Lung cancer ranks the most common cancer and leading cause of tumour-related death globally (1). According to Global cancer statistics 2018, >2 million new lung cancer cases occur, causing nearly 1.7 million deaths worldwide (2). Non-small cell lung cancer (NSCLC) accounts for ~85% of all new lung cancer cases (3). Although noteworthy developments regarding cancer diagnosis and therapies have been realized over the past decades, patients with NSCLC still present with poor clinical outcomes (4,5). NSCLC can spread to lymph nodes, other pulmonary lobes or distant organs as a result of its aggressive characteristics (6). The 5-year survival rate of patients with NSCLC with local or distant metastasis is <5%, according to the data from 2017 (7). A number of factors, including heredity, air contamination and smoking, are considered to be involved in NSCLC pathogenesis (8); however, their mechanisms of action are diverse and complex and are still largely unknown. Therefore, efforts to illuminate the molecular events facilitating NSCLC progression would help in the development of effective diagnostic and therapeutic targets.Long non-coding RNAs (lncRNAs) belong to a large family of RNA molecules whose transcripts are >200 nucleotides long and lack protein-coding ability (9). lncRNAs have previously been regarded as non-functional; however, a series of studies identified the critical actions of lncRNAs in fundamental biological mechanisms, such as differentiation, stress response, growth, immunity, inflammation and tumorigenesis (10–12). Numerous lncRNAs are differentially expressed in NSCLC, with a notable association with NSCLC genesis and progression (13–15). lncRNAs are involved in the modulation of pathological processes in NSCLC cells, where they execute anti-oncogenic or pro-oncogenic roles (16).MicroRNAs (miRNAs/miRs) represent single-stranded and short non-coding RNA molecules, usually composed of 17–22 nucleotides. By directly binding to the 3′-untranslated regions of their target genes, miRNAs can promote translation suppression or degrade mRNAs, thereby regulating gene expression (17). Additionally, miRNAs have an effect on the oncogenesis and progression of NSCLC by controlling a wide range of biological behaviours (18–20). lncRNAs can operate as competitive endogenous RNAs (ceRNAs) by competitively binding to miRNAs and weakening the miRNA-mediated inhibitory effects on mRNA expression, thereby controlling gene expression at the posttranslational level (21–23). Therefore, uncovering the mechanisms of NSCLC-related lncRNAs is important to fully understand cancer pathogenesis and for the identification of promising anticancer therapies for NSCLC.SLC25A25 antisense RNA 1 (SLC25A25-AS1) is downregulated in colorectal cancer and has antitumour activity during cancer progression (24). However, to the best of our knowledge, there are no defined functions or underlying mechanisms of SLC25A25-AS1 in the control of NSCLC tumorigenesis and progression at present. The present study aimed to measure SLC25A25-AS1 expression in NSCLC, to elucidate the exact roles of SLC25A25-AS1 in regulating aggressive phenotypes, and to determine the possible working mechanism. Collectively, the present study examined the interaction among SLC25A25-AS1, miR-195-5p and integrin α2 (ITGA2) in NSCLC and demonstrated the involvement of the SLC25A25-AS1/miR-195-5p/ITGA2 signalling pathway during NSCLC progression.
Materials and methods
Tissue samples
Tumour tissues and matched adjacent healthy tissues were acquired from 48 patients with NSCLC (31 males, 17 females; age range, 38–72 years; median age, 53 years; mean age, 59 years) that were treated with surgery resection at Weifang People's Hospital (Weifang, China) between June 2014 to November 2015. Adjacent healthy tissues were obtained 3 cm away from tumor tissues. The inclusion criteria were as follows: i) Diagnosed with NSCLC; and ii) had not received preoperative anticancer treatments. The exclusion criteria were as follows: i) Diagnosed with other humancancer types; ii) did not agree to take part the current research; iii) had been treated with radiotherapy, chemotherapy or other anticancer treatments. Surgical tissues were kept in liquid nitrogen (−196°C) until use. The present study was approved by the Ethics Committee of Weifang People's Hospital (approval no. EC-WFPH.20150602; Weifang, China). Prior to their enrolment in the present study, all participants provided written informed consent.
Cell lines
A human nontumorigenic bronchial epithelial cell line (BEAS-2B; American Type Culture Collection) was cultured in Bronchial Epithelial Cell Growth Medium (Lonza Group, Ltd.). The humanA549 and H460NSCLC cell lines were purchased from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences (Shanghai, China) and cultured in F-12K and RPMI-1640 media containing 10% FBS (all from Gibco; Thermo Fisher Scientific, Inc.) and 1% Glutamax was added to increase superfluity for A549 cells. SK-MES-1 (American Type Culture Collection) and H522 (American Type Culture Collection) NSCLC cell lines were maintained in 10% FBS-supplemented Minimum Essential Medium and RPMI-1640 medium, respectively (Gibco; Thermo Fisher Scientific, Inc.). Furthermore, 100 U/ml penicillin and 100 g/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) was added to all cell culture media. All cell lines were cultured at 37°C in an incubator with 5% CO2.
TCGA
TCGA data (https://portal.gdc.cancer.gov/) were used to examine SLC25A25-AS1 expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues.
