MicroRNA-664 targets paired box protein 6 to inhibit the oncogenicity of pancreatic ductal adenocarcinoma

Retraction in: /10.3892/ijo.2022.5380

  • Authors:
    • Qi Wang
    • Jiaqi Wang
    • Songtao Niu
    • Songsong Wang
    • Yibin Liu
    • Xiaoya Wang
  • View Affiliations

  • Published online on: March 19, 2019     https://doi.org/10.3892/ijo.2019.4759
  • Pages: 1884-1896
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Abstract

The abnormal expression of microRNAs (miRNAs or miRs) with oncogenic or tumor‑suppressive roles in pancreatic ductal adenocarcinoma (PDAC) has been widely reported in recent years, and these dysregulated miRNAs are implicated in the formation and progression of PDAC. Therefore, an investigation into the functional roles of miRNAs in PDAC may facilitate the identification of effective therapeutic targets. miRNA‑664 (miR‑664) has been found to be aberrantly expressed and to play crucial roles in several human cancer types. However, the expression pattern and functional roles of miR‑664 in the malignant capacity of PDAC have yet to be elucidated. In this study, the results revealed that miR‑664 was clearly downregulated in PDAC tissues and cell lines. The low miR‑664 expression was strongly associated with pathological T stage and lymph node metastasis of the patients with PDAC. Patients with PDAC with a low miR‑664 expression had a poorer overall survival and a worse disease‑free survival than those patients with a high miR‑664 level. Functional experiments suggested that exogenous miR‑664 expression suppressed the growth and metastasis of PDAC cells in vitro, whereas miR‑664 downregulation exerted the opposite effects. In addition, miR‑664 suppressed the tumor growth of PDAC cells in vivo. Mechanistically, paired box protein 6 (PAX6) was identified as a direct target gene of miR‑664 in PDAC cells. Furthermore, PAX6 was upregulated in PDAC tissues, and its upregulation inversely correlated with miR‑664 levels. Moreover, the silencing of PAX6 mimicked the effects of miR‑664 upregulation in PDAC cells, and the recovered expression of PAX6 eliminated the effects of miR‑664 on PDAC cells. Notably, miR‑664 could inhibit the activation of PI3K/Akt pathway in PDAC cells in vitro and in vivo. Cumulatively, these results indicate an important role of the miR‑664/PAX6 pathway in suppressing the aggressiveness of PDAC cells, suggesting that miR‑664 may be an attractive therapeutic target for the treatment of patients with this fatal disease.

Introduction

Pancreatic carcinoma, one of the most lethal solid tumors, is the fifth most common cancer and the second leading cause of cancer-associated mortality worldwide (1). Pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer, accounts for approximately 90% of all pancreatic carcinoma cases (2). The majority of patients with PDAC with locally advanced or metastatic disease are diagnosed at the time of presentation, as this disease usually causes no symptoms at its early stage (3,4). Although substantial developments have been achieved in surgery, chemotherapy and radiotherapy, the overall prognosis of patients with advanced PDAC remains very poor, with a 5-year survival rate of <7% and a median survival time of only 6 months (5,6). More than half of patients with PDAC experience tumor recurrence or metastasis even after surgical resection (7); however, little is known concerning the reasons for the aggressive behavior of PDAC. Therefore, a better understanding of PDAC occurrence and development may lead to the development of novel therapeutic techniques for improving the prognosis of patients with PDAC.

MicroRNAs (miRNAs or miRs) are a group of noncoding, evolutionarily conserved, and short RNA molecules of 21-25 nucleotides in length (8). miRNAs downregulate protein expression by translational inhibition or by mRNA degradation through a direct interaction with the 3′-untranslated regions (3′-UTRs) of their target genes in a base-pairing manner (9). One particular miRNA can regulate the expression of multiple human genes simultaneously; therefore, miRNAs play crucial roles in biological and pathological processes, including cell differentiation, proliferation, cycle, apoptosis, metastasis, metabolism and immune response (10-12). Increasing evidence strongly indicates that the altered expression of miRNAs is a common and important feature of human malignant tumors (13-15). In recent years, a variety of miRNAs have been shown to be aberrantly expressed in PDAC, such as miR-155 (16), miR-184 (17), miR-454 (18) and miR-506 (19). These dysregulated miRNAs participate in the carcinogenesis and progression of PDAC by functioning as oncogenes or tumor suppressors (20-22). Thus, further investigation of the functional roles of miRNAs in PDAC is likely to provide novel and effective therapeutic targets for patients with this lethal disease.

