HOTAIR mediates cisplatin resistance in nasopharyngeal carcinoma by regulating miR-106a-5p/SOX4 axis

ABSTRACT This study explored the function and mechanisms of HOX transcript antisense RNA (HOTAIR) in the drug resistance of nasopharyngeal carcinoma (NPC). Quantitative PCR, Western blotting, MTT assay, flow cytometry, Transwell assay, and luciferase assay were performed. HOTAIR expression levels were upregulated in cisplatin (DDP)-resistant NPC tissues and cells. Knockdown of HOTAIR in DDP-resistant NPC cells increased cell sensitivity of DDP, as well as decreased cell viability, expression of chemoresistance-related proteins, migration and invasion, increased cell apoptosis. In addition, downregulation of microRNA 106a-5p (miR-106a-5p) expression and upregulation of SRY-box transcription factor 4 (SOX4) expression were observed in DDP-resistant NPC tissues and cells. MiR-106a-5p targets HOTAIR and SOX4; thus, silencing of HOTAIR significantly increased miR-106a-5p expression. The overexpression of miR-106a-5p significantly reversed the increase in SOX4 expression induced by HOTAIR lentivirus (Lv-HOTAIR). Knockdown of SOX4 reduced the drug resistance of DDP caused by the silencing of miR-106a-5p expression. In summary, HOTAIR enhanced DDP resistance in NPC cells by regulating the miR-106a-5p/SOX4 axis.


Introduction
Nasopharyngeal carcinoma (NPC) is a highly malignant neoplasm originating from nasopharyngeal mucosa [1]. The majority of patients diagnosed with NPC is between 40 and 60 years of age. The age-specific incidence of NPC in China is relatively high among the elderly population, and the agespecific incidence in males is significantly higher than in females [2]. At present, NPC is usually treated in various of ways, including radiotherapy, surgery, chemotherapy, targeted therapy, and immunotherapy [3]. However, chemotherapeutic drug resistance remains a big challenge [4]. Therefore, it is urgent to decipher and explore the underlying molecular mechanisms of drug resistance of NPC and to find novel therapeutic targets.
Long non-coding RNAs (lncRNAs) is a type of non-coding RNA molecule with a transcript length of more than 200 nucleotides, and together with short-chain non-coding RNA, it forms a noncoding RNA population [5,6]. The expression of lncRNAs is developmentally regulated, and shows tissue and cell specificity [7]. LncRNAs perform important biological functions. The lncRNA HOX transcript antisense RNA (HOTAIR) is abnormally expressed in a variety of cancers [8], such as breast cancer [9], lung cancer [10], and hepatocellular carcinoma (HCC) [11]. In addition, HOTAIR mediates cisplatin (DDP)-resistant nonsmall cell lung cancer by regulating the Wnt signaling pathway [12]; however, the role of HOTAIR in DDP resistance in NPC is not clearly understood.
MicroRNAs (miRNAs), approximately 22 nucleotides in length, could target the posttranscriptional level of the gene [13]. And miRNAs regulate many cellular pathways and functions including cell proliferation, differentiation, apoptosis, and ontogenesis [14,15]. Moreover, present studies have shown that miRNAs play key regulatory roles in drug resistance in many cancers [16]. MiR-106a-5p is abnormally expressed in many cancer types, including NPC [17], HCC [18], and colon cancer [19]. SRY-box transcription factor 4 (SOX4) has important biological functions including transcriptional regulation, intracellular material transport, and chromosome remodeling, and participates in various biological processes including cell differentiation, growth and development, stress responses, and disease occurrence and development. Thus, SOX4 may play an important role in the progression of NPC [20,21].
The objective of this study was to assess the role of HOTAIR in DDP-resistant NPC, and to understand the mechanisms underlying cell proliferation, invasion, and apoptosis. And this study aimed to explore the role of miR-106a-5p/SOX4 axis in NPC drug resistance and to demonstrate the central role played by HOTAIR. It is significant for looking for novel therapeutic targets in the treatment of NPC drug resistance.

Patient methods
We collected the tumor tissues surgically removed from all patients and selected 10 cases of DDPsensitive and 10 cases of DDP-resistant tissues according to the Huvos scoring system at The Second Hospital of Anhui Medical University between 2014 and 2018. Participation was required to sign written informed consent. Proof of the ethical approvals was verified by the local Ethics Committee (SL-YX2019-061).

