MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway

Retraction in: /10.3892/ijmm.2022.5134

  • Authors:
    • Xiaoxia Tan
    • Lihua Jiang
    • Xia Wu
    • Wen Feng
    • Qingfang Lin
  • View Affiliations

  • Published online on: May 20, 2019     https://doi.org/10.3892/ijmm.2019.4203
  • Pages: 346-356
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Abstract

Numerous microRNAs (miRs) are dysregulated in non‑small cell lung cancer (NSCLC), serving pivotal roles in its formation and progression. miR‑625 is dysregulated in several types of human cancer, but its involvement in the formation and development of NSCLC remains poorly understood. In the present study, we aimed to investigate miR‑625 expression in NSCLC and its role in regulating NSCLC cell behavior. miR‑625 expression in NSCLC tissues and cell lines was detected using reverse transcription‑quantitative polymerase chain reaction. The effects of miR‑625 overexpression on NSCLC cell proliferation, apoptosis, migration and invasion in vitro were assessed using an MTT assay, flow cytometry, and cell migration and invasion assays, respectively. The effects of miR‑625 upregulation on NSCLC growth were evaluated in an in vivo xenograft model. The molecular mechanisms underlying the tumor‑suppressing roles of miR‑625 in NSCLC were explored in detail. miR‑625 expression was determined to be downregulated in NSCLC tissues and cell lines. This decreased expression was associated with advanced clinical features and poor overall survival of patients with NSCLC. Exogenous miR‑625 expression suppressed NSCLC cell proliferation, migration and invasion, and induced apoptosis in vitro. miR‑625 upregulation hindered NSCLC tumor growth in vivo. Homeobox B5 (HOXB5) was proposed to be the direct target gene of miR‑625 in NSCLC cells. The tumor‑suppressing effects of HOXB5 silencing were similar to those of miR‑625 overexpression in NSCLC cells. In rescue experiments, HOXB5 overexpression partially reversed the inhibitory effects of miR‑625 in NSCLC cells. miR‑625 upregulation directly targeted HOXB5 to deactivate the Wnt/β‑catenin signaling pathway in NSCLC cells in vitro and in vivo. miR‑625 was determined to be associated with HOXB5 suppression and Wnt/β‑catenin pathway deactivation, which in turn inhibited the aggressive behavior of NSCLC cells in vitro and in vivo.

Introduction

Lung cancer is one of the most common types of malignancies in humans and a leading cause of cancer-associated mortali-ties among males and females worldwide (1). Lung cancer can be divided into two main types: Non-small cell lung cancer (NSCLC) and small cell lung cancer (2). NSCLC is an aggressive type of lung cancer and accounts for ~85% of all lung cancer cases (3). Among NSCLCs, 32-40% are adenocarcinomas, 25-30% are squamous type and 8-16% are large cell cancers (4). Despite considerable advances in NSCLC treatment, the prognosis of patients with NSCLC remains poor, with a 5-year survival rate of <16% (5). Tumor recurrence and metastasis are frequent, posing considerable challenges during treatment (6,7). Therefore, the development of novel effective therapeutic strategies for treating NSCLC is urgent. Thus, elucidation of the molecular mechanisms underlying the pathogenesis of NSCLC is imperative, and may provide novel insight into the diagnosis, treatment and prevention of disease.

MicroRNAs (miRNAs/miRs) are a class of endogenous short (~21-25 nucleotides), single-stranded non-coding RNAs (8). miRNAs negatively regulate gene expression by directly binding to the 3′-untranslated region (3′-UTR) of their target genes, promoting their degradation and inhibiting translation (8,9). Several miRNAs can directly target one mRNA, whereas a single miRNA can bind to various mRNAs (10). miRNAs are reportedly involved in various biological activities, including cell proliferation, division, apoptosis and differentiation, generation, migration, invasion and metastasis (11-13). Increasing evidence has suggested that miRNAs act as key regulators in the development and progression of human cancers (14-16). Recently, alterations in the expression of miRNAs have been wildly reported in nearly all human cancers, including NSCLC (17-19). miRNAs can serve as tumor-suppressing or tumor-promoting miRNAs, depending on the functional characterization of their target genes (20). Therefore, a detailed investigation into cancer-associated miRNAs in NSCLC may indicate novel prognostic biomarkers and therapeutic targets for NSCLC.

miR-625 dysregulation has been reported in several human malignancies (21-24); however, whether miR-625 is involved in the formation and development of NSCLC remains poorly understood at present. Hence, the present study aimed to investigate miR-625 expression in NSCLC and its role in the regulation of NSCLC cell behavior. We examined miR-625 expression in NSCLC tissues and cell lines, and overexpressed miR-625 in NSCLC cells to determine its effects on cancer cell behavior. In addition, we explored in detail the molecular mechanisms underlying the tumor-suppressing roles of miR-625 in this disease.