Several studies have revealed that miR-664 is aberrantly expressed and plays crucial roles in several human cancer types (23-26). However, the expression pattern and functional roles of miR-664 in the malignant capacity of PDAC have yet to be elucidated. To the best of our knowledge, this study is the first to demonstrate that PAX6 is a direct target of miR-664 and that miR-664 inhibits the aggressive phenotypes by targeting PAX6 in PDAC cells. Our findings provide a novel target for the therapy of patients with PDAC.

Materials and methods

Ethics statement

The current study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) and was conducted according to The Declaration of Helsinki. In addition, written informed consent was obtained from all patients participating in this research.

Tissue samples

A total of 49 patients with PDAC, who underwent surgical resection from June, 2012 to August, 2017 at The First Affiliated Hospital of Zhengzhou University, were enrolled in this study. None of these patients had been treated with chemotherapy, radiotherapy or other anticancer therapy. Specimens of PDAC and corresponding adjacent normal tissues were obtained from these patients, frozen in liquid nitrogen, and stored at −80°C.

Cell lines

Four human PDAC cell lines (Aspc-1, Bxpc-3, Panc-1 and SW1990) and a normal human pancreatic cell line, HPDE6c7, were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone/GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cell cultures were maintained at 37°C in a humidified incubator containing 5% CO2.

Cell transfection

miR-664 mimics, negative control miRNA mimics (miR-NC), miR-664 inhibitor and negative control inhibitor (NC inhibitor) were chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). To knockdown PAX6 expression, a small interfering RNA (siRNA) against PAX6 (si-PAX6) and a negative control siRNA (si-NC) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The si-PAX6 sequence was 5′-GUAGGUAUCAUAACUCCGCCCA UTT-3′ and the si-NC sequence was 5′-UUCUCCGAACGUGU CACGUTT-3′.To restore PAX6 expression, the PAX6 overexpression plasmid, pcDNA3.1-PAX6, and the empty pcDNA3.1 plasmid were generated by GeneCopoeia Co. Ltd. (Guangzhou, China). Prior to transfection, 6×105 cells were plated into 6-well plates until a cell density of 70-80% was achieved. Cell transfection was performed using Lipofectamine™ 2000 (Invitrogen/Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. At 6 h post-transfection, the cells were washed with phosphate-buffered saline (PBS; Gibco/Thermo Fisher Scientific, Inc.), and fresh DMEM containing 10% FBS was added to each well.

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

Total RNA was isolated from the patient tissues and cultured cells using TRIzol® regent (Invitrogen/Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. For the quantification of miR-664 expression, complementary DNA (cDNA) was synthesized using the miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). The temperature protocols for reverse transcription were as follows: 37°C for 60 min, 95°C for 5 min and kept at 4°C. Subsequently, the Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific, Inc.) was used to carry out quantitative PCR with the miScript SYBR-Green PCR kit (Qiagen GmbH). The thermocycling conditions were as follows: 95°C for 2 min, 95°C for 10 sec, 55°C for 30 sec and 72°C for 30 sec, for 40 cycles. To detect PAX6 mRNA expression, cDNA synthesis was performed with the PrimeScript RT reagent kit, and the synthesized cDNA was subjected to PCR using the SYBR Premix Ex Taq (both from Takara Biotechnology, Inc., Dalian, China). The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec. The thermocycling conditions for qPCR were performed with cycling conditions as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 small nuclear RNA and β-actin were used as internal references for miR-664 and PAX6 mRNA expression, respectively. All reactions were performed in triplicate and repeated 3 times under similar conditions. All data were calculated using the 2−∆∆Cq method (27). The primers were designed as follows: miR-664, 5′-TACAACACCGGTCACTAACGCATTG-3′ (forward) and 5′-GTATCACCTCCTCCAGCAACTAACA-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); PAX6, 5′-AGACACAGCCCTCACAAAC-3′ (forward) and 5′-ATCATAACTCCGCCCATTC-3′ (reverse); and β-actin, 5′-CAGGGCGTGATGGTGGGCA-3′ (forward) and 5′-CAA ACATCATCTGGGTCATCTTCTC-3′ (reverse).