Small interfering RNA (siRNA/si), vector and oligonucleotide transfection
For loss-of-function experiments, SLC25A25-AS1 expression was silenced using siRNAs for SLC25A25-AS1 (si-SLC25A25-AS1s; Shanghai GenePharma Co., Ltd.), and a negative control siRNA (si-NC) served as the comparison. The sequences of siRNAs are shown in Table I. The ITGA2 overexpression vector pcDNA3.1-ITGA2 (Shanghai GeneChem Co., Ltd.) was synthesized to upregulate endogenous ITGA2 expression. miRNA oligonucleotides, including miR-195-5p mimic and miR-195-5p inhibitor (Guangzhou RiboBio Co., Ltd.), were used to alter miR-195-5p levels. The miRNA mimic negative control (NC mimic) and NC inhibitor were used as controls for the miR-195-5p mimic and miR-195-5p inhibitor, respectively. The miR-195-5p mimic sequence was 5′-CGGUUAUAAAGACACGACGAU-3′ and the NC mimic sequence was 5′-UUGUACUACACAAAAGUACUG-3′. The miR-195-5p inhibitor sequence was 5′-GCCAAUAUUUCUGUGCUGCUA-3′ and the NC inhibitor sequence was 5′-ACUACUGAGUGACAGUAGA-3′.
For cell transfection, A549 and SK-MES-1 cells were seeded into 6-well plates with an initial density of 6×105 cells/well. When cells reached 70–80% confluence, the siRNAs (100 pmol), miRNA oligonucleotides (100 pmol) and vector (4 µg) were transfected into NSCLC cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). All transfection experiment was conducted at room temperature for 6 h and culture medium was then replaced with fresh culture medium. Twenty-four hours later, Cell Counting Kit-8 (CCK-8) assay was performed. Flow cytometry analysis, Transwell migration and invasion assays, reverse transcription-quantitative PCR (RT-qPCR) and western blotting were carried out after 48 h.
Reverse transcription-quantitative PCR (RT-qPCR)
The extraction of small RNA from NSCLC tissues, matched adjacent healthy tissues, tumour xenografts, NSCLC cells (A549 and SK-MES-1) was performed using RNAiso for small RNA (Takara Biotechnology Co., Ltd.). A NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) and 1% agarose gel electrophoresis were employed to assess the purity, concentration and integrity of total RNA. To analyse miRNA expression, complementary DNA was generated by conducting reverse transcription using a Mir-X miRNA First-Strand Synthesis Kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions for reverse transcription were as follows: 37°C for 60 min, and 85°C for 5 sec. Subsequently, PCR amplification was performed on the obtained complementary DNA using the Mir-X miRNA RT-qPCR TB Green® Kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions were as follows: 95°C for 10 sec; 95°C for 5 sec and 60°C for 20 sec, for 40 cycles; 95°C for 60 sec, 55°C for 30 sec and 95°C for 30 sec. Small nuclear RNA U6 was used as a control for the measurement of miRNA expression.To quantify SLC25A25-AS1 and ITGA2 expression, total RNA was extracted NSCLC tissues, matched adjacent healthy tissues, tumour xenografts, BEAS-2B cells, NSCLC cells (A549, H460, SK-MES-1, H522) using RNAiso Plus (Takara Biotechnology Co., Ltd.) and reverse transcribed into complementary DNA with a PrimeScript reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). The thermocycling conditions for reverse transcription were as follows: 37°C for 15 min, and 85°C for 5 sec. Subsequently, qPCR was performed using TB Green® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.). The thermocycling conditions were as follows: initial denaturation at 95°C for 30 sec; 40 cycles of amplification at 95°C for 3 sec, and annealing for 30 sec at 60°C and extension at 72°C for 30 sec. GAPDH was considered an internal parameter for SLC25A25-AS1 and ITGA2 expression. Gene expression was calculated using the 2−ΔΔCq method (25). The primer sequences are shown in Table II.
Table II.
Primer sequences used for reverse transcription-quantitative PCR.
The transfected cells were seeded into 96-well plates with a density of 2,000 cells/well, followed by 0, 24, 48, or 72 h cultivation. Cell proliferation was monitored every day until day 4. A total of 10 µl CCK-8 reagent (Dojindo Molecular Technologies, Inc.) was added. After continuous cultivation of cells in a standard environment for 2 h, the absorbance (450 nm) was determined using a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Inc.).
Flow cytometry analysis
Cells under different transfection conditions were incubated with trypsin without EDTA, and centrifugation at 1,000 × g at room temperature for 5 min was conducted to collect transfected cells. Apoptosis was assessed with an Annexin V-FITC/PI apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.). After rinsing with PBS twice, transfected cells were centrifuged at 1,000 × g at room temperature for 5 min and the supernatant was carefully removed. The obtained cells were incubated in the dark with 5 µl Annexin V-FITC and 5 µl propidium iodide diluted in 500 µl binding buffer. Culture was continued for 15 min at ambient temperature. Finally, early + late apoptotic cells were detected using a FACSCalibur flow cytometer (BD Biosciences), and analysed with the CellQuest software v.2.9 (BD Biosciences).