Materials and methods

Tissue samples

NSCLC tissue samples paired with normal adjacent tissues (NATs) were obtained from 53 patients with NSCLC (21 males, 32 females; age range, 49-72 years) who underwent surgical resection at The Third People's Hospital of Linyi (Linyi, China) between May 2013 to March 2018. All patients were divided into low or high expression subgroups according to the median value of miR-625 expression in the NSCLC tissues. The tumor, node and metastasis (TNM) staging system (25) was employed in the current study to classify the tumor stages of patients. NATs were ≥2-cm distal to tumor margins. None of the patients had been treated with adjuvant chemotherapy, radiotherapy or other approaches prior to surgery. The samples were immediately frozen in liquid nitrogen and maintained at -80°C until further use for total RNA isolation. The present study was approved by the Ethics Committee of The Third People's Hospital of Linyi (Linyi, China), and all patients provided written informed consent.

Cell culture and transfection

The non-tumorigenic bronchial epithelium cell line BEAS-2B and five human NSCLC cell lines (H460, H522, A549, SK-MES-1 and H1299) were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) and 1% v/v penicillin-streptomycin (Sigma-Aldrich; Merc KGaA, Darmstadt, Germany). The cells were grown at 37°C in a humidified atmosphere with 5% CO2.

miR-625 agomir (agomir-625) and agomir negative control (agomir-NC) were chemically produced by Shanghai GenePharma Co., Ltd. (Shanghai, China). The agomir-625 sequence was 5′-AGG GGG AAA GUU CUA UAG UCC-3′ and the agomir-NC sequence was 5′-UUC UCC GAA CGU GUC ACG UTT-3′. Small interfering RNA (siRNA) for homeobox 5 (HOXB5) silencing (si-HOXB5) and control siRNA (si-ctrl) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). The si-HOXB5 sequence was 5′-GGA UGG ACC UCA GCG UCA ATT-3′ and the si-ctrl sequence was 5′-UUC UCC GAA CGU GUC ACG UTT-3′. Full-length HOXB5 without 3′-UTR was synthesized by Synbio Technologies (Suzhou, China) and then inserted into pcDNA3.1(+) (Invitrogen; Thermo Fisher Scientific, Inc.); the generated plasmid was defined as pcDNA3.1-HOXB5 (pc-HOXB5). An empty vector served as the control in the case of HOXB5 overexpression. Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transient transfection and cotransfection, performed in accordance with the manufacturer's protocols. After incubation at 37°C for 8 h, the culture medium was replaced with fresh DMEM supplemented with 10% FBS.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the tissues and cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and its concentration was quantified using an ND-2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) following the manufacturer's protocol. miR-625 expression was detected via RT using a TaqMan MicroRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature protocol for RT was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. The cDNA was subjected to qPCR using a TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature protocol for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec. To quantify HOXB5 mRNA expression, cDNA was prepared from total RNA using a PrimeScript RT Reagent kit and subjected to qPCR using a SYBR Premix Ex Taq™ kit (both from Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for RT was as follows: 37°C for 15 min and 85°C for 5 sec. The thermocycling conditions for qPCR were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. The 7900HT Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform all reactions. U6 small nuclear RNA and GAPDH served as internal references for the expression of miR-625 and HOXB5 mRNA, respectively. Relative gene expression was analyzed according to the 2−∆∆Cq method (26).

The primers were designed as follows: miR-625, 5′-AGGGGGAAAGTTCTATAGTCC-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); HOXB5, 5′-TCAGTGCAAAATGTCTTCTG-3′ (forward) and 5′-TGACCCAGACTATCCCCATAT-3′ (reverse); and GAPDH, 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward) and 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse).