Cell counting kit-8 (CCK-8) and colony formation assays

The transfected cells were collected after 24 h of incubation at 37°C and seeded, in triplicate, in 96-well plates at an initial density of 3×103 cells/well. Following culture at 37°C for 0, 24, 48 and 72 h, the CCK-8 assay (Dojindo, Kumamoto, Japan) was performed to evaluate cellular proliferation. At each time point, the culture medium was exchanged for 100 µl of medium and 10 µl of CCK-8 solution. Following 2 h of incubation at 37°C with 5% CO2, the optical density (OD) value of each well was read at 450 nm using an EnSpire™ 2300 Multilabel Reader (PerkinElmer, Inc., Waltham, MA, USA).

For the colony formation assays, the transfected cells were inoculated in 6-well plates at 1,000 cells/well and cultured at 37°C, in the presence of 5% CO2, for 14 days. On day 15, the colonies were fixed with 4% paraformaldehyde and stained with methyl violet (Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 30 min. The number of colonies was counted under an Olympus light microscope (Olympus IX83; Olympus Corporation, Tokyo, Japan).

Flow cytometric analysis of apoptotic cells

Following transfection for 48 h, the cells were harvested and washed 3 times with ice-cold PBS. Thereafter, an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Biolegend, San Diego, CA, USA) was used to determine the apoptotic rate. Briefly, Annexin V-FITC and propidium iodide were added to a 100-µl cell suspension in binding buffer. Cells were incubated at room temperature in darkness for 15 min and, then, analyzed using a flow cytometer (FACScan™; BD Biosciences, Franklin Lakes, NJ, USA) within 1 h of staining. Data analysis was performed using the CellQuest software version 5.1 (BD Biosciences).

In vitro Transwell assays

Cell migration and invasion were addressed using Transwell chambers (24-well insert; 8 µm pore size; Corning Inc., Corning, NY, USA). For invasion assays, the chambers were coated with Matrigel (BD Biosciences). A total of 5×104 transfected cells suspended in 200 µl FBS-free medium were plated in the top compartments of the Transwell chambers. Meanwhile, the lower compartments were covered with 500 µl culture medium containing 20% FBS as a chemoattractant. Following culture at 37°C for 24 h, cells remaining on the top surface of the Transwell chambers were gently wiped off using a cotton swab. Cells that had migrated into or invaded the lower surface of the chambers were fixed with 4% paraformaldehyde at room temperature for 30 min, stained with 0.05% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 30 min, and washed thrice with PBS. Five random fields were selected for quantification under an Olympus light microscope. The average number of migrated or invading cells counted in 5 random fields was used as the final result.

In vivo xenograft tumor model

A total of 8 BALB/c nude mice, 4-6 weeks old (weighing 20 g), were purchased from Beijing Vital River Laboratory (Beijing, China) and were maintained under pathogen-free conditions (25°C, 50% humidity, 10-h light/14-h dark cycle). Cells transfected with miR-664 mimics or miR-NC were injected subcutaneously into the rear flanks of nude mice (n=4 for each group). Two weeks after the injection, the volume of the xenografts was calculated using the following formula: Volume = 1/2 (length x width2). All nude mice were sacrificed at the study endpoint (4 weeks after injection). The xenografts were excised and weighed. All the in vivo experiments were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and were performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Bioinformatics analysis

The putative targets of miR-664 were predicted using TargetScan (http://www.targetscan.org/vert_71/) and microRNA (http://www.microrna.org/microrna/home.do).