Transwell migration and invasion assays
Transfected cells were resuspended in serum-free culture medium (RPMI-1640 for A549, Minimum Essential Medium for SK-MES-1), and adjusted to a final concentration of 5×105 cells per ml. For the migration assay, 200 µl cell suspension was added to the upper chamber of Transwell inserts (pore size, 8 µm; BD Biosciences). A total of 600 µl of 10% FBS-supplemented complete culture medium was seeded into the lower chamber, which was used as the nutritional attractant. After a 24-h incubation at 37°C, the nonmigrating cells were gently removed using a cotton bud. The migrated cells were stained with 0.5% crystal violet at room temperature for 20 min and fixed in 50% methanol at room temperature for 20 min, followed by imaging under a light microscope. For the invasion assay, Matrigel (BD Biosciences) was utilized to pre-coat the upper chambers, and polymerized by cultivating at 37°C for 2 h. The other experimental processes were the same as those for the migration assay.
In vivo animal experiments
All animal experiments were implemented under the approval of the Animal Care and Use Committee of Weifang People's Hospital (approval nos. ACUC-WFPH.20191106; Weifang, China). Lentiviruses were produced using a second-generation lentiviral system. To obtain NSCLC cells with stable SLC25A25-AS1 silencing, short hairpin RNA (shRNA/sh) against SLC25A25-AS1 (sh-SLC25A25-AS1) and negative control shRNA (sh-NC) were acquired from Shanghai GenePharma Co., Ltd. After insertion into the pLKO.1 plasmid (Addgene Inc.), the yield plasmids together with psPAX2 and pMD2.G were transduced into 293T cells (Cell Bank of Type Culture Collection of The Chinese Academy of Sciences). The transfection duration is 6 h, at which time the medium was replaced with complete culture medium. 293T cells were grown in DMEM containing 10% heat-inactived FBS, 1% Glutamax, 1% non-essential Amino Acids and 1% Sodium Pyruvate 100 mM Solution (all from Gibco; Thermo Fisher Scientific, Inc.). All plasmids were diluted to a concentration of 1 µg/µl, and total of 30 µg (psPAX2:pMD2G: pLKO.1 =1:1:2) were added into each 10-cm dish for lentivirus packaging. Subsequently, the lentiviruses stably expressing sh-SLC25A25-AS1 or sh-NC were collected at 48 h post-transfection and mixed with polybrene (5 µg/ml; Sigma-Aldrich; Merck KGaA) and RPMI-1640 medium. Thereafter, the mixture was added into A549 cells with MOI=5 for lentivirus infection. Stably SLC25A25-AS1-deficient cells were selected by treatment with puromycin (2 µg/ml). The maintenance concentration of antibiotics used was 0.4 µg/ml.Male BALB/c nude mice (n=6; 20 g), aged 4–6 weeks, were acquired from Charles River Laboratories, Inc. All mice were housed under specific pathogen-free conditions at 25°C and 50% humidity, with a 10:14 light/dark cycle and ad libitum access to food and water. A total of 2×106 A549 cells stably transfected with sh-SLC25A25-AS1 or sh-NC were collected, resuspended in 100 µl phosphate buffer saline and subcutaneously inoculated into the left flank of nude mice. Each group contained three nude mice. The size of the formed tumour xenografts was recorded every 5 days, and tumour volume was detected by applying the following formula: Volume = 0.5 × length × width2. At day 30 post tumour cell injection, all mice were euthanized via cervical dislocation. The resected tumour xenografts were weighed and processed for molecular detection.
Subcellular fractionation
The Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek Corp.) was used to segregate the cytoplasmic and nuclear fractions of NSCLC cells, and was implemented according to the manufacturer's protocol. After RNA extraction, RT-qPCR was conducted to assess the expression levels of SLC25A25-AS1, GAPDH and U6 in both fractions, which was performed as aforementioned.
Bioinformatics analysis and luciferase reporter assay
StarBase (version 3.0; http://starbase.sysu.edu.cn/), an online bioinformatics database, was used to search for the putative targets of SLC25A25-AS1. The targets of miR-195-5p were identified using StarBase and TargetScan (Release 7.2: March 2018; http://www.targetscan.org).The SLC25A25-AS1 sequences possessing wild-type (wt) or mutant (mut) miR-195-5p binding sites were synthesized and inserted into the psiCHECK™-2 vector (Promega Corporation), generating the SLC25A25-AS1-wt and SLC25A25-AS1-mut fusion plasmids. The fusion reporter plasmids ITGA2-wt and ITGA2-mut were generated according to the same experimental process. The obtained wt or mut fusion reporter plasmids (Shanghai GenePharma Co., Ltd.) together with miR-195-5p mimic or NC mimic (Guangzhou RiboBio Co., Ltd.) were introduced into NSCLC cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The miR-195-5p mimic sequence was 5′-CGGUUAUAAAGACACGACGAU-3′ and the NC mimic sequence was 5′-UUGUACUACACAAAAGUACUG-3′. Luciferase activity was detected at 48 h post-transfection using the dual-luciferase reporter assay system (Promega Corporation). Renilla luciferase activity was utilized to normalize the firefly luciferase activity.