Apoptosis assay
Apoptosis analysis was performed by using flow cytometer. Cells were collected and washed with PBS. Then resuspended (3 × 10 3 cells/ well) in 500 μL binding buffer and stained with 5 μL annexin V-fluorescence isothiocyanate and 10 μL propidium iodide for 5-15 min at room temperature. Flow cytometry was used to detect cell apoptosis [24].

Cell counting Kit-8 assays
The cells were seeded (2 × 10 3 cells/96 well) and cultured in a 100 μL medium containing 10% FBS for 72 h. Then, 10 μL CCK-8 solution was added to each well of the plate for 2-h incubation, and the absorbance was measured at 490.

Quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen). The extracted RNA was quantified and reversely transcribed into complementary deoxyribose nucleic acid (cDNA), followed by PCR using SYBR Green method. The relative level was calculated by 2 −ΔΔCT method.

Transwell assay
The migration and invasion of cells were measured using Transwell plates (pore filter size, 8 µm; Corning Inc., Corning, NY, USA). The Transwell filter chamber was pre-coated with 50 mg/L Matrigel at a ratio of 1:8 at 4°C. After incubation for 24 h, cells (5,000 cells /well) on the upper chamber surface were removed, and cells adhering to the bottom side of the Transwell insert were stained with 0.1% crystal violet and counted.

RNA immunoprecipitation
To further confirm the target, RNA immunoprecipitation was performed with RNA Immunoprecipitation Kit (Guangzhou Geneseed biotech, Co.Ltd, China) as instructed. 1 × 10 5 cells at 75% confluence were transfected. At posttransfection of 48 h, the cell was lysed in RIP lysis buffer. The supernatant was prepared through centrifugation and incubated with 10 µL anti-Ago2 antibodies and normal IgGs at 4°C for 2 h following another 2 h incubation with 40 µL protein A/G beads. Successful immunoprecipitation was evaluated by RT-qPCR.

Statistical analysis
Values are expressed as mean ± standard deviation (mean ± SD). Student t-test was used to indicate the difference between two groups while one-way ANOVA was performed for the difference between three or multiple groups. P < 0.05 indicated statistical significance.

Results
Our results demonstrated that HOTAIR was overexpressed in DDP-resistant NPC tissues and cells compared with normal tissue and cells. Inhibition of HOTAIR expression decreased the resistance of NPC cells to DDP by mediating miR-106a-5p/SOX4 axis activity, mainly shown as suppressing cell viability, invasion and migration, and promoting apoptosis in DDP-resistant NPC cells.

Down-regulation HOTAIR expression decreases the resistance of NPC cells to DDP
To investigate the effects of HOTAIR on DDP resistance in NPC, HOTAIR expression levels were measured using qPCR. The results showed that HOTAIR expression levels were significantly increased in NPC tissues, especially DDP-resistant tissues compared with normal tissues ( Supplementary  Fig.S1A). Also, HOTAIR expression levels were also upregulated in DDPresistant NPC cells compared with their normal cell lines (Supplementary Fig.1SB).

Downregulation of HOTAIR suppresses cell viability, invasion and migration, and promotes apoptosis in DDP-resistant NPC cells
Si-HOTAIR inhibited cell viability in C666-1/DDP and CNE2/DDP cells, migration and invasion in DDP-resistant NPC cells. But the apoptosis was increased in si-HOTAR transfected DDP-resistant NPC cells (Figure 2(a-e)).

Low expression of miR-106a-5p in DDP-resistant NPC tissues and cells and downregulated HOTAIR promotes miR-106a-5p expression
Our results showed that miR-106a-5p was markedly decreased in DDP-resistant NPC tissues and cells (Figure 3(a,b)). As shown in Figure 4(c), miR-106a-5p was determined to be the target site of HOTAIR. The results of dual luciferase reporter assay were confirmed that HOTAIR was the target of miR-106a-5p using wild-type and mutant-binding sites (Figure 3(d)). RIP assay showed the significant enrichment of miR-106a-5p and HOTAIR using Ago2 antibody compared with IgG antibody (Figure 3(e)). In addition, decreased HOTAIR expression dynamically increased the miR-106a-5p expression in DDP-resistant NPC cells (figure 3(f)).