Luciferase reporter assay

The 3′-UTR fragments of PAX6 with a wild-type (wt) or mutant (mut) miR-664 binding sites were chemically synthesized by Shanghai GenePharma Co., Ltd. The fragments were cloned into a pMIR-REPORT™ Luciferase plasmid (Promega Corporation, Madison, WI, USA) to generate the luciferase reporter plasmids pMIR-wt-PAX6-3′-UTR and pMIR-mut-PAX6-3′-UTR. One day prior to transfection, cells were inoculated into 24-well plates at a density of 1×105 cells/well. Luciferase reporter plasmids along with either miR-664 mimics or miR-664 inhibitor were co-transfected into cells using Lipofectamine™ 2000, according to the manufacturer’s instructions. Luciferase activity was measured at 48 h following transfection, at 37°C, using a Dual-Luciferase® Reporter Assay System (Promega Corporation) according to the manufacturer’s instructions. Renilla luciferase activity was used for normalization of the Firefly luciferase activity.

Western blot analysis

Cultured cells or tissue specimens were lysed using a ProteoPrep® Total Extraction Sample kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). The concentration of total protein was determined using an Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were loaded, separated by 10% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology). Subsequently, the membranes were blocked in 5% non-fat milk powder, at room temperature for 2 h, and then incubated at 4°C overnight with the following primary antibodies: Rabbit anti-human monoclonal PAX6, rabbit anti-human monoclonal p-PI3K, mouse anti-human monoclonal PI3K, mouse anti-human monoclonal GAPDH (cat. nos. ab109233, ab182651, ab86714 and ab9484, respectively; Abcam, Cambridge, UK), mouse anti-human monoclonal p-Akt, and mouse anti-human monoclonal Akt (cat. no. sc-81433 and sc-56878, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. All these primary antibodies were used at a 1:1,000 dilution. Following 3 washes, the membranes were further incubated with horseradish peroxidase-conjugated immunoglobulin G goat anti-mouse (cat. no. ab205719; 1:5,000 dilution; Abcam) or goat anti-rabbit (cat. no. ab205718; 1:5,000 dilution) (both from Abcam) secondary antibodies at room temperature for 2 h. The detection of specific protein bands was performed with an ECL detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). The density of the protein signals was quantified using Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and normalized to that of GAPDH.

Statistical analysis

All experimental data are presented as the means ± standard deviation. A Student’s t-test was carried out for two-group comparisons, while one-way analysis of variance followed by Tukey’s post-hoc test was used for multiple group comparisons. The Chi-square test was used to investigate the association between miR-664 and the clinicopathological characteristics of the patients with PDAC. The correlation between miR-664 and PAX6 expression in the PDAC tissues was assessed using the Spearman’s correlation analysis. Kaplan-Meier analysis was utilized to estimate the survival rate. The log-rank test was used to determine the association between miR-664 expression and overall survival as well as disease-free survival. All functional assays were repeated at least 3 times to improve accuracy. All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 16.0 (SPSS, Inc., Chicago, IL, USA), and a P-value inferior to 0.05 was considered to indicate a statistically significant difference.

Results

miR-664 is frequently downregulated in PDAC and is associated with a poor prognosis of patients with PDAC

The expression of miR-664 in 49 pairs of PDAC tissues and corresponding adjacent normal tissues was detected by RT-qPCR. The results revealed that the expression of miR-664 was decreased in PDAC tissues relative to that in the corresponding adjacent normal tissues (P<0.05; Fig. 1A). In addition, the expression level of miR-664 was explored in 4 human PDAC cell lines, Aspc-1, Bxpc-3, Panc-1 and SW1990. A normal human pancreatic cell line, HPDE6c7, was used as a control. Consistently, miR-664 was downregulated in all 4 PDAC cell lines tested in comparison with the HPDE6c7 cells (P<0.05; Fig. 1B).

To evaluate the clinical significance of miR-664 in PDAC, all patients with PDAC were divided into either the miR-664-low or miR-664-high expression groups, with the median value of miR-664 as the cut-off. As shown in Table I, the low expression of miR-664 was notably associated with pathological T stage (P=0.015) and lymph node metastasis (P=0.030) in the patients with PDAC. However, there was no association between miR-664 and other characteristics, such as age, sex, maximum tumor diameter and differentiation (all P>0.05; Table I). In addition, patients with PDAC with a low miR-664 expression exhibited a poorer overall survival (P=0.047; Fig. 1C) and a worse disease-free survival (P=0.0005; Fig. 1D) than those patients with a high miR-664 expression. Thus, we proposed that miR-664 may be closely related with the development of PDAC.

Table I

Association between miR-664 expression levels and the clinical characteristics of patients with PDAC.

