriptional, posttranscriptional, and epigenetic levels [7]. Mechanistically, these lncRNAs can control the onset and progression of tumors through epigenetic modifcation, chromatin recruitment, and alternative splicing and represent promising diagnostic and therapeutic targets in many cancers [8]. Specifc lncRNAs have been reported to be associated with the development of chemoresistant characteristics in breast cancer [9] and cutaneous squamous cell carcinoma [10]. Tere is also evidence for the ability of certain lncRNAs to shape tumor cell resistance to TMZ [11,12]. Eforts to target these lncRNAs may thus represent a novel TMZ sensitization strategy. In prior reports, the lncRNA HOXA-AS2 was found to target the miR-885-5p/ RBBP4 axis to infuence GBM malignancy [13]. However, the role this lncRNA plays in the context of TMZ chemoresistance or the underlying mechanisms governing such activity remain poorly understood.
Here, HOXA-AS2 was found to target miR-302a-3p and thereby indirectly promote IGF1 expression and signaling activity in GBM cells, thereby enhancing resistance to TMZ, as confrmed through analyses of GBM tissue samples and cell lines. Accordingly, these fndings suggest that targeting HOXA-AS2 may represent an efective means of overcoming GBM resistance to TMZ treatment.

Patients Samples.
In total, 264 glioma tissue samples were collected from individuals at the First People's Hospital of Wenling that had undergone surgery and chemotherapy. Te Ethics Committee of the First People's Hospital of Wenling approved this study, and all patients provided written informed consent.

Cell
Culture. Te U87 and U251 human GBM cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) with TMZ-resistant substrains of these cell lines (U87TR and U251TR) having been established by our laboratory. Cells were grown in DMEM (Invitrogen) supplemented with 10% FBS (Hyclone, UT, USA) and penicillin/streptomycin in a 37°C 5% CO 2 humidifed incubator (Termo Scientifc, MA, USA). TMZ resistance was maintained by alternating the culture of U87TR and U251TR cells in media supplemented with TMZ (Sigma, CA, USA).

Viability and TMZ Chemosensitivity Analyses.
After addition to 96-well plates, GBM cells were treated for 48 h with TMZ. Media was then exchanged for media supplemented with 10% CCK-8 solution for 2 h (Dojindo, Kumamoto, Japan), after which absorbance at 450 nm was assessed via ultra multifunctional microplate reader (Tecan, Switzerland), with IC50 values being determined as a measurement of cell sensitivity to TMZ treatment.

RNA Immunoprecipitation.
A magna RIP RNA-binding protein immunoprecipitation kit (Bersinbio, Guangzhou, China) was used based on provided directions to complete all RNA immunoprecipitation (RIP) assays. Briefy, RIP lysis bufer was used to lyse cells which were then separated into equal volumes and incubated overnight with 5 μg of human antiargonaute2 (AGO2) (Millipore, MA, USA) or control IgG (Millipore) at 4°C with constant shaking. RNA was then purifed for qPCR analysis. Te purifed RNA was used to detect the expression levels of the genes of interest by RT-qPCR.
2.5. qPCR. Trizol (Takara Bio, Shiga, Japan) was used based on provided directions to extract total RNA from appropriate samples, with RNA yield and quality being determined through measurements of absorbance values at 260 and 280 nm. Te M-MLV reverse transcriptase (Promega, WI, USA) was used to prepare cDNA for HOXA-AS2 analyses, whereas the prime script TM RT reagent kit (Takara Bio, Shiga, Japan) was used to prepare cDNA for analyses of mRNA expression. All qPCR reactions were performed with the SYBR GREEN PCR master mix (Takara Bio, Shiga, Japan) and a 7500 fast real-time PCR instrument (Applied Biosystem, CA, USA).

Statistical Analysis.
Data are means ± SD from triplicate analyses and were compared using Student's t-tests or one-way ANOVAs with Tukey's posthoc test. Mann-Whitney U-tests were used to compare data for tumor samples. Te relationship between HOXA-AS2 expression and patient survival was examined using Kaplan-Meier curves. All analyses were conducted using SPSS 19.0 (SPSS Inc, IL, USA) and GraphPad Prism 7.0 (GraphPad Software, Inc, CA, USA).