HOTAIR mediated miR-106a-5p/SOX4 axis activity to regulate DDP resistance, cell viability, invasion and apoptosis in NPC DDP-resistant cells
Further to explore the relationship of the miR-106a-5p/ SOX4 axis with HOTAIR in NPC DDP-resistant cells, we found that si-SOX4 inhibited the increased IC 50 values induced by miR-106a-5p inhibitors (Figure 6(a,  b)), as well as the increased apoptosis ( Figure 6(c)). miR-106a-5p inhibitor could increase the invasion and migration of cells, and si-SOX4 increased those effects in NPC DDP-resistant cells (Figure 6(d,e)).

Discussion
The most significant shortcoming of DDP therapy is that tumor cells are prone to becoming drugresistant [26,27]. There are two main forms of (d-f) Suppression of tumor growth was observed after HOTAIR or SOX4 knockdown in CNE2/DDP cell line. Tumor size was enlarged by transfecting with miR-106a-5p inhibitor, tumor volume was retrieved in si-HOTAIR group on addition of miR-106a-5p inhibitor. Tumor growth was measured every other day after 7 days of injection, and tumors were then harvested on day 28 and weighed. Actual tumor size after the harvest was shown in the medium panel. a compared with NC group, p < 0.05. b compared with si-HOTAIR group, p < 0.05. c compared with miR-106a-5pinhibitor group, p < 0.05. DDP resistance: inherent and acquired resistance [28]. The mere elaboration of the mechanism of NPC resistant to DDP alone has important therapeutic value. In this study, we aimed to detect the role of HOTAIR in DDP resistance in NPC, and the underlying molecular mechanisms.
LncRNAs (more than 200bp in length) are most highly conserved in sequence, which constructs an intricate network of interplays with various diverse biomolecules (DNA, RNA or protein), and thusly their perturbation exhibits profound regulatory influences and plays a new role on cancer [29]. Dysfunction of lncRNA is closely associated with the occurrence and development of numerous diseases, including cancer [30]. HOTAIR can regulate the resistance of lung cancer cells to DDP by reducing the expression of p21WAF1/CIP1 [31]. In addition, HOTAIR activates the PI3K/AKT/ MRP1 signaling pathway by targeting miR-126, to promote DDP resistance in gastric cancer [32]. In this study, we observed increased HOTAIR expression in NPC tissue, especially DPPresistant tissues. Interestingly, HOTAIR expression was increased in DPP-resistant NPC cells. Our results showed that knockdown of HOTAIR could significantly downregulate DDP resistance, accompanied by decreased resistance to the expression of related genes in DDP-resistant NPC cells. Silencing HOTAIR expression decreased cell viability, migration and invasion in DDP-resistant NPC cells, and upregulated cell apoptosis.
MiRNAs have been identified as important posttranscriptional regulators involved in various biological and pathological processes of cells in tumor with DDP-resistance [33,34]. The expression of miR-26b significantly inhibits Jagged canonical notch ligand 1 expression in DDP-resistant NPC [35]. In addition, miR-106a-5p has been shown to regulate Fas-activated serine/threonine kinase, which has suppressive effects on astrocytoma [36]. Moreover, miR-106a-5p targets high mobility group AT-hook 2 and inhibits osteosarcoma progression [37]. Here, we also observed the decreased miR-106a-5p expression in DDPresistant NPC tissues and cells. Moreover, miR-106a-5p mimics suppressed the DDP resistance induced by HOTAIR up-regulation. SOX4 was determined to be the target of miR-106a-5p, and silencing of SOX4 increased DDP sensitivity in DDP-resistant NPC cells.
In conclusion, this study demonstrates the role of HOTAIR in DDP-resistant NPC, and this study explores the role of the miR 106a-5p/SOX4 axis in drug-resistant NPC.

Conclusion
Taken together, our results revealed increased expression of HOTAIR and SOX4, and decreased miR-106a-5p levels, in DDP-resistant NPC tissues and cells. Inhibition of HOTAIR expression decreased the resistance of NPC cells to DDP by mediating miR-106a-5p/SOX4 axis activity, mainly shown as suppressing cell viability, invasion and migration, and promoting apoptosis in DDP-resistant NPC cells.

Availability of data and materials
The data used to support the findings of this study are included in the article.

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

Funding
The study was supported by the Cooperative research promotion plan of basic medicine and clinical medicine of Anhui Medical University (2020XKjT036).