RNA immunoprecipitation (RIP)
NSCLC cells were harvested using trypsin and lysed in RIP cell lysis buffer (MilliporeSigma). The obtained supernatant was used for the RIP assay with the help of the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (cat. no. 03-110; MilliporeSigma). The input was defined as 10% of the cell extract, and 100 μl cell extract was incubated at 4°C with 50 µl magnetic beads Protein A/G conjugated with anti-argonaute2 (AGO2) antibody (5 µl) or normal mouse IgG (5 µl) antibody (both from cat. no. 03-110; MilliporeSigma) overnight. The magnetic beads were harvested by centrifugation at 1,000 × g at room temperature for 2 min and rinsed with 1 ml RIP buffer. After detachment with proteinase K at 55°C for 30 min, the immunoprecipitated RNA was extracted and analysed by RT-qPCR, which was performed as aforementioned.
Western blotting
Total protein content was isolated from cultured cells (A549 and SK-MES-1) or tumour xenografts using RIPA lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) supplemented with phenylmethylsulfonyl fluoride (Nanjing KeyGen Biotech Co., Ltd.). After quantification using a BCA Protein Assay Kit (Nanjing KeyGen Biotech Co., Ltd.), 10% SDS-PAGE was performed to separate equal amounts of protein (30 µg/lane). The separated proteins were transferred onto PVDF membranes, followed by blocking at room temperature with 5% non-fat milk for 2 h and incubation at 4°C with primary antibodies overnight. A total of two primary antibodies, rabbit monoclonal anti-ITGA2 (1:1000; cat. no. ab133557) and rabbit monoclonal anti-GAPDH (1:1,000; cat. no. ab128915), were purchased from Abcam. A goat anti-rabbit IgG HRP-labelled secondary antibody (1:5,000; cat. no. ab205718; Abcam) was used to treat the membranes at room temperature for 1 h. The target proteins were detected using an enhanced chemiluminescence kit (Nanjing KeyGen Biotech Co., Ltd.), and protein quantification was performed using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control.
Statistical analysis
All experiments were repeated three times, and each experiment was performed in triplicate. All data are presented as the mean ± standard deviation and were analysed with SPSS 18.0 software (SPSS, Inc.). Normal distribution of data was evaluated using the Shapiro-Wilk normality test. Student's t-test (both paired and unpaired) were used for comparisons between two groups. One-way ANOVA followed by Tukey's post hoc test was performed for comparing the differences among ≥3 groups. Utilizing the median value (3.32) of SLC25A25-AS1 in NSCLC tissues as the cut-off line, all patients were divided into either low-SLC25A25-AS1 or high-SLC25A25-AS1 groups. Overall survival was examined using Kaplan-Meier analysis and the log-rank test. Pearson's correlation analysis was applied to determine the gene expression correlation. P<0.05 was considered to indicate a statistically significant difference.
Results
High SLC25A25-AS1 expression is associated with poor prognosis in NSCLC
First, TCGA data were used to examine SLC25A25-AS1 expression in NSCLC tissues. The analysis revealed a marked upregulation of SLC25A25-AS1 expression in LUAD and LUSC compared with normal tissues (Fig. 1A). In line with the results from TCGA, SLC25A25-AS1 expression was upregulated in NSCLC tissues compared with in adjacent tissues (Fig. 1B). Furthermore, compared with that in BEAS-2B cells, SLC25A25-AS1 expression was upregulated in NSCLC cell lines (Fig. 1C). In addition, Kaplan-Meier analysis and a log-rank test demonstrated that patients with NSCLC with high SLC25A25-AS1 expression had poor overall survival compared with patients with low SLC25A25-AS1 expression (Fig. 1D). These results suggested that upregulation of SLC25A25-AS1 expression was markedly associated with poor clinical outcomes of patients with NSCLC, implying an important role of SLC25A25-AS1 in NSCLC progression.
Figure 1.
SLC25A25-AS1 expression is upregulated in NSCLC. (A) SLC25A25-AS1 expression in LUAD and LUSC tissues was determined utilizing The Cancer Genome Atlas. **P<0.01 compared with normal tissues. (B) RT-qPCR was performed to quantify SLC25A25-AS1 expression in NSCLC tissues and adjacent normal tissues. **P<0.01 compared with normal tissues. (C) SLC25A25-AS1 expression in NSCLC cell lines was determined using RT-qPCR. **P<0.01 compared with BEAS-2B. (D) Kaplan-Meier analysis was performed to assess the association between SLC25A25-AS1 expression and overall survival in NSCLC. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; SLC25A25-AS1, SLC25A25 antisense RNA 1.