TMZ-Resistant GBM Tissues and Cells Exhibit
HOXA-AS2 Upregulation. When comparing TMZ-sensitive and TMZ-resistant GBM patient tissue samples, the expression of HOXA-AS2 was increased in the context of TMZ resistance (Figure 1(a)), with such resistance also being correlated with signifcantly decreased patient's overall survival ( Figure 1(b)). To validate these fndings, GBM cell lines were next interrogated, revealing higher HOXA-AS2 expression levels in TMZ-resistant cell lines (U87TR and U251TR) relative to corresponding parental cells (Figure 1(c)). As expected, these U87TR and U251TR cell lines exhibited superior TMZ resistance (Figure 1(d)), and more detailed analyses revealed HOXA-AS2 to be preferentially enriched in the cytosol relative to the nuclear fraction ( Figure 1(e)). Receiver operating characteristic (ROC) curves suggested that HOXA-AS2 expression may ofer value as a biomarker capable of predicting GBM patient resistance to TMZ treatment ( Figure 1(f )).

Knocking down HOXA-AS2 Suppresses the Ability of GBM Cells to Resist TMZ in Vitro.
Next, a knockdown approach was used to suppress HOXA-AS2 expression in U87TR and U251TR, as confrmed via qPCR (Figure 2(a)). Subsequent viability and drug sensitivity analyses indicated that TMZ IC50 values were signifcantly reduced following treatment with a HOXA-AS2-specifc siRNA rin both tested TMZ-resistant cell lines as compared to control si-NC transfection (Figure 2(b)). Accordingly, these data confrmed the ability of HOXA-AS2 silencing to suppress GBM cell resistance to TMZ in vitro.
3.3. HOXA-AS2 targets miR-302a-3p to Enhance GBM Cell TMZ Resistance. Predictive bioinformatics analyses suggested miR-302a-3p to be a putative HOXA-AS2 target miRNA (Figure 3(a)). Consistently, miR-302a-3p overexpression in a luciferase reporter assay system was sufcient to suppress the activity of the wild-type HOXA-AS2 reporter, whereas the mutated version of this reporter was unafected (Figure 3(b)). Consistently, RIP assays confrmed the identity of miR-302a-3p as a HOXA-AS2 target (Figure 3(c)) and TMZ-resistant GBM cells exhibited lower miR-302a-3p expression (Figure 3(d)), while such expression was enhanced following the silencing of HOXA-AS2 ( Figure 3(e)). In addition, relative to tissue samples from TMZ-sensitive GBM patients, those from individuals resistant to TMZ exhibited lower levels of miR-302a-3p (Figure 3(f )). Moreover, HOXA-AS2 and miR-302a-3p expression levels were negatively correlated in TMZresistant GBM patient tissue samples (Figure 3(g)). When miR-302a-3p expression was knocked down with an inhibitor constructed in TMZ-resistant cells (Figure 3(h)), this reversed the efect of HOXA-AS2 knockdown on the TMZ sensitivity of these cells (Figure 3(i)). Accordingly, these results confrmed that the ability of HOXA-AS2 to enhance the resistance of GBM cells to TMZ treatment was related to the ability of this lncRNA to interact with miR-302a-3p.

miR-302a-3p Targets IGF1 to Reduce GBM Cell
Resistance to TMZ. Te TargetScan database identifed IGF1 as a putative miR-302a-3p target (Figure 4(a)). Consistently, the cotransfection of 293T cells with miR-302a-3p mimic constructs and luciferase reporter vectors harboring WT or mutated versions of the 3'-UTR of IGF1 revealed that miR-302a-3p was only able to efectively bind the WT version of this sequence to modulate luciferase activity (Figure 4(b)). Accordingly, TMZ-resistant cells were found to exhibit increased IGF1 expression as detected via qPCR and ELISA (Figures 4(c) and 4(d)), while miR-302a-3p inhibitor transfection enhanced IGF1 levels in these TMZ-resistant cells, and miR-302a-3p mimic transfection had the opposite efect (Figures 4(e)-4(f )). Marked upregulation of IGF1 was evident in tumor tissues from TMZ-resistant patients with GBM (Figure 4(g)), with a corresponding negative correlation between IGF1 and miR-302a-3p in this cohort of patient samples (Figure 4(h)). Further analysis of the ability of this miR-302a-3p/IGF1 regulatory axis to control TMZ resistance revealed that knocking down IGF1 in TMZresistant GBM cells (Figures 4(i) and 4(j)) reversed the effects of miR-302a-3p inhibitor treatment on the TMZ IC50 value for these cells (Figure 4(k)). Tese fndings thus confrmed the ability of miR-302a-3p to target IGF1 and to thereby overcome GBM cell chemoresistance.

