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Introduction

Renal cell carcinoma (RCC), the most common type of human kidney cancer, is the most lethal urological malignancy worldwide (1). In total, 295,000 novel cases of RCC and 134,000 mortalities due to RCC are estimated globally every year (2). RCC may be divided into three histological subtypes: Clear cell RCC (ccRCC), papillary RCC and chromophobe RCC (3); these subtypes exhibit distinct structural and cytogenetic characteristics (4). ccRCC, the most aggressive and frequent subtype, accounts for ~80% of all diagnosed RCC cases (5). Radical nephrectomy is the primary therapeutic method used in patients with ccRCC who have been diagnosed at an early stage, whereas, patients with local or distant metastasis are not suitable for surgery (6,7). In addition, patients with ccRCC benefit minimally from chemotherapy and radiotherapy treatments (8). Despite significant progress in therapeutic strategies, the long-term prognosis of patients with metastatic ccRCC remains poor, with a median survival period of 1.5 years (9). Therefore, it is urgent to elucidate the molecular mechanism underlying ccRCC occurrence and progression, in order to identify novel therapeutic targets for the improvement of prognosis in patients with this malignancy.

MicroRNAs (miRNAs) are a group of endogenous, single stranded and non-coding RNA molecules with a length of 19–24 nucleotides (10). Mature miRNAs mediate gene expression by inducing mRNA degradation and/or translation inhibition by binding to the complementary sequences in the 3′-untranslated regions (3′-UTRs) of their target genes (11). Over one-half of the characterized miRNAs are located in genomic regions associated with oncogenes, suggesting that miRNAs may serve important roles in carcinogenesis and cancer progression (12). An increasing number of previous studies have demonstrated that miRNAs are dysregulated in approximately all types of human malignancy, including ccRCC (13–15). miRNA (miR)-19a (16), miR-224 (17), miR-1274a (18) and miR-543 (19) were upregulated in ccRCC, whereas, miR-30a (20), miR-200a (21), miR-384 (22) and miR-411 (23) were downregulated. These aberrantly expressed miRNAs are implicated in the oncogenesis and progression of ccRCC, and regulate various crucial biological processes, including cell proliferation, apoptosis, metastasis and angiogenesis (13,24). Further investigation on the miRNAs in ccRCC may provide valuable therapeutic strategies for treating patients with this aggressive disease.

miR-663 was upregulated in nasopharyngeal carcinoma (25,26), lung cancer (27) and castration-resistant prostate cancer (28); however, it was downregulated in papillary thyroid carcinoma (29), pancreatic cancer (30), glioblastoma (31) and gastric cancer (31). However, little is known regarding the expression pattern and roles of miR-663 in ccRCC. The present study investigated miR-663 expression in ccRCC and examined its effects on ccRCC progression. Additionally, the mechanisms underlying the action of miR-663 in ccRCC were investigated.

p21 activated kinase 4 (PAK4) is a member of the serine/threonine protein kinase family. PAK4 is located at the locus 19q13.2 and it was described as a downstream effector of Ras-related C3 botulinum toxin substrate 1 and cell division control protein 42 homolog (32). PAK4 is upregulated in multiple human cancer types, including colorectal cancer (33), non-small cell lung cancer (34), glioblastoma (35) and gastric cancer (36). PAK4 was implicated in the carcinogenesis and cancer progression by affecting numerous biological processes, including cell proliferation, apoptosis, metastasis and cytoskeletal organisation (37–39). Notably, it is highly expressed in ccRCC, and serves crucial roles in the initiation and development of ccRCC (38,40–42). Therefore, inhibition of PAK4 may represent a potential therapeutic approach to treat patients with ccRCC.

Materials and methods

Tissue collection

A total of 31 ccRCC tissues and their matched normal renal tissues were collected from patients at The China-Japan Union Hospital of Jilin University (Changchun, China) between June 2015 and March 2017. Patients that had received chemotherapy, radiotherapy or other treatments prior to surgical resection were excluded from this research. The patients (18 males and 13 females; age range, 48–71 years) received surgical resection, and the fresh tissues were quickly frozen in liquid nitrogen and subsequently stored at −80°C. The present study was approved by the Research Ethics Committee of The China-Japan Union Hospital, and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of The China-Japan Union Hospital. Written informed consent was obtained from all the patients for the use of their clinical tissues.