Table I

Association between miR-664 expression levels and the clinical characteristics of patients with PDAC.

CharacteristicsmiR-664 expression status
P-value
LowHigh
Age (years)0.656
 <601210
 ≥601314
Sex0.686
 Male1715
 Female89
Maximum tumor diameter (cm)0.644
 <4119
 ≥41415
Differentiation0.322
 Well and moderate1612
 Poor912
Pathological T stage0.015a
 T1+T2816
 T3+T4178
Lymph node metastasis0.030a
 Negative1017
 Positive157

a Indicates statistical significance (P<0.05).

miR-664 plays a suppressive role in PDAC cell growth and metastasis in vitro

Among the 4 PDAC cell lines tested, the Panc-1 and SW1990 cells were selected for use in subsequent functional experiments as they expressed relatively lower and higher levels of miR-664, respectively. To examine whether miR-664 plays a tumor-suppressive role in PDAC progression, gain- and loss-of-function assays were performed in the Panc-1 and SW1990 cells. The Panc-1 cells were transfected with miR-664 mimics or miR-NC, whereas the SW1990 cells were transfected with miR-664 inhibitor or NC inhibitor. RT-qPCR analysis indicated that transfection with miR-664 mimics markedly increased the expression levels of miR-664 in the Panc-1 cells, whereas the miR-664 inhibitor reduced miR-664 expression in the SW1990 cells (P<0.05; Fig. 2A). CCK-8 and colony formation assays were employed to examine the effects of miR-664 on the proliferative and colony-forming abilities of the PDAC cells. The results revealed that the upregulation of miR-664 restricted the proliferation and colony formation of the Panc-1 cells, whereas miR-664 downregulation exerted the opposite effect on the SW1990 cells (P<0.05; Fig. 2B and C). To examine whether the change in proliferation was due to cell apoptosis, we further investigated the effects of miR-664 on PDAC cell apoptosis. Compared with the respective controls, transfection with miR-664 mimics clearly enhanced the apoptosis of the Panc-1 cells, and the apoptosis of the SW1990 cells was reduced following transfection with miR-664 inhibitor (P<0.05; Fig. 2D). Furthermore, the effects of miR-664 on PDAC cell metastasis were determined using in vitro Transwell assays. The ectopic expression of miR-664 suppressed the migration and invasion of the Panc-1 cells, whereas miR-664 inhibition accelerated the migration and invasion of the SW1990 cells in vitro (P<0.05; Fig. 2E and F). These data suggested that miR-664 suppresses the progression and development of PDAC.

PAX6 is a direct target of miR-664 in PDAC cells

To elucidate the mechanisms underlying the carcinostatic activity of miR-664 in PDAC cells, bioinformatics analysis was performed to predict miR-664 putative targets. As shown in Fig. 3A, PAX6 harbors a potential miR-664 binding site. PAX6 was selected for further investigation as this well-known oncogene has been well documented to be involved in the genesis and development of PDAC (28,29). A luciferase reporter assay was applied to determine whether the 3′-UTR of PAX6 is directly targeted by miR-664 in PDAC cells. The data revealed that miR-664 overexpression decreased the luciferase activity when this reporter plasmid included the wt 3′-UTR in Panc-1 cells (P<0.05). However, the downregulation of miR-664 notably increased the luciferase activity of the plasmid harboring the wt 3′-UTR in SW1990 cells (P<0.05). Of note, the luciferase activity was not altered when the seed region of miR-664 in the 3′-UTR of PAX6 was mutated (Fig. 3B). Furthermore, the mRNA and protein expression levels of PAX6 were downregulated by the overexpression of miR-664 in the Panc-1 cells (P<0.05; Fig. 3C and D, respectively), whereas the expression of PAX6 was increased when miR-664 was knocked down in the SW1990 cells (P<0.05; Fig. 3C and D, respectively), as demonstrated by RT-qPCR and western blot analysis. Thus, PAX6 is a direct target of miR-664 in PDAC cells.

