Background
Cervical cancer (CC) is the second most common female cancer worldwide, and is the leading cause of mortality from cancer among females in developing countries [
1]. Though great advances aimed at the development of therapy for preventing CC in recent decades, about 20–25% patients remain suffer from treatment failure because of distant metastasis and recurrence [
2,
3]. Cisplatin (DDP) is a widely used chemotherapeutic drug, and is often used to combination with radiotherapy for the treatment of patients with advanced or recurrent CC [
4]. However, the development of resistance to DDP-based chemotherapy has become a major limiting factor for its efficacy as an anticancer drug [
5].
Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides in length and are not translated into protein [
6]. Nevertheless, emerging evidence has revealed that lncRNAs involve in multiple biological activities, such as gene regulation, RNA transport and protein synthesis [
7,
8]. Recently, lncRNAs were evidently discovered to modulate drug transport and ultimate resistance [
9]. Many lncRNAs are reported to mediate DDP resistance in cancers. For example, lncRNA XIST promoted DDP resistance of human lung adenocarcinoma cells via let-7i/BAG-1 axis [
10]. LncRNA UCA1 promoted proliferation and DDP resistance of oral squamous cell carcinoma by suppressing miR-184 expression [
11]. LncRNA CCAT1/miR-130a-3p axis increased DDP resistance in non-small-cell lung cancer cell line by targeting SOX4 [
12]. In recent, LncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1), located in the chromosome 5p12 region, has been reported to be highly expressed in drug-resistant NSCLC tissues and cells, and promote the resistance of NSCLC cells to DDP through the MAPK/Slug signaling pathway [
13], indicating NNT-AS1 may be related to DDP-resistant. Additionally, NNT-AS1 was verified to function as a tumor promoter in CC via promoting cell proliferation and invasion [
14]. However, the relationship between NNT-AS1 and DDP resistance in CC has not been reported.
MicroRNAs (miRNAs), a class of small noncoding RNAs with 22 nucleotides in size, are able to bind to the 3′-untranslated region (3′UTR) of most protein-coding transcripts to lead translational repression, mRNA decay and mRNA deadenylation [
15,
16]. Recently, increasing evidence has revealed that aberrantly expressed miRNAs involve in the development of drug resistance [
17,
18]. MiR-186, a member of miRNAs, has also been found that can induce sensitivity of cancer cells to DDP in several tumors, including ovarian cancer, glioblastoma and lung cancer [
19‐
21]. All the findings indicated the regulatory role of miR-186 in DDP resistance. High mobility group box 1 (HMGB1) protein is a ubiquitous chromatin component expressed in nucleated mammalian cells. HMGB1 has been identified to be involved in transcription regulation of many cancer genes, including BRCA1, E-selectin, TNF-α and insulin receptor [
22]. Interestingly, recent evidence demonstrated that HMGB1 was overexpressed in CC and promoted cell invasion and migration in vitro [
23]. Besides that, highly expressed HMGB1 contributed to the DDP resistance in human CC cells [
24]. Nevertheless, the mechanisms underlying HMGB1 affects DDP resistance in CC remains unclear.
In the present study, we explored the expression patterns of NNT-AS1 in CC DDP-resistant tissues and cell lines and investigated the effects of NNT-AS1 on CC patients DDP resistance in vivo and vitro. Besides that, we also explored the potential molecular mechanisms underlying the function of NNT-AS1 on DDP resistance. This study may contribute to provide a potential therapeutic approach for CC treatment.
Materials and methods
Patients and specimens
The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consents were collected from all patients and hospitals. Cervical cancer tissues and adjacent normal tissues were collected from 58 CC patients undergoing surgical resection in the First Affiliated Hospital of Zhengzhou University and all cancer tissue samples were diagnosed as CC by pathological examination. All fresh samples were snap-frozen and preserved in liquid nitrogen until further experiments. 58 CC patients were classified into two groups depending on the sensitivity of CC patients to chemotherapy drugs: chemotherapy-sensitive group (tumor remission after 6 cycles of chemotherapy, Chemosensitive group, N = 24) and chemotherapy-resistant group (tumor stabilization or progression after 6 cycles of chemotherapy, Chemoresistant group, N = 34). Additionally, 58 patients were divided into two groups based on the expression of NNT-AS1 to calculate the overall survival of all participants at the different periods (0, 20, 40, 60 month) after cisplatin treatment.