HOXA-AS2 Promotes IGF1 Upregulation Via Seques-
tering miR-302a-3p. Given the above data demonstrating the ability of HOXA-AS2 to bind miR-302a-3p and the ability of this miRNA to in turn suppress IGF1 expression, a fnal set of experiments was performed to examine the ability of HOXA-AS2 to indirectly regulate IGF1 by serving as a competing endogenous RNA (ceRNA) specifc for miR-302a-3p. GBM patient tissues exhibited positive correlations between HOXA-AS2 and IGF1 levels ( Figure 5(a)), and the knockdown of HOXA-AS2 in TMZ-resistant cell lines resulted in a corresponding drop in IGF1 expression at the mRNA and protein levels, while this was reversed by miR-302a-3p inhibitor treatment (Figures 5(b) and 5(c)). Overall, these results were consistent with the ability of HOXA-AS2 to bind miR-302a-3p and thereby indirectly modulate the expression of IGF1.

Discussion
Resistance to TMZ represents a major roadblock to the efective treatment of GBM, highlighting a critical need for further therapeutic innovation. Here, the association between HOXA-AS2 expression and such chemoresistance was analyzed in GBM patients and cells. Te knockdown of HOXA-AS2 in vitro ultimately revealed that it functions to promote resistance to TMZ in GBM cells along the miR-302a-3p/IGF1 axis. Te upregulation of HOXA-AS2 was evident in both TMZ-resistant GBM patient samples and cells. Tese data align with prior evidence that HOXA-AS2 can serve as a regulator of GBM progression [13], and with other research suggesting that lncRNAs can act as critical modulators of chemoresistance in head and neck squamous cell carcinoma [14], colorectal cancer [15], and nonsmall cell lung cancer [16]. Tese fndings thus suggest that HOXA-AS2 may represent a novel mediator of resistance to TMZ in GBM. While initially disregarded as a form of transcriptional noise, more recent work has characterized the importance of lncRNAs as physiological regulators in diverse contexts. Mechanistically these noncoding RNAs can act as ceRNAs for particular miRNAs, can interact with specifc proteins, and can regulate transcriptional activity and splicing [17][18][19]. Te ceRNA mechanism of action has been a focus of key research interest in recent years, ofering a pathway whereby these lncRNAs can infuence therapeutic responsiveness. Lnc-TALC, for example, was found to bind to miR-20b-3p in a competitive manner and thereby drive the expression of O6-methylguanine-DNA methyltransferase through c-Met pathway regulation [20], with the NEAT1 lncRNA that can regulate the let-7g-5p/MAP3K1 pathway to control the malignancy of glioblastoma stem cells and their ability to resist TMZ treatment [11].
Here, HOXA-AS2 was identifed as an inducer of the onset of TMZ resistance owing to its ability to competitively bind miR-302a-3p and thereby upregulate IGF1. Tese fndings are consistent with prior evidence for the ability of miR-302a-3p to serve as an oncogenic mediator in testicular germ cell tumors [21]. However, this miRNA has not previously been reported to act as a regulator of chemoresistance. IGF1 has been shown to enhance melanoma cell resistance to cisplatin [22], in line with the prior data. Indeed, TMZ-resistant GBM patient tissue samples and cells exhibited increased IGF1 expression, and the knockdown of HOXA-AS2 or the upregulation of miR-302a-3p was suffcient to reduce IGF1 levels in TMZ-resistant cells. Overall, these fndings thus support the existence of a HOXA-AS2/ miR-302a-3p/IGF1 regulatory axis that controls the ability of GBM cells to resist TMZ. Te mechanisms whereby IGF1 controls such resistance and the clinical relevance of this regulatory pathway, however, will necessitate further study and experimental validation.
In summary, the present fndings indicate that HOX-A-AS2 overexpression is evident in GBM patients in the context of TMZ resistance, with the upregulation of this lncRNA representing an important biomarker associated with poor prognostic outcomes. From a mechanistic perspective, knocking down HOXA-AS2 was sufcient to alleviate GBM cell resistance to TMZ via the miR-302a-3p/IGF1 axis. Together these data ofer new insight regarding chemoresistance in GBM, potentially highlighting novel avenues for future therapeutic intervention.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.