MTT assay

The transfected cells were harvested 24 h after transfection and inoculated into 96-well plates at a density of 3,000 cells/well. Cells were then incubated at 37°C for 0, 24, 48 or 72 h. At each time point, 20 µl MTT solution (5 mg/ml, Sigma-Aldrich; Merck KGaA) was added to each well, and cells were incubated for an additional 4 h at 37°C. The culture medium was removed, and 150 µl dimethyl sulf-oxide (Sigma-Aldrich; Merck KGaA) was added to each well. Finally, a microplate reader (iMark™; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the optical density at 490 nm.

Flow cytometry

After 48 h transfection, cells were harvested by 0.25% trypsinization at room temperature for 5 min, washed with ice-cold PBS (Gibco, Thermo Fisher Scientific, Inc.), and collected by centrifugation (12,000 × g for 10 min at 4°C). Cell apoptosis was detected with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, Inc., San Diego, CA, USA). The collected cells were resuspended in 100 µl of binding buffer, and 5 µl of Annexin V-FITC and 5 µl of propidium iodide were added prior to incubation in the dark for 15 min at room temperature. The proportion of apop-totic cells was analyzed using a flow cytometer (FACScan™, BD Biosciences, Franklin Lakes, NJ, USA). CellQuest-Pro software program (BD Biosciences) was used for analysis.

Cell migration and invasion assays

Cell migration and invasion were assessed using Transwell chambers (8-µm pore size; BD Biosciences, San Jose, CA, USA). For the migration assay, transfected cells were harvested at 48 h after transfection, washed with PBS, and suspended in FBS-free DMEM. Then, 200 µl of cell suspension containing 5×104 cells was added into the upper compartments. The lower compartments were covered with 500 µl DMEM containing 20% FBS (Gibco, Thermo Fisher Scientific, Inc.) to serve as a chemoattractant. After incubation at 37°C for 24 h, non-migrated cells were carefully wiped away with cotton wool. Migrated cells were fixed in 4% paraformaldehyde at room temperature for 30 min, stained with 0.5% crystal violet at room temperature for 30 min, imaged, and counted in five randomly selected fields under a light microscope at ×200 magnification. The experimental procedures for the cell invasion assay were similar to those for the migration assay; however, the Transwell chambers were precoated with Matrigel (BD Biosciences).

In vivo xenograft model analysis

All in vivo studies were approved by the Ethics Review Committee of The Third People's Hospital of Linyi. All experiments were performed in accordance with the 'Animal Protection Law of the People's Republic of China-2009' for experimental animals. A total of 8 BALB/c nude mice (4-weeks-old) were obtained from Charles River Laboratories, Inc. (Beijing, China) and maintained under specific pathogen-free conditions. Cells transfected with agomir-625 or agomir-NC were suspended in 100 µl culture medium and subcutaneously inoculated into the dorsal flank of each mouse (n=4 for each group). After 2 weeks of injection, the width and length of tumor xenografts were measured using Vernier calipers, and tumor volume was estimated as tumor volume (mm3)=[width2 (mm2) x length (mm)]/2. After 4 weeks of inoculation, the mice were sacrificed by cervical dislocation, and the tumor xenografts were resected and weighed.

Bioinformatic predication and luciferase reporter assay

TargetScan (Release 7.2; http://www.targetscan.org/), miRanda (August 2010 Release; http://www.microrna.org/microrna/), and miRDB (http://mirdb.org/) were used for predicting the targets of miR-625. The 3′-UTR fragments of HOXB5 containing the predicted wild-type (WT) and mutant (MUT) binding sites of miR-625 were amplified by Shanghai GenePharma Co., Ltd. and cloned into the pmirGLO luciferase reporter vector (Promega Corporation, Madison, WI, USA). The generated luciferase reporter plasmids were labeled pmirGLO- HOXB5-WT-3′-UTR and pmirGLO-HOXB5-MUT-3′-UTR, respectively. For the reporter assay, cells were seeded into 24-well plates and co-transfected with agomir-625 or agomir-NC, and pmirGLO-HOXB5-WT-3′-UTR or pmirGLO-HOXB5-MU3′-UTR using Lipofectamine 2000. After 48 h, luciferase activity was assessed using a Dual-Luciferase® Reporter Assay system (Promega Corporation) and normalized to that of Renilla.