SLC25A25-AS1 deficiency restricts NSCLC cell proliferation and metastasis and increases apoptosis
To decipher the detailed roles of SLC25A25-AS1, A549 and SK-MES-1 cell lines, which expressed the highest levels of SLC25A25-AS1 among the four NSCLC cell lines, were used in subsequent experiments. A total of two siRNAs, both si-SLC25A25-AS1#1 and si-SLC25A25-AS1#2, were used to silence endogenous SLC25A25-AS1 expression in NSCLC cells. RT-qPCR was performed to confirm that the transfection was successful (Fig. 2A). The biological effect of SLC25A25-AS1 depletion on NSCLC cell proliferation was tested using a CCK-8 assay. NSCLC cell proliferation was markedly restricted by transfection with si-SLC25A25-AS1 (Fig. 2B). Additionally, silencing of SLC25A25-AS1 markedly facilitated NSCLC cell apoptosis (Fig. 2C). Furthermore, compared with those in the si-NC group, the migratory (Fig. 2D) and invasive (Fig. 2E) abilities of NSCLC cells were suppressed in the si-SLC25A25-AS1 groups. Therefore, SLC25A25-AS1 performed pro-oncogenic actions in NSCLC cells.
Figure 2.
SLC25A25-AS1 interference suppresses the aggressiveness of NSCLC cells. (A) Efficiency of SLC25A25-AS1 silencing in NSCLC cells following transfection with si-SLC25A25-AS1 was verified using reverse transcription-quantitative PCR. (B) A Cell Counting Kit-8 assay revealed the effect of si-SLC25A25-AS1 transfection on NSCLC cell proliferation. (C) si-SLC25A25-AS1-transfected and si-NC-transfected cells were subjected to flow cytometry analysis for apoptosis determination. (D) Transwell migration and (E) invasion assays were performed to analyse the migratory and invasive properties of SLC25A25-AS1-deficient NSCLC cells (magnification, ×200). **P<0.01 compared with si-NC. NC, negative control; NSCLC, non-small cell lung cancer; si, small interfering RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1.
SLC25A25-AS1 acts as a ceRNA by sponging miR-195-5p in NSCLC
To elucidate the mechanisms mediating the pro-oncogenic roles of SLC25A25-AS1, the location of SLC25A25-AS1 in NSCLC cells was determined, with data from a subcellular fractionation assay indicating that SLC25A25-AS1 was primarily distributed in the cell cytoplasm (Fig. 3A). Accordingly, it was hypothesized that SLC25A25-AS1 may promote the oncogenicity of NSCLC via a ceRNA. The StarBase platform was used for bioinformatic analysis, and a total of 14 miRNAs (Fig. 3B) were predicted to have complementary sequences for SLC25A25-AS1. Notably, according to data from TCGA, miR-497-5p and miR-195-5p were downregulated in LUSC and LUAD (Fig. 3C and D); thus, the two miRNAs were selected for further verification. After SLC25A25-AS1 silencing, miR-195-5p expression was markedly upregulated, whereas miR-497-5p expression remained unchanged following si-SLC25A25-AS1 transfection (Fig. 3E). Furthermore, miR-195-5p expression was downregulated in NSCLC tissues (Fig. 3F) and negatively correlated with SLC25A25-AS1 expression (Fig. 3G).
Figure 3.
miR-195-5p is sponged by SLC25A25-AS1 in NSCLC cells. (A) Subcellular localization of SLC25A25-AS1 in A549 and SK-MES-1 cells. (B) Putative target miRNAs of SLC25A25-AS1 predicted by StarBase. (C and D) miR-497-5p and miR-195-5p in LUSC and LUAD was analysed utilizing TCGA database. (E) miR-195-5p and miR-497-5p expression in NSCLC cells after SLC25A25-AS1 interference was detected by RT-qPCR. **P<0.01 compared with si-NC. (F) miR-195-5p expression in NSCLC tissues was examined by RT-qPCR. **P<0.01 compared with normal tissues. (G) Pearson's correlation analysis revealed the expression relationship between SLC25A25-AS1 and miR-195-5p in NSCLC tissues. (H) wt and mut binding sites of miR-195-5p targeting SLC25A25-AS1. (I) Transfection efficiency of miR-195-5p mimic in NSCLC cells was verified by RT-qPCR. **P<0.01 compared with NC mimic. (J) Either SLC25A25-AS1-wt or SLC25A25-AS1-mut reporter plasmids alongside miR-195-5p mimic or NC mimic were introduced into A549 and SK-MES-1 cell. The reporter activity was quantified after 48 h of cultivation. **P<0.01 compared with NC mimic. (K) RNA immunoprecipitation was implemented to assess the association between SLC25A25-AS1 and miR-195-5p in NSCLC cells. **P<0.01 compared with IgG. AGO2, argonaute2; miRNA/miR, microRNA; mut, mutant; NC, negative control; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1; wt, wild-type.