Cell culture

In total, one normal renal cell line (HK-2) and three human ccRCC cell lines (Caki-1, 786-O and A498) were purchased from The American Type Culture Collection (Manassas, VA, USA). HK-2 cells were maintained in keratinocyte serum-free growth medium containing bovine pituitary extract (0.05 mg/ml) and human recombinant epidermal growth factor (5 ng/ml) (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The ccRCC cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 100 µl/ml penicillin and 100 mg/ml streptomycin sulphates (all from Gibco; Thermo Fisher Scientific, Inc.). All cells were incubated at 37°C in a humidified incubator containing 5% CO2.

Transfection assay

Chemically synthesized human miR-663 mimics and negative control miRNA mimics (miR-NC) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The PAK4 overexpression plasmid pcDNA3.1-PAK4 and empty pcDNA3.1 plasmid were provided by The Chinese Academy of Sciences (Changchun, China). A total of 5×105 786-O and A498 cells were seeded into 6-well culture plates and transfected with miRNA mimics (100 pmol) or plasmid (4 µg) employing Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The miR-663 mimics sequence was 5′-AGGCGGGGCGCCGCGGGACCGC-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The transfection efficiency was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Transfected cells were harvested and used for the subsequent functional assays. RT-qPCR and cell invasion assay was performed at 48 h post-transfection. Cell Counting Kit-8 (CCK-8) assays and western blot analysis were performed after 24 and 72 h of incubation, respectively.

Extraction of total RNA and RT-qPCR

An RT-qPCR assay was conducted to measure miR-663 and PAK4 mRNA expression levels in ccRCC tissues and cells. Total RNA was isolated from tissues or cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For miR-663 detection, total RNA was subjected to RT reactions using a TaqMan miRNA 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 amplified by qPCR using a TaqMan miRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR was performed using the following thermocycling conditions: 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 1 min.

To quantify PAK4 mRNA expression, cDNA was synthesised using the Moloney Murine Leukemia Virus RT system (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Subsequently, a SYBR-Green PCR master mix (Takara Bio, Inc., Otsu, Japan) was used to perform qPCR. The thermocycling conditions for qPCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. U6 small nuclear RNA and GAPDH were used as internal reference genes for miR-663 and PAK4 mRNA, respectively. RT-qPCR analysis was performed in triplicate, and the data were analyzed based on the 2−ΔΔCq method (43). The primers were: miR-663, 5′-TGCGGAGGCGGGGCGCCGCGGG-3′ (forward) and 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); PAK4, 5′-AGGGAAGGCGGGAGATGAG-3′ (forward) and 5′-TCAGTTGCTTGTTCGTGC-3′ (reverse); and GAPDH, 5′-GAAGGCGGGAGATGAG-3′ (forward) and 5′-TCAGTTGCTTGTTCGTGC-3′ (reverse).

CCK-8 assay

Cells were harvested, resuspended and plated into 96-well plates at a density of 3×103 cells each well, 24 h following transfection. Cells were subsequently incubated at 37°C in a humidified incubator containing 5% CO2 for 0, 24, 48 and 72 h. At every time point, transfected cells were subjected to a CCK-8 assay to measure cell proliferation. In total, 10 µl CCK-8 solution was added into each well, followed by incubation at 37°C for 2 h. The optical density was determined at 450 nm on the BioTek™ ELx800™ spectrophotometer (BioTek China, Beijing, China).

Cell invasion assay

Cell invasive ability was assessed using Transwell chambers (8.0 µm pore size) that were precoated with Matrigel (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A total of 5×104 transfected cells suspended in 200 µl FBS-free DMEM was added to the upper compartments of the Transwell chambers, 48 h following transfection. The lower compartments of the chambers were filled with 500 µl DMEM containing 20% FBS. Following an incubation of 24 h, the non-invasive cells remaining on the upper membrane were gently removed using a cotton swab. The invasive cells were fixed with 95% ethanol at room temperature for 30 min and stained with 0.5% crystal violet at room temperature for 30 min. The number of invasive cells was counted in five randomly chosen visual fields using an inverted light microscope (IX83; Olympus Corporation, Tokyo, Japan; magnification, ×200).

miR-663 target prediction and luciferase reporter assay

A total of two publically available databases, TargetScan (http://www.targetscan.org/vert_71/) and microRNA (http://www.microrna.org/microrna/home.do), were used to predict the potential targets of miR-663. The 3′-UTR of PAK4 containing putative wild-type (Wt) miR-663-binding sites or mutant (Mut) miR-663-binding sites were purchased from Shanghai GenePharma Co., Ltd. and cloned into pmirGLO luciferase reporter vector (Promega Corporation). The Wt and Mut luciferase plasmids were defined as pmirGLO-Wt-PAK4-3′-UTR and pmirGLO-Mut-PAK4-3′-UTR, respectively. 786-O and A498 cells (1×105) were inoculated into 24-well plates in triplicate one night prior to transfection. Cotransfection with pmirGLO-Wt-PAK4-3′-UTR or pmirGLO-Mut-PAK4-3′-UTR and miR-663 mimics or miR-NC into cells was conducted using Lipofectamine® 2000, according to the manufacturer's protocol. Luciferase activity was measured after 48 h of incubation using the Dual-Luciferase® Reporter assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Western blot analysis