Western blot analysis

Total protein was extracted from cells, tissues and tumor xenografts using radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was quantified using a BCA assay (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China). Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After 2 h blocking at room temperature in TBST containing 5% non-fat dry milk, the membranes were incubated overnight at 4°C with primary antibodies against HOXB5 (1:1,000 dilution; sc-81099; SantaCruz Biotechnology, Inc., Dallas, TX, USA), phosphorylated (p)-β-catenin (1:1,000 dilution; sc-57534; Santa Cruz Biotechnology, Inc.), β-catenin (1:1,000 dilution; sc-59737; Santa Cruz Biotechnology, Inc.), cyclin D1 (1:1,000 dilution; sc-450; Santa Cruz Biotechnology, Inc.), and GADPH (1:1,000 dilution; sc-166574; Santa Cruz Biotechnology). Furthermore, the membranes were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; sc-516102; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Finally, the protein bands were visualized using an enhanced chemiluminescence detection kit (Sigma-Aldrich; Merck KGaA), and analyzed using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).

Statistical analysis

Each experiment was repeated at least three times. Data are presented as mean + standard error. SPSS version 19.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Differences between two groups were analyzed using Student's t-test, and differences among three or more groups were analyzed using one-way analysis of variance with Student-Newman-Keuls post-hoc test. Associations between miR-625 expression and the clinicopathological characteristics of patients with NSCLC were assessed using χ2 test. Kaplan Meier and log-rank tests applied to assess patient outcome. A log-rank test was employed to determine the association between miR-625 expression and overall survival of patients with NSCLC. Spearman correlation analysis was applied to examine the association between miR-625 and HOXB5 mRNA expression levels in NSCLC tissues. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-625 is downregulated in NSCLC tissues and cell lines

The expression profiles of miR-625 were assessed using RT-qPCR in 53 pairs of NSCLC tissue and NAT samples. miR-625 expression in NSCLC tissues was significantly lower than in NATs (P<0.05; Fig. 1A). miR-625 expression was investigated in five NSCLC cell lines (H460, H522, A549, SK-MES-1 and H1299) and the non-tumorigenic bronchial epithelium cell line BEAS-2B. miR-625 expression was significantly down-regulated in the NSCLC cell lines compared with in BEAS-2B cells (P<0.05; Fig. 1B).

The clinical value of miR-625 for patients with NSCLC was explored by dividing the patients into low and high expression subgroups according to the median value of miR-625 expression in the NSCLC tissues. Decreased miR-625 expression was significantly associated with tumor size (P=0.039), lymph node metastasis (P=0.018), and TNM stage (P=0.026) of NSCLC (Table I). Furthermore, patients with low miR-625 expression exhibited poorer overall survival compared with those exhibiting high miR-625 expression (P=0.0008; Fig. 1C). These results suggested that miR-625 may be a prognostic biomarker for NSCLC.

Table I

Association between miR-625 expression and the clinicopathological characteristics of patients with NSCLC.

Table I

Association between miR-625 expression and the clinicopathological characteristics of patients with NSCLC.

CharacteristicsmiR-625 expression
P-value
Lowhigh
(n=27)(n=26)
Age (years)0.500
 <651517
 ≥65129
Sex0.122
 Male138
 Female1418
Tumor size (cm)0.039a
 <5815
 ≥51911
Lymph node metastasis0.018a
 Negative1119
 Positive167
TNM stage0.026a
 I1421
 II + III135
Distant metastasis0.088
 Negative1621
 Positive115

a P<0.05. NSCLC, non-small cell lung cancer; TNM, tumor, node and metastasis.

miR-625 inhibits NSCLC growth and metastasis in vitro

As miR-625 was expressed at low levels in NSCLC tissues and cell lines, we speculated that miR-625 suppressed the development and progression of NSCLC. To investigate this hypothesis, H460 and H522 cell lines, which exhibited the lowest miR-625 levels among the cell lines examined, were used for functional experiments. Cells were transfected with agomir-625 or agomir-NC. RT-qPCR analysis revealed that compared with agomir-NC, agomir-625 significantly increased miR-625 expression in H460 and H522 cells (P<0.05; Fig. 2A). MTT assay revealed that H460 and H522 cell proliferation significantly decreased with miR-625 overexpression at 48 and 72 h compared with the control group (P<0.05; Fig. 2B). Flow cytometry revealed that the apoptotic rate in H460 and H522 cells significantly increased following transfection with agomir-625 compared with the control (P<0.05; Fig. 2C). In the cell migration and invasion assays, ectopic miR-625 expression significantly reduced migration and invasion of H460 and H522 cells compared with the control group (P<0.05; Fig. 2D). Collectively, these results demonstrated that miR-625 exerted a tumor-suppressing role by inhibiting NSCLC growth and metastasis in vitro.