Fig. 3H shows the wt and mut binding sites of miR-195-5p within the sequence of SLC25A25-AS1. RT-qPCR was used to verify that the transfection of miR-195-5p mimic in NSCLC cells was successful (Fig. 3I). The luciferase reporter assay demonstrated that exogenous miR-195-5p expression markedly suppressed the luciferase activity of SLC25A25-AS1-wt; however, the regulatory effect of miR-195-5p overexpression on luciferase activity was abrogated after the binding sequences were mutated (Fig. 3J). In the RIP assay, SLC25A25-AS1 and miR-195-5p were markedly enriched in AGO2-containing beads compared with the IgG control (Fig. 3K). Therefore, SLC25A25-AS1 may act as a sponge for miR-195-5p in NSCLC cells via direct interaction.
miR-195-5p directly targets ITGA2, and SLC25A25-AS1 positively regulates it by decoying miR-195-5p
miR-195-5p has been reported to be an antioncogenic miRNA in NSCLC (26–28). To elucidate the downstream target of miR-195-5p, bioinformatics prediction was conducted, and ITGA2 harboured a putative binding site for miR-195-5p (Fig. 4A). Compared with adjacent healthy tissues, a high ITGA2 expression was confirmed in NSCLC tissues (Fig. 4B). An inverse expression relationship was verified between ITGA2 and miR-195-5p in NSCLC tissues (Fig. 4C). Additionally, ITGA2 mRNA and protein expression (Fig. 4D and E) was reduced following miR-195-5p mimic transfection. Furthermore, transfection with miR-195-5p mimic markedly decreased the luciferase activity of ITGA2-wt, whereas no obvious alteration in the luciferase activity of NSCLC cells was observed after miR-195-5p mimic and ITGA2-mut co-transfection (Fig. 4F).
Figure 4.
ITGA2 is controlled by SLC25A25-AS1/miR-195-5p in NSCLC cells. (A) wt and mut binding sites between miR-195-5p and the ITGA2 3′ untranslated region. (B) Expression levels of ITGA2 in NSCLC tissues and adjacent tissues were measured by RT-qPCR. **P<0.01 compared with normal tissues. (C) Negative correlation between miR-195-5p expression and ITGA2 expression in NSCLC tissues as revealed using Pearson's correlation analysis. (D) RT-qPCR and (E) western blotting were performed to measure ITGA2 expression in miR-195-5p-overexpressing NSCLC cells. **P<0.01 compared with NC mimic. (F) ITGA2-wt or ITGA2-mut plasmids in parallel with miR-195-5p mimic or NC mimic were co-transfected into NSCLC cells, followed by a luciferase reporter assay for reporter activity quantification. **P<0.01 compared with NC mimic. (G) Transfection efficiency of the miR-195-5p inhibitor was determined via RT-qPCR. **P<0.01 compared with NC inhibitor. The regulatory effects of si-SLC25A25-AS1 on ITGA2 expression were assessed by (H) RT-qPCR and (I) western blotting. **P<0.01 compared with si-NC. SLC25A25-AS1-silenced NSCLC cells were further transfected with miR-195-5p inhibitor or NC inhibitor, followed by the determination of ITGA2 (J) mRNA and (K) protein expression. **P<0.01 compared with si-NC and si-SLC25A25-AS1 + miR-195-5p inhibitor. (L) A positive correlation between SLC25A25-AS1 expression and ITGA2 expression in NSCLC tissues was revealed by Pearson's correlation analysis. (M) RNA immunoprecipitation was performed to explore direct interaction among SLC25A25-AS1, miR-195-5p and ITGA2 in NSCLC. **P<0.01 compared with IgG. AGO2, argonaute2; ITGA2, integrin α2; miR, microRNA; mut, mutant; NC, negative control; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1; wt, wild-type.
The present study examined whether SLC25A25-AS1 was involved in regulating ITGA2 in NSCLC cells. The measurement of miR-195-5p inhibitor transfection efficiency revealed that miR-195-5p was considerably downregulated in miR-195-5p inhibitor-transfected NSCLC cells (Fig. 4G). Silencing of SLC25A25-AS1 markedly decreased ITGA2 expression (Fig. 4H and I) in NSCLC cells, whereas the introduction of a miR-195-5p inhibitor offset the silencing effect of si-SLC25A25-AS1 on ITGA2 expression (Fig. 4J and K). In addition, a positive correlation was identified between ITGA2 and SLC25A25-AS1 expression in the 48 NSCLC tissues (Fig. 4L). Furthermore, SLC25A25-AS1, miR-195-5p and ITGA2 were significantly enriched in AGO2-containing beads compared with the IgG group (Fig. 4M). Therefore, SLC25A25-AS1 acted as a ceRNA in NSCLC cells and could increase ITGA2 expression by sequestering miR-195-5p.
miR-195-5p/ITGA2 axis is responsible for SLC25A25AS1-induced actions in NSCLC cells
Rescue experiments were performed to evaluate whether miR-195-5p/ITGA2 is implicated in si-SLC25A25-AS1-induced antitumour activity in NSCLC. miR-195-5p inhibitor or NC inhibitor and si-SLC25A25-AS1 were transfected into NSCLC cells, and cell experiments were performed. Co-transfection of the miR-195-5p inhibitor restored the proliferative ability of NSCLC cells, which was impeded by silencing of SLC25A25-AS1 (Fig. 5A). As demonstrated by flow cytometry analysis, si-SLC25A25-AS1 treatment significantly promoted the apoptosis of NSCLC cells, and application of miR-195-5p inhibitor reversed the effect (Fig. 5B). Additionally, migration and invasion were hindered in si-SLC25A25-AS1-treated NSCLC cells; however, inhibition of miR-195-5p reversed the decrease in migration (Fig. 5C) and invasion (Fig. 5D) induced by SLC25A25-AS1 knockdown.