Radioimmunoprecipitation Assay Lysis and Extraction Buffer (Thermo Fisher Scientific, Inc.) was used to extract total protein from tissues or cells. The concentration of total protein was measured using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). An equal amount of protein (30 µg/lane) was separated by 10% SDS-PAGE gels and subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h and incubated at 4°C overnight with primary antibodies against PAK4 (cat. no. ab62509; 1:500; Abcam, Cambridge, UK) or GAPDH (cat. no. ab181603; 1:500; Abcam). Following three washes with TBST, the membranes were incubated at room temperature with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; 1:5,000; Abcam) for 2 h. The protein signals were detected using an enhanced chemiluminescent Substrate kit (Pierce™; Thermo Fisher Scientific, Inc.). GAPDH served as the internal control to normalize PAK4 protein expression. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for densitometry.

Statistical analysis

All data are presented as the mean ± standard deviation from three independent experiments and were analyzed with SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). Student's t-test was used to evaluate the differences between two groups. One-way analysis of variance combined with Student-Newman-Keuls test as the post hoc test, was used to compare the difference between multiple groups. The correlation between expression levels of miR-663 and PAK4 mRNA was determined using Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-663 is downregulated in ccRCC tissues and cell lines

To investigate the expression pattern of miR-663 in ccRCC, miR-663 expression in 31 paired ccRCC tissues and corresponding matched normal renal tissues was measured by RT-qPCR. The miR-663 expression was significantly downregulated in ccRCC tissues compared with their matched normal renal tissues (Fig. 1A). Subsequently, miR-663 expression in three ccRCC cell lines (Caki-1, 786-O and A498) and one normal renal cell line (HK-2) was determined using RT-qPCR. The expression level of miR-663 was decreased in ccRCC cell lines compared with the HK-2 cell line (Fig. 1B). The results suggested that miR-663 expression was downregulated in ccRCC, and its expression level may be associated with the development of ccRCC.

Figure 1. miR-663 expression is decreased in ccRCC tissues and cell lines. (A) RT-qPCR analysis was used to detect relative miR-663 expression in 31 ccRCC tissues and in their matched normal renal tissues. *P<0.05. (B) miR-663 expression was determined in three ccRCC cell lines (Caki-1, 786-O and A498) and in one normal renal cell line (HK-2) by RT-qPCR. *P<0.05 vs. HK-2. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR-663, microRNA-663; ccRCC, clear cell renal cell carcinoma.

miR-663 restoration inhibits the proliferation and invasion of 786-O and A498 cells

Among the three ccRCC cell lines (Caki-1, 786-O and A498) investigated, 786-O and A498 cell lines expressed the lowest level of miR-663 and were selected for the following experiments. To investigate the roles of miR-663 in ccRCC, miR-663 mimics or miR-NC was transfected into 786-O and A498 cells. Following transfection, RT-qPCR analysis was used to evaluate transfection efficiency. miR-663 expression level was significantly increased in 786-O and A498 cells transfected with miR-663 mimics compared with the miR-NC (Fig. 2A).

Figure 2. miR-663 upregulation inhibits cell proliferation and invasion of 786-O and A498 cell lines. (A) 786-O and A498 cells were transfected with miR-663 mimics or miR-NC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to determine miR-663 expression levels 48 h following transfection. (B) Cell Counting Kit-8 assay was used to investigate the proliferation of 786-O and A498 cells following transfection with miR-663 mimics or miR-NC. (C) Cell invasion assay was conducted to evaluate the effect of miR-663 overexpression on the invasive ability of 786-O and A498 cells (magnification, ×200). *P<0.05 vs. respective miR-NC. miR-663, microRNA-663; miR-NC, negative control microRNA; OD, optical density.

A CCK-8 assay was performed to determine the effect of miR-663 overexpression on the proliferative ability of ccRCC cells. Upregulation of miR-663 led to a significant decrease in cell proliferation in 786-O and A498 cells at 48 and 72 h (Fig. 2B). The invasive ability of 786-O and A498 cells transfected with miR-663 mimics or miR-NC was determined by a cell invasion assay. As depicted in Fig. 2C, miR-663 overexpression suppressed the invasive ability of 786-O and A498 cells compared with cells transfected with miR-NC. The present results suggested that miR-663 may serve tumor-suppressive roles in ccRCC progression.