Cell culture and transfection
Cervical cancer cell lines HeLa and SiHa were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The normal cervical epithelial cell line HaCaT was obtained from institute of Biochemistry and Cell Biology (Shanghai, China). HeLa and SiHa cells were cultured in increasing concentrations of cisplatin (Sigma, St. Louis, MO, USA) for over 6 months to establish cisplatin-resistant cell lines, HeLa/DDP and SiHa/DDP. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 U/mL streptomycin (SigmaAldrich, Shanghai, China) at 37 °C with 5% CO2 in a humidified atmosphere.
The short hairpin RNA (shRNA) targeting NNT-AS1 (sh-NNT-AS1) and shRNA scramble control (sh-NC), pcDNA and pcDNA-NNT-AS1 overexpression vector (NNT-AS1), pcDNA-HMGB1 overexpression vector (HMGB1) were synthesized by Genepharma (Shanghai, China). The miR-186 mimic (miR-186), mimic negative control (miR-NC), miR-186 inhibitor (anti-miR-186) and inhibitor negative control (anti-NC) were purchased from RIBOBIO (Guangzhou, China). The transfection of miRNA mimics (10 nM) or vectors was performed using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA), when the HeLa/DDP and SiHa/DDP cells reached 50–60% confluence. Then cells were harvested for 48 h for the subsequent analysis.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from CC cells and tissues using TRIzol reagents (Invitrogen). RNA was reversely transcribed into complementary DNA (cDNA) with the help of AMV reverse transcription kits (Takara, Dalian, China). Fluorescence qRT-PCR was performed using an SYBR Premix Ex Taq II kit (Takara) according to the manufacturer’s introduction. GAPDH or U6 was as internal control and the fold change was assessed using the 2−ΔΔCt method. The specific primer sequences were listed as follows: NNT-AS1, forward, 5′-ACGTGCAGACAACATCTACCT-3′, reverse, 5′-TACAACACCTTCCCGCAT-3′; miR-186, forward, 5′-CGCGGATCCGGTTTACAGAACACCCATCAT-3′, reverse 5′-CCGCTCGAGGTGTTGACATTCACATGCTTC-3′; HMGB1, forward: 5′-GGAGAGATGTGGAATA-3′, reverse, 5′-GGGAGTGAGTTGTGTA-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-ACGCTTCACGAATTTGCGT-3′; GAPDH, forward 5′-AACGGATTTGGTCGTATTGG-3′, reverse 5′-TTGATTTTGGAGGGATCTCG-3′.
Cell viability assay
Cell viability was determined using the 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di-phenytetrazoliumromide (MTT, Beyotime, Shanghai, China) assay. Briefly, transfected DDP-resistant cells were seeded into 96-well plate with a density of 5 × 103 cells/well and incubated with different doses of DDP. At different time points, 20 μL of MTT solution was added to each well for 4 h, followed by the addition of DMSO to resolve the generated formazan. Finally, the absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
Cell migration and invasion assay
The migration and invasive capacities of the DDP-resistant cells in vitro were detected by transwell assay. Transfected DDP-resistant cells were seeded in the upper chamber of a 24-well plate with or without matrigel (Becton–Dickinson, Franklin Lakes, NJ, USA) in DMEM without serum and complete medium with containing 10% FBS was added into the lower compartment of each well as a chemoattractant. After incubation for 24 h at 37 °C, the non-motile cells on the upper surface were removed using a dry cotton swab. Then cells attached to the bottom were fixed with methanol and stained with 0.5% crystal violet for 30 min. The cells from five randomly selected fields were counted with an inverted microscope.