Figure 5.
Downregulation of miR-195-5p is sufficient to abolish the regulatory activities of si-SLC25A25-AS1 in NSCLC cells. si-SLC25A25-AS1 and miR-195-5p inhibitor or NC inhibitor were transfected into NSCLC cells. (A) Cell proliferation and (B) apoptosis were analysed using a Cell Counting Kit-8 assay and flow cytometry, respectively. (C) Migratory and (D) invasive abilities of the aforementioned cells were investigated using Transwell migration and invasion assays (magnification, ×200). *P<0.05 and **P<0.01. miR, microRNA; NC, negative control; NSCLC, non-small cell lung cancer; si, small interfering RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1.
The present study also evaluated whether si-SLC25A25-AS1-mediated actions are dependent on ITGA2. Western blotting demonstrated the efficiency of pcDNA3.1-ITGA2 in upregulating ITGA2 expression (Fig. 6A). Similarly, ITGA2 overexpression counteracted the si-SLC25A25-AS1-induced repressing effect in NSCLC cell proliferation (Fig. 6B). In addition, the promotive effect of SLC25A25-AS1 knockdown on NSCLC cell apoptosis was abolished by pcDNA3.1-ITGA2 treatment (Fig. 6C). Furthermore, the decreased NSCLC cell migration and invasion caused by SLC25A25-AS1 depletion was recovered after ITGA2 overexpression (Fig. 6D and E). Overall, SLC25A25-AS1 knockdown suppressed the oncogenicity of NSCLC cells by modulating the miR-195-5p/ITGA2 axis.
Figure 6.
Antitumour activity of si-SLC25A25-AS1 in NSCLC cells may be due to ITGA2 downregulation. (A) Western blotting was performed to verify the efficiency of pcDNA3.1-ITGA2 transfection in NSCLC cells. **P<0.01 compared with pcDNA3.1. (B-E) A549 and SK-MES-1 cells were transfected with si-SLC25A25-AS1 in combination with pcDNA3.1-ITGA2 or pcDNA3.1. (B) A Cell Counting Kit-8 assay, (B) flow cytometry analysis, and (D) Transwell migration and (E) invasion assays (magnification, ×200) were performed to explore cell proliferation, apoptosis, migration and invasion, respectively. **P<0.01 compared with si-NC and si-SLC25A25-AS1+ pcDNA3.1-ITGA2. *P<0.05 compared with si-SLC25A25-AS1 + pcDNA3.1-ITGA2. ITGA2, integrin α2; NC, negative control; NSCLC, non-small cell lung cancer; si, small interfering RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1.
Targeting SLC25A25-AS1 suppresses in vivo tumour growth
Subsequently, in vivo animal experiments were performed to examine the effect of SLC25A25-AS1 deficiency on in vivo tumour growth of NSCLC cells. The sh-SLC25A25-AS1 stably transfected cells generated markedly smaller tumour xenografts (Fig. 7A), in agreement with the in vivo growth curve (Fig. 7B) and tumour weights (Fig. 7C). In addition, SLC25A25-AS1 (Fig. 7D) and ITGA2 (Fig. 7E) expression in the sh-SLC25A25-AS1 group was decreased, while miR-195-5p expression was increased (Fig. 7F). Overall, the aforementioned results suggested that SLC25A25-AS1 deficiency reduced the in vivo tumour growth of NSCLC cells.
Figure 7.
SLC25A25-AS1 silencing impairs in vivo tumour growth of non-small cell lung cancer cells. (A) Images of excised tumour xenografts. (B) After cell injection, tumour volume measurement was performed every 5 days, and a tumour growth curve was generated. (C) Tumour xenografts were removed from nude mice, and tumour weight was calculated. (D) SLC25A25-AS1 expression in excised tumour xenografts. (E) ITGA2 protein expression in excised tumour xenografts. (F) miR-195-5p expression in removed tumour tissues. **P<0.01 compared with sh-SLC25A25-AS1. ITGA2, integrin α2; miR, microRNA; NC, negative control; sh, short hairpin RNA; SLC25A25-AS1, SLC25A25 antisense RNA 1.