Expression level of miR-664 inversely correlates with that of PAX6 in patients with PDAC

PAX6 expression in PDAC tissues and corresponding adjacent normal tissues from 49 patients with PDAC was measured by RT-qPCR and western blot analysis. RT-qPCR analysis revealed a marked upregulation of PAX6 mRNA in the PDAC tissues compared with that in adjacent normal tissues (P<0.05; Fig. 4A). Additionally, PAX6 protein was notably overexpressed in PDAC tissues in comparison with adjacent normal tissues (P<0.05; Fig. 4B and C). Furthermore, an evidently negative correlation was identified between the expression levels of miR-664 and PAX6 mRNA in PDAC tissues (r=−0.5298, P<0.0001; Fig. 4D).

PAX6 silencing and miR-664 upregulation exhibit similar effects in PDAC cells

Following the identification of PAX6 as a direct target gene of miR-664, we then attempted to explore the biological functions of PAX6 in PDAC cells. To this end, endogenous PAX6 expression was knocked down in both the Panc-1 and SW1990 cells with a specific siRNA against the expression of PAX6 (si-PAX6) (P<0.05; Fig. 5A). The results of CCK-8 and colony formation assays revealed that PAX6 silencing suppressed the proliferation and colony formation of the Panc-1 and SW1990 cells in vitro (P<0.05; Fig. 5B and C). Flow cytometric analysis verified that the downregulation of PAX6 significantly increased the percentage of apoptotic Panc-1 and SW1990 cells (P<0.05; Fig. 5D). Furthermore, the migration and invasion of the Panc-1 and SW1990 cells was also attenuated by PAX6 knockdown (P<0.05; Fig. 5E and F). These results demonstrated that PAX6 knockdown exerted similar effects to those induced by miR-664 overexpression, suggesting that PAX6 is a downstream target of miR-664 in PDAC cells.

PAX6 is required for miR-664-associated phenotypes in PDAC cells

A series of rescue experiments were carried out to explore the role of PAX6 in miR-664-regulated PDAC progression. Panc-1 cells overexpressing miR-664 were transfected with the PAX6 overexpression plasmid pcDNA3.1-PAX6, while the SW1990 cells were co-transfected with si-PAX6 and miR-664 inhibitor. Western blot analysis was used to evaluate PAX6 protein expression in the rescue experiment. The protein expression level of PAX6 was notably upregulated in the Panc-1 and SW1990 cells following transfection with pcDNA3.1-PAX6 (P<0.05; Fig. 6A). In the Panc-1 cells, co-transfection with miR-664 mimics and pcDNA3.1-PAX6 restored PAX6 protein expression which had been decreased by miR-664 mimics (P<0.05; Fig. 6B). Similarly, the increased PAX6 protein level in the SW1990 cells induced by the miR-664 inhibitor was reduced following co-transfection with si-PAX6 (P<0.05; Fig. 6B). Functional experiments revealed that the proliferation (P<0.05; Fig. 6C), colony formation (P<0.05; Fig. 6D), apoptosis (P<0.05; Fig. 6E), migration (P<0.05; Fig. 6F), and invasion (P<0.05; Fig. 6G) of the Panc-1 and SW1990 cells, affected by the gain and loss of miR-664, was markedly ‘rescued’ by the restoration of PAX6 expression. The proliferation, colony formation, migration and invasion of the cells was restored, and cell apoptosis was decreased following the restoration of PAX6 expression. Accordingly, these results suggest that miR-664 may exert its anticancer effects in PDAC, at least partly, via the direct regulation of PAX6 expression.

miR-664 inactivates the PI3K/Akt pathway in PDAC cells

Previous studies have reported that the PI3K/Akt pathway is negatively regulated by PAX6 (30-32). Therefore, we hypothesized that miR-664 may be involved in the regulation of the PI3K/Akt pathway in PDAC cells by inhibiting PAX6 expression. Hence, the Panc-1 cells were co-transfected with miR-664 mimics and pcDNA3.1-PAX6 or pcDNA3.1, while miR-664 inhibitor in combination with si-PAX6 or si-NC was introduced into the SW1990 cells. Western blot analysis revealed that p-PI3K and p-Akt protein expression in the Panc-1 cells was inhibited by miR-664 overexpression and increased by miR-664 downregulation in the SW1990 cells. In addition, the change in miR-664 expression did not affect the total expression of PI3K and Akt protein in either the Panc-1 or the SW1990 cells. Furthermore, the re-introduction of PAX6 and the silencing of PAX6 abolished the regulatory effects of miR-664 on the p-PI3K and p-Akt protein levels in the Panc-1 and SW1990 cells, respectively (Fig. 7). These results revealed that miR-664 inhibits the activation of the PI3K/Akt pathway in the PDAC cells via the inhibition of PAX6.