miR-625 directly targets HOXB5 in NSCLC

miRNAs can directly bind to the 3′-UTR of their target genes and act as inhibitors, reducing the expression of these genes (8,9). Bioinformatic analysis was applied to predict possible molecular mechanisms by which miR-625 acted on NSCLC growth and metastasis, with a particular focus on HOXB5 (Fig. 3A)-a candidate oncogene for the development and progression of NSCLC (27). A luciferase reporter assay was performed to determine whether the 3′-UTR of HOXB5 was directly targeted by miR-625 in NSCLC cells. Resumption of miR-625 expression efficiently reduced luciferase activity in H460 and H522 cells that were transfected with a luciferase plasmid harboring the WT HOXB5 binding site (P<0.05). Conversely, no significant alterations in the luciferase activity of cells cotransfected with agomir-625 and the MUT luciferase plasmid were observed (Fig. 3B). RT-qPCR and western blotting revealed that miR-625 overexpression significantly decreased HOXB5 expression at the mRNA and protein levels in H460 and H522 cells (P<0.05; Fig. 3C and D). The correlation between miR-625 and HOXB5 expression in NSCLC was further examined using RT-qPCR to measure HOXB5 expression in 53 pairs of NSCLC tissues and NATs. HOXB5 mRNA expression was significantly upregulated in NSCLC tissues compared with in NATs (P<0.05; Fig. 3E). The inverse correlation between miR-625 and HOXB5 mRNA expression was confirmed by Spearman correlation analysis (R2=0.3902, P<0.0001; Fig. 3F). These results suggested that miR-625 directly targeted HOXB5 in NSCLC.

HOXB5 suppression attenuates NSCLC cell proliferation, migration and invasion, and promotes cell apoptosis in vitro

After confirming HOXB5 as the direct target gene of miR-625, a loss-of-function assay was used to clarify its functions in NSCLC cells by transfecting H460 and H522 cells with si-HOXB5 or si-ctrl. Evaluation of the transfection efficiency using western blotting confirmed that HOXB5 protein expression was significantly downregulated in H460 and H522 cells transfected with si-HOXB5 (P<0.05; Fig. 4A). MTT assay and flow cytometry revealed that HOXB5 knockdown significantly suppressed H460 and H522 cell proliferation at 48 and 72 h compared with the control (P<0.05; Fig. 4B), and promoted their apoptosis (P<0.05; Fig. 4C) compared with si-ctrl transfection. Cell migration and invasion assays revealed that H460 and H522 cells transfected with si-HOXB5 exhibited significantly reduced migration and invasion compared with cells transfected with si-ctrl (P<0.05; Fig. 4D). These findings indicated that HOXB5 downregulation exerted effects similar to those induced by miR-625 overexpression on NSCLC cells, further suggesting that HOXB5 was a downstream target of miR-625 in these cells.

HOXB5 restoration partially reverses the tumor-suppressing effects of miR-625 overexpression in NSCLC cells

Rescue experiments were performed to confirm whether HOXB5 downregulation was essential for the inhibition of NSCLC cell growth and metastasis via miR-625 overexpression. HOXB5 protein expression was restored in H460 and H522 cells transfected with agomir-625 by cotransfecting them with a HOXB5-overexpressing plasmid pc-HOXB5 (P<0.05; Fig. 5A). An MTT assay demonstrated that miR-625 upregulation suppressed H460 and H522 cell proliferation, but this suppressive effect was rescued by HOXB5 restoration (P<0.05; Fig. 5B). Similarly, restored HOXB5 expression significantly abolished the effects of miR-625 overexpression on H460 and H522 cell apoptosis (P<0.05; Fig. 5C), migration and invasion (P<0.05; Fig. 5D). These results suggested that HOXB5, at least partly, mediated the tumor-suppressing roles of miR-625 in NSCLC cells.