Cell apoptosis analysis
Transfected DDP-resistant cells were harvested for 48 h. Then Cell apoptosis rate was analyzed using Annexin V-FITC/PI apoptosis detection kit (Solarbio, Beijing, China) according to the instructions of manufacture. All samples were performed in triplicate.
Western blot assay
Western blot assay was conducted as previous described [
12]. Immunoblot assays were performed using antibodies against HMGB1, Bax, Bcl-2, Cleaved caspase 3, N-cadherin, Vimentin, E-cadherin, as well as GAPDH.
Luciferase reporter assay
The NNT-AS1 mRNA and HMGB1 3′-UTR containing wild-type (WT) or mutant (MUT) binding sequence of miR-186 were cloned into the psiCHECK™-2 luciferase plasmid (Promega, Shanghai, China), respectively. Then, HeLa/DDP and SiHa/DDP cells were seeded in 24-well plates, followed co-transfected with NNT-AS1-WT or NNT-AS1-MUT or HMGB1-WT or HMGB1-MUT with miR-186 mimics or miR-NC respectively using Lipofectamine 2000 (Invitrogen). After transfection for 48 h, a dual luciferase assay kit (Promega) was used to analyze the luciferase activity following the manufacturer’s instructions.
RNA pull-down assay
For the RNA pull-down assay, 3vitro-biotinylated miRNA mimic was transfected into DDP-resistant cells. Subsequently, cells were lysed and cultured with streptavidin-coupled beads to generate biotin-miRNA-lncRNA complexes. Finally. RNA was isolated and analyzed using qRT-PCR.
Murine xenograft assay
Female BALB/c mice (aged 4–6 weeks) were bought from Vital River Laboratory Animal Technology (Beijing, China). The experiment was permitted by the Animal Research Committee of the First Affiliated Hospital of Zhengzhou University and performed in accordance with the guidelines of the National Animal Care and Ethics Institution. SiHa/DDP cells (5 × 106) transfected with the sh-NNT-AS1 or sh-NC were subcutaneously injected into the backs of nude mice under sterile conditions. After 7-day administration, mice were treated with DDP or PBS followed by the examination of tumor sizes every 3 d. After 30 d, all mice were sacrificed and tumor samples were weighted and used for further molecular analysis.
Statistical analysis
All statistical analyses were performed with GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). The data were expressed as the mean ± SD. The correlation among NNT-AS1, miR-186 and HMGB1 was analyzed by Pearson’s correlation analysis. Kaplan–Meier survival curves were plotted and the difference in survival between two groups was analyzed by the log-rank test. The significant group differences were assessed by Student’s t test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
Discussion
Since its discovery, DDP is considered as a pivotal drug in chemotherapy for cancers [
25]. Nevertheless, it has been an impediment in using DDP for cancer treatment because of the chemoresistance in cancers [
9]. Unsurprisingly, the treatment of CC is also plagued by drug resistance. Recently, numerous studies have identified the association between the dysregulation of lncRNAs and DDP resistance in cancers, including CC, and many lncRNAs has been investigated to be implicated in the DDP resistance. For instance, lncRNA UCA1 promotes DDP resistance in CC by involving in signaling pathways modulating cell apoptosis and proliferation [
26]. LncRNA GAS5, acts as a tumor suppressor, can block DDP resistance in CC via miR-21 [
27]. LncRNA ZFAS1 is upregulated in CC tissues, and its high expression indicates a poor prognosis and enhances DDP chemoresistance [
2]. All the studies revealed that lncRNAs participate in the DDP resistance in CC. NNT-AS1, a newly detected lncRNA, has been identified to play important roles in tumor progression by affecting cell proliferation, metastasis, apoptosis and tumorigenesis in many cancers via targeting downstream signaling pathway, including CC [
14,
28‐
30]. While the effects of NNT-AS1 on CC prognosis and DDP resistance remain unknown.