Discussion
Increasing evidence has highlighted lncRNAs as critical regulators of NSCLC progression and revealed the close association between their dysregulation and NSCLC oncogenesis and cancer progression (29–31). Given the importance of lncRNAs in NSCLC, identifying novel lncRNAs associated with NSCLC and exploring their exact roles is of great value for anticancer therapy development. Although numerous lncRNAs are encoded by the human genome, the majority of lncRNA expression patterns and detailed functions are not completely understood. Therefore, the present study aimed to investigate whether SLC25A25-AS1 is involved in the aggressiveness of NSCLC and the possible underlying mechanism.SLC25A25-AS1 expression is downregulated in colorectal cancer tissues and serum samples (24). It exerts antitumorigenic effects in colorectal cancer, and controls cell proliferation, clonality, chemoresistance and epithelial-mesenchymal transition (24). However, to the best of our knowledge, the exact roles of SLC25A25-AS1 in NSCLC are still unclear, and more studies are required to explore the biological, prognostic and molecular classifications of SLC25A25-AS1 in NSCLC. In the present study, SLC25A25-AS1 expression was identified to be upregulated in NSCLC, as verified by data from both TCGA and our cohort. High SLC25A25-AS1 expression was associated with poor prognosis in patients with NSCLC. SLC25A25-AS1 depletion suppressed proliferation, migration and invasion but increased apoptosis in NSCLC cells. After SLC25A25-AS1 interference, the tumour growth of NSCLC cells in vivo was also impaired. Therefore, SLC25A25-AS1 may be a promising target for NSCLC diagnosis, prognosis and management.The mechanisms mediating the pro-oncogenic actions of SLC25A25-AS1 are unknown and warrant further investigation. lncRNAs function through a variety of different mechanisms. lncRNAs are capable of epigenetically silencing mRNA expression and regulating genes at the transcriptional level (32). At the posttranscriptional level, the ceRNA theory (33) has attracted increasing attention and serves an important role in the study of lncRNAs (34). According to the ceRNA theory, lncRNAs contain miRNA response elements and are capable of competitively binding with specific miRNAs, ultimately resulting in decreased regulatory activities of miRNAs on their target mRNAs (34). Therefore, the present study examined whether SLC25A25-AS1 acted as a ceRNA by verifying its subcellular distribution in NSCLC cells. The outcomes of the subcellular fractionation assay identified SLC25A25-AS1 as a cytoplasmic lncRNA in NSCLC.In the present study, miR-195-5p was predicted to be a binding partner of SLC25A25-AS1. The function of SLC25A25-AS1 sponging miR-195-5p in NSCLC cells was verified by experimental observations. First, downregulation of SLC25A25-AS1 increased miR-195-5p expression in NSCLC cells. Second, an inverse relationship between SLC25A25-AS1 and miR-195-5p expression was demonstrated in NSCLC tissues. Finally, a luciferase reporter assay and RIP collectively corroborated the direct binding and interaction between SLC25A25-AS1 and miR-195-5p. After confirming SLC25A25-AS1 as a miR-195-5p sponge in NSCLC, additional mechanical experiments were performed. The present results suggested that miR-195-5p could reduce ITGA2 expression by directly targeting it and that SLC25A25-AS1 knockdown decreased ITGA2 expression in NSCLC, potentially by sequestering miR-195-5p. These three RNAs, SLC25A25-AS1, miR-195-5p and ITGA2, comprise a novel ceRNA regulatory network in NSCLC cells.miR-195-5p is aberrantly expressed in multiple humancancer types, including NSCLC (35,36). miR-195-5p has tumour-inhibiting capacities in weakening the aggressive phenotype of NSCLC cells (35–37). In the present study, ITGA2, an important collagen receptor on platelets and epithelial cells, was demonstrated to be negatively controlled by miR-195-5p in NSCLC. Therefore, the present study inferred that inhibiting ITGA2 is essential for SLC25A25-AS1-induced effects in NSCLC cells. The present results demonstrated that overexpression of ITGA2 abrogated the antitumour effects of SLC25A25-AS1 silencing. Similarly, downregulation of miR-195-5p restored the malignant behaviours suppressed by the loss of SLC25A25-AS1. Specifically, miR-195-5p/ITAG2 was the downstream mediator of SLC25A25-AS1 in NSCLC.Recently, lncRNAs have gained increasing attention in targeted therapy of humancancer (38–40). At present, targeting lncRNAs can be achieved through multiple different approaches, such as transcription block, degradation and gene-editing technology (41,42). Furthermore, abolishment of the interaction between lncRNAs and their downstream targets utilizing competitive binding is an additional method targeting lncRNAs (43). Despite a lack of satisfactory lncRNA-targeting antitumour drugs, rapid developments in medical treatment technology may offer the possibility of a feasible therapy targeting lncRNAs inside humancancer cells. The present research elucidating the role of SLC25A25-AS1 in NSCLC may aid in the development of effective treatment strategies.The present study had two limitations. First, 6–10 mice are usually used in in vivo animal experiments; however, the present study only used three mice in each group. Second, SLC25A25-AS1 was demonstrated to be a miR-195-5p sponge in NSCLC; however, SLC25A25-AS1 may also act as ceRNA for other miRNAs. These limitations will be addressed in further experiments.In conclusion, the present study revealed increased expression levels of SLC25A25-AS1 in NSCLC tissues, which was notably associated with poor patient prognosis. SLC25A25-AS1 acted as a ceRNA for miR-195-5p in NSCLC cells and thereby positively controlled ITGA2 expression, consequently exerting oncogenic activity. Therefore, the SLC25A25-AS1/miR-195-5p/ITAG2 signalling pathway might be an attractive target for future therapeutic options in NSCLC.
Authors: Hany S Zinad; Chanachai Sae-Lee; Maria Ascensión Ariza-Mateos; Grace Adamson; Mushtaq Mufleh Khazeem; Amber Knox; Git Chung; Jelena Mann; Andreas Werner Journal: Noncoding RNA Date: 2022-02-11
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