miR-625 deactivates the Wnt/β-catenin pathway by targeting HOXB5 in NSCLC cells

The Wnt/β-catenin pathway is involved in the genesis and progression of cancer (28,29). In addition, this signaling pathway may be regulated by HOXB5 (27). Therefore, we explored whether miR-625 inhibited the Wnt/β-catenin pathway in NSCLC cells via regulation of HOXB5. After cotransfecting H460 and H522 cells with agomir-625 and pc-HOXB5 or pcDNA3.1, the expression of important molecules associated with the Wnt/β-catenin pathway was assessed using western blotting. The protein levels of p-β-catenin and cyclin D1 were significantly downregulated in H460 and H522 cells upon miR-625 overexpression (P<0.05), whereas no significant alterations in total β-catenin levels were observed (Fig. 6). Restoration of HOXB5 expression significantly alleviated the downregulation of p-β-catenin and cyclin D1 induced by miR-625 upregulation (P<0.05). These results indicated that miR-625 directly targeted HOXB5 to inactivate the Wnt/β-catenin pathway in NSCLC cells.

miR-625 inhibits NSCLC tumor growth in vivo

The contribution of miR-625 to tumor growth was investigated in an in vivo xenograft model developed by inoculating H460 cells transfected with agomir-625 or agomir-NC into the dorsal flanks of nude mice. The volume (Fig. 7A and B) and weight (Fig. 7C) of tumors were significantly lower in the agomir-625 group than the agomir-NC group (P<0.05). RT-qPCR revealed clear miR-625 upregulation in tumor xenografts in the agomir-625 group compared with the agomir-NC group (P<0.05; Fig. 7D). Western blotting confirmed that miR-625 overexpression suppressed HOXB5, p-β-catenin and cyclin D1 protein expressions in tumor xenografts (P<0.05; Fig. 7E), which is consistent with results in vitro. These findings suggested that miR-625 reduced NSCLC tumor growth in vivo by inhibiting the Wnt/β-catenin pathway via HOXB5 targeting.

Discussion

Increasing evidence has demonstrated that numerous miRNAs are dysregulated in NSCLC and that this dysregulation is a hallmark of NSCLC (30-32). Recently, miRNAs have been identified as a novel player in gene regulation in NSCLC, with aberrant miRNAs expression serving pivotal roles in formation and progression of NSCLC (33-35). A detailed investigation of cancer-associated miRNAs in NSCLC is therefore crucial for identifying effective therapeutic targets for this disease. Previous studies have focused on miRNAs expression profiles and functions during the development of NSCLC (36-38); however, the association between miR-625 and NSCLC requires further investigation. To the best of our knowledge, the present study is the first to assess miR-625 expression in NSCLC and investigated its clinical value in patients with NSCLC. Specifically, we explored in detail the potential functions of miR-625 in NSCLC and the mechanisms underlying its activity.

miR-625 expression is decreased in colorectal cancer, and this decrease is significantly correlated with lymph node and liver metastases (21-24). Patients with colorectal cancer and low miR-625 expression exhibited poor overall survival and unfavorable prognosis compared with those exhibiting high miR-625 expression (21). In addition, miR-625 was determined to be downregulated in breast cancer, which was closely associated with estrogen receptor and epidermal growth factor receptor 2 levels, as well as clinical stage. Kaplan-Meier and multivariate analyses have suggested miR-625 expression as an independent predictor of unfavorable prognosis in patients with breast cancer (22). Additionally, low miR-625 expression levels have been reported in laryngeal squamous cell carcinoma (23), gastric cancer (24), esophageal cancer (39), hepatocellular carcinoma (40), malignant melanoma (41) and glioma (42); however, the expression profile of miR-625 in NSCLC and its significance remains largely unknown. In the present study, we demonstrated that miR-625 was significantly downregulated in NSCLC tissues and cells, and that this downregulation was associated with unfavorable clinicopathological characteristics of patients with NSCLC. In addition, NSCLC patients with low miR-625 expression exhibited poorer overall survival compared with those exhibiting increased expression. These findings suggest that miR-625 could be a potential biomarker for the diagnosis and prognosis of NSCLC.