In the present study, we demonstrated that NNT-AS1 was highly expressed in CC tissues and cells, which was consistent with previous study. In the meanwhile, especially elevated of NNT-AS1 expression in DDP-resistant tumors and cell lines was also detected, suggesting the potential regulatory role of NNT-AS1 in DDP resistance of CC. The resistance towards DDP is multifaceted since it implicates multiple cellular pathways. Therefore, proliferation, migration, invasion and apoptosis abilities of DDP-resistant CC cells was evaluated and we found knockdown of NNT-AS1 could attenuate DDP resistance by inhibiting proliferation, metastasis and but inducing apoptosis in DDP-resistant CC cells. Additionally, the acquisition of EMT features has been investigated that contributes to the DDP-resistance in cancer cells including CC. EMT was revealed to play an important role in promoting chemoresistance [
31,
32]. Thus, we also illustrated the expression levels of EMT related protein in CC DDP-resistant cells and an inhibition of N-cadherin and Vimentin expression but enhancement of E-cadherin expression induced by NNT-AS1 deletion in DDP cells, indicating NNT-AS1 stimulated EMT and induced DDP resistance in CC cells. Besides that, a murine xenograft model was established using stably transfected SiHa/DDP cells and the results showed that NNT-AS1 knockdown significantly prevented tumor volume and weight, suggesting NNT-AS1 deletion antagonized DDP resistance in vivo, which was consistent with the results in vitro. In consideration of the above-described biological behaviors of NNT-AS1, the relationship between NNT-AS1 and overall survival was evaluated and highly expressed NNT-AS1 was identified to predicate poor prognosis in CC patients.
It has been reported that lncRNA could act as competing endogenous RNA (ceRNA) of miRNAs to indirectly regulate gene expression implicated in various biological processes, including cancer [
33]. MiR-186 was found to be a target of NNT-AS1 using bioinformatics prediction program and then NNT-AS1 directly binding to miR-186 was verified by luciferase reporter assay and RNA pull down assay. Subsequently, miR-186 expression was analyzed and miR-186 was demonstrated to be down-regulated in CC tissues and cell lines, especially in DDP-resistant tumors and cell lines. Immediately, a perfect negative correlation between miR-186 and NNT-AS1 expression in CC patients was identified. In all, all the results indicated that miR-186 might relate to NNT-AS1-mediated DDP-resistant.
MiR-186 has been investigated to act as a tumor suppressor to regulate proliferation, metastasis, apoptosis and EMT in CC cells [
34,
35]. Nevertheless, the role of miR-186 in DDP resistance in CC remains unclear. Thus, to verify whether miR-186 related to NNT-AS1-mediated DDP-resistant, rescue experiments were performed and the data showed that miR-186 mimic transfection could restore NNT-AS1 deletion induced inhibition of DDP resistance in DDP-resistant CC cells. Therefore, NNT-AS1 improved DDP resistance in DDP-resistant CC cells by targeting miR-186.
An increasing number of findings suggests that aberrantly expressed miRNAs promote the development of drug resistance through interfering with the expression of target proteins which may be drug targets, drug transporters, or cell-cycle- and cell apoptosis-related components, causing cells with different degrees of resistant to chemotherapeutic drugs [
36]. HMGB1 has been identified that may function as a DDP-resistant regulator in DDP resistance and inhibiting the cytoplasmic location of HMGB1 can reverse DDP resistance in human CC cells [
37]. In this study, bioinformatics prediction program and luciferase reporter assay predicted and validated that HMGB1 was a target of miR-186. Moreover, expression analysis showed that NNT-AS1 could regulate HMGB1 expression via modulating miR-186 expression in vivo and vitro. Thus, we hypothesized HMGB1 might associated with NNT-AS1/miR-186 mediated DDP-resistant. Immediately, rescue experiments was performed and we found knockdown of NNT-AS1 could antagonize DDP resistance in DDP-resistant CC cells via regulating HMGB1. Therefore, NNT-AS1 knockdown might improve DDP-sensitivity of CC cells via blocking HMGB1 expression by competitive interaction with miR-186.
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