miR-664 inhibits tumor growth of PDAC cells in vivo

The function of miR-664 in the in vivo tumor growth of PDAC cells was analyzed using an in vivo xenograft model. Panc-1 cells transfected with miR-664 mimics or miR-NC were injected subcutaneously into the rear flanks of nude mice. The volume (P<0.05; Fig. 8A and B) and weight (P<0.05; Fig. 8C) of the xenograft were significantly reduced in the miR-664 mimics group when compared with those in the miR-NC group. RT-qPCR was further performed to detect miR-664 expression in the xenografts and to confirm that miR-664 overexpression was responsible for the in vivo tumor growth inhibition of PDAC. As expected, miR-664 was still upregulated in the xenografts derived from the miR-664 mimics group (P<0.05; Fig. 8D). Furthermore, western blot analysis indicated that the tumor xenografts arising from the miR-664 mimics-transfected Panc-1 cells exhibited decreased PAX6, p-PI3K and p-AKt protein expression levels (Fig. 8E). Therefore, the upregulation of miR-664 hindered the tumor growth of PDAC cells in vivo by inhibiting PAX6 expression and deactivating the PI3K/Akt pathway.

Discussion

In recent years, the abnormal expression of miRNAs with oncogenic or tumor-suppressive roles in PDAC has been widely reported in a variety of studies, and these dysregulated miRNAs have been implicated in the formation and progression of PDAC (33-35). The analysis of miRNA expression in clinical follow-up samples has provided novel insight into the identification of attractive prognostic biomarkers (36). Therefore, an investigation into the functional roles of miRNAs in PDAC may promote the development of promising therapeutic targets for managing patients with this aggressive malignant tumor. The present study measured, for the first time, to the best of our knowledge miR-664 expression in PDAC, evaluated its clinical significance, and explored its effects on PDAC progression. Additionally, the molecular mechanisms underlying the regulatory roles of miR-664 in PDAC progression were examined.

The expression level of miR-664 is decreased in cutaneous malignant melanoma tissues and cell lines. Patients with cutaneous malignant melanoma with a low miR-664 expression exhibit a shorter overall survival period than those patients with high miR-664 expression (23). miR-664 expression has been found to be downregulated in cervical cancer, and its downregulation has been shown to be associated with lymphatic invasion, distant metastasis, the International Federation of Gynecology and Obstetrics (FIGO) stage, and histological grade, as well as with a shorter overall survival of patients with cervical cancer (24). Multivariate analysis also validated miR-664 expression as an independent biomarker for predicting the overall survival in these patients (24). miR-664 has also been shown to be expressed at low levels in colorectal (25) and breast (26) cancers. On the contrary, miR-664 has been shown to be upregulated in T-cell acute lymphoblastic leukemia (37), osteosarcoma (38,39) and lung cancer (40). These inconsistent observations arouse our interest to illustrate the expression pattern of miR-664 in PDAC. In this study, RT-qPCR analysis indicated that miR-664 was expressed at low levels in PDAC tissues and cell lines. The low expression of miR-664 was significantly associated with pathological T stage and lymph node metastasis of patients with PDAC. Patients with PDAC harboring a low miR-664 level exhibited a poorer overall survival and a worse disease-free survival than those patients with a high miR-664 level. These findings suggest that the expression status of miR-664 exhibits tissue specificity and that miR-664 may be an effective biomarker for the diagnosis and prognosis of patients with PDAC.

miR-664 targets proteolipid protein 2, functioning as a tumor-suppressive miRNA in cutaneous malignant melanoma by regulating cell proliferation, anchorage-independent growth, cell cycle arrest in vitro, and tumor growth in vivo (23). The upregulation of miR-664 blocks the migratory capacity and improves chemosensitivity to cisplatin in cervical cancer cells (41). In breast cancer, the ectopic expression of miR-664 suppresses cell proliferation and invasion via blockade of insulin receptor substrate 1 (26). Conversely, miR-664 was identified as an oncogene in osteosarcoma (38,39), lung cancer (40) and T-cell acute lymphoblastic leukemia (37). However, whether miR-664 is involved in the malignant phenotypes of PDAC cells remains unknown. Herein, we demonstrated that the exogenous expression of miR-664 inhibited PDAC cell growth and metastasis in vitro and attenuated tumor growth in vivo, while miR-664 inhibition exerted the opposite effects. Collectively, these findings suggest that miR-664 may serve as a valuable target for the anticancer therapy of patients with the abovementioned cancer types.