Several studies have shown that miR-625 can suppress the genesis and progression of cancer. For instance, the ectopic miR-625 expression evidently inhibited cell growth, metastasis and epithelial-mesenchymal transition in laryngeal squamous cell carcinoma (23). Furthermore, miR-625 expression attenuated the invasion and metastasis of gastric cancer in vitro and in vivo (24). In malignant melanoma, miR-625 upregulation restricted cell proliferation, wound healing and metastasis in vitro, and tumor growth in vivo (41). In glioma, restored expression of miR-625 inhibited cell proliferation and promoted cell cycle arrest and apoptosis in vitro; tumorigenicity was inhibited in vivo. In addition, miR-625 upregulation augmented the chemosensitivity of glioma cells to temozolomide (42); however, whether miR-625 serves a role in the progression of NSCLC in vitro and in vivo requires further investigation. In the present study, miR-625 overexpression was proposed to inhibit NSCLC development by regulating cell proliferation, apoptosis, migration and invasion in vitro, and tumor growth in vivo. These results indicated that miR-625 regulates the aggressive behavior of NSCLC.

Previous studies have identified various direct target genes of miR-625, including SRY-box 4 (SOX4) in laryngeal squamous cell carcinoma (23), high mobility group AT-hook 1 in breast cancer (22), integrin-linked protein kinase in gastric cancer (24), SOX2 in esophageal cancer (39), insulin-like growth factor 2 in hepatocellular carcinoma (40), and AKT2 in glioma (42). However, the molecular mechanisms underlying the action of miR-625 in NSCLC remain unknown. In the present study, HOXB5, a member of the HOX gene family (43), was determined to be the direct downstream target of miR-625 in NSCLC cells. HOXB5 is upregulated in several types of human cancer, including NSCLC (27), retinoblastoma (44), gastric cancer (43), breast cancer (45) and oral squamous cell carcinoma (46). HOXB5 silencing inhibited NSCLC cell proliferation, migration, invasion and epithelial-mesenchymal transition in vitro, and tumor growth in vivo (27). In the present study, miR-625 was reported to directly target HOXB5 to inactivate the Wnt/β-catenin pathway, which in turn suppressed the malignant development of NSCLC cells. Thus, miR-625 overexpression-mediated HOXB5 suppression and Wnt/β-catenin pathway inactivation may be effective therapeutic strategies in treating patients with NSCLC.

In our study, miR-625 was demonstrated to be significantly downregulated in NSCLC. miR-625 was also reported to serve as a suppressor of NSCLC development, possibly by directly targeting HOXB5 and inhibiting the Wnt/β-catenin pathway. These observations may potentially be applied in the treatment of NSCLC to inhibit tumor growth and metastasis in NSCLC.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

QL made substantial contributions to the design of the present study. XT performed the CCK-8 assay and in vivo xenograft model analysis. Flow cytometry, cell migration and invasion assays, and western blot analysis were conducted by LJ and XW. Bioinformatic predictions and the luciferase reporter assay were conducted by WF. All authors read and approved the final draft.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The Third People's Hospital of Linyi, and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of The Third People's Hospital of Linyi. All patients provided written informed consent.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

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July-2019
Volume 44 Issue 1

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Online ISSN:1791-244X

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Spandidos Publications style
Tan X, Jiang L, Wu X, Feng W and Lin Q: MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway Retraction in /10.3892/ijmm.2022.5134. Int J Mol Med 44: 346-356, 2019
APA
Tan, X., Jiang, L., Wu, X., Feng, W., & Lin, Q. (2019). MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway Retraction in /10.3892/ijmm.2022.5134. International Journal of Molecular Medicine, 44, 346-356. https://doi.org/10.3892/ijmm.2019.4203
MLA
Tan, X., Jiang, L., Wu, X., Feng, W., Lin, Q."MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway Retraction in /10.3892/ijmm.2022.5134". International Journal of Molecular Medicine 44.1 (2019): 346-356.
Chicago
Tan, X., Jiang, L., Wu, X., Feng, W., Lin, Q."MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway Retraction in /10.3892/ijmm.2022.5134". International Journal of Molecular Medicine 44, no. 1 (2019): 346-356. https://doi.org/10.3892/ijmm.2019.4203