It is well known that miRNAs can contribute to the carcinogenesis and cancer progression by regulating the expression of their targets (42). PAX6, a member of the PAX gene family (43), was identified as a direct target gene of miR-664 in PDAC cells. It is a highly conserved transcription factor and was observed to be upregulated in multiple human cancer types (30,44-47). PAX6 was also overexpressed in PDAC. The high expression of PAX6 is closely related with PDAC progression and development, and it has been described to affect a multitude of biological behaviors, including cell growth, cycle status, differentiation and metastasis (28,29).

Previous studies have reported that the PAX6 is implicated in the regulation of the PI3K/Akt pathway (30-32), which is a core regulator of cell metabolism, differentiation, growth and survival, and is implicated in the tumorigenesis and development of PDAC (48,49). This study revealed that PAX6 silencing was able to inhibit the aggressive phenotypes of PDAC cells, and the downregulation of PAX6 was essential for the tumor-suppressive role of miR-664. Considering the important roles of PAX6 and miR-664 in PDAC, miR-664 restoration and PAX6 inhibition may be potential therapeutic approaches for patients with PDAC.

Overall, the present study demonstrated that miR-664 is an important tumor suppressor in PDAC progression. The upregulation of miR-664 suppresses the malignant behaviors of PDAC cells in vitro and in vivo by directly targeting PAX6 and inactivating PI3K/Akt pathway, while miR-664 downregulation promotes these behaviors. Based on these results, we propose that the miR-664/PAX6 pathway has potential therapeutic applications in the antineoplastic therapy of patients with PDAC.

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

QW, XW and JW designed this study, and performed RT-qPCR, CCK-8, and colony formation assays. Flow cytometric analysis and luciferase reporter assay was conducted by SN. SW and YL carried out the in vitro Transwell assays, in vivo xenograft tumor model analysis and western blot analysis. All authors have read and approved the final draft.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University, and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of The First Affiliated Hospital of Zhengzhou University. In addition, written informed consent was obtained from all patients participating in this research. In addition, all the in vivo animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University and were performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of The First Affiliated Hospital of Zhengzhou University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

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May-2019
Volume 54 Issue 5

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Wang Q, Wang J, Niu S, Wang S, Liu Y and Wang X: MicroRNA-664 targets paired box protein 6 to inhibit the oncogenicity of pancreatic ductal adenocarcinoma Retraction in /10.3892/ijo.2022.5380. Int J Oncol 54: 1884-1896, 2019
APA
Wang, Q., Wang, J., Niu, S., Wang, S., Liu, Y., & Wang, X. (2019). MicroRNA-664 targets paired box protein 6 to inhibit the oncogenicity of pancreatic ductal adenocarcinoma Retraction in /10.3892/ijo.2022.5380. International Journal of Oncology, 54, 1884-1896. https://doi.org/10.3892/ijo.2019.4759
MLA
Wang, Q., Wang, J., Niu, S., Wang, S., Liu, Y., Wang, X."MicroRNA-664 targets paired box protein 6 to inhibit the oncogenicity of pancreatic ductal adenocarcinoma Retraction in /10.3892/ijo.2022.5380". International Journal of Oncology 54.5 (2019): 1884-1896.
Chicago
Wang, Q., Wang, J., Niu, S., Wang, S., Liu, Y., Wang, X."MicroRNA-664 targets paired box protein 6 to inhibit the oncogenicity of pancreatic ductal adenocarcinoma Retraction in /10.3892/ijo.2022.5380". International Journal of Oncology 54, no. 5 (2019): 1884-1896. https://doi.org/10.3892/ijo.2019.4759