PAK4 is a direct target of miR-663 in ccRCC cells

A bioinformatics analysis was performed to predict the putative targets of miR-663 and to elucidate the mechanisms underlying the inhibitory effects of miR-663 in ccRCC. Among the candidates, PAK4 (Fig. 3A) was selected for further identification since it is known to serve crucial roles in the tumor formation and progression (38,40–42). To investigate whether PAK4 is a target of miR-663, a luciferase reporter assay was performed in 786-O and A498 cells following cotransfection of miR-663 mimics or miR-NC and pmirGLO-Wt-PAK4-3′-UTR or pmirGLO-Mut-PAK4-3′-UTR. miR-663 overexpression caused a significant decrease in luciferase activity in 786-O and A498 cells transfected with the plasmid carrying the Wt binding sites. However, the luciferase activity in cells transfected with the plasmid carrying the mutated binding sites was unaffected (Fig. 3B). To further investigate this hypothesis, the role of miR-663 in regulating endogenous PAK4 expression in ccRCC cells was examined. RT-qPCR and western blot analysis demonstrated that the mRNA (Fig. 3C) and protein (Fig. 3D) expression levels of PAK4 in 786-O and A498 cells transfected with miR-663 mimics were significantly decreased compared with the respective miR-NC. The present results demonstrated that PAK4 was a direct target gene of miR-663 in ccRCC cells.

Figure 3. miR-663 directly targets PAK4 in 786-O and A498 cells. (A) Putative Wt miR-663 targeting site in the 3′-UTR of PAK4 and the generated Mut 3′-UTR. (B) Luciferase activity in 786-O and A498 cells cotransfected with miR-663 mimics or miR-NC and luciferase plasmids containing Wt or Mut 3′-UTR of PAK4. PAK4 (C) mRNA expression level and (D) protein expression level in 786-O and A498 cells transfected with miR-663 mimics or miR-NC were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. *P<0.05 vs. respective miR-NC. miR-663, microRNA-663; miR-NC, negative control microRNA; Wt, wild-type; Mut, mutant; UTR, untranslated region; PAK4, p21 activated kinase 4.

miR-663 expression is negatively correlated with PAK4 expression in ccRCC tissues

To further test the regulatory effects of miR-663 on PAK4 in ccRCC, the mRNA expression level of PAK4 in 31 paired ccRCC tissues and their corresponding matched normal renal tissues was measured by RT-qPCR. The PAK4 mRNA expression level was significantly increased in ccRCC tissues compared with matched normal renal tissues (Fig. 4A). The correlation analysis for miR-663 and PAK4 demonstrated that the expression level of miR-663 was inversely correlated with PAK4 expression level in ccRCC tissues (Fig. 4B). Furthermore, the protein expression level of PAK4 was upregulated in ccRCC tissues compared with normal renal tissues (Fig. 4C).

Figure 4. miR-663 expression is inversely correlated with PAK4 expression in ccRCC tissues. (A) PAK4 mRNA expression level was significantly increased in ccRCC tissues compared with the corresponding matched normal renal tissues. *P<0.05. (B) Spearman's correlation analysis was used to assess the correlation between miR-663 and PAK4 mRNA expression levels in ccRCC tissues. r=−0.5359; P=0.0019. (C) Western blot analysis was used to determine PAK4 protein expression level in three cases of ccRCC tissues compared with their matched normal renal tissues. PAK4, p21 activated kinase 4; ccRCC, clear cell renal cell carcinoma; miR-663, microRNA-663.

PAK4 upregulation attenuates the tumor-suppressing roles of miR-663 in ccRCC cells

The PAK4 role in mediating the suppressive effects of miR-663 in ccRCC cells was investigated by performing rescue experiments. 786-O and A498 cells were cotransfected with miR-663 mimics and PAK4 overexpression plasmid or empty pcDNA3.1 plasmid, as the NC. The western blot analysis demonstrated that the PAK4 protein expression level in 786-O and A498 cells, which was decreased by miR-663 mimics, was re-established by cotransfection of PAK4 expression plasmid (Fig. 5A). Subsequent functional experiments demonstrated that PAK4 overexpression attenuated the inhibitory effects of miR-663 overexpression on the proliferative (Fig. 5B and C) and invasive ability (Fig. 5D) of 786-O and A498 cells. The present results suggested that miR-663 may serve as a tumor suppressor in ccRCC partly by downregulating PAK4 expression.

Figure 5. PAK4 restoration attenuates the effects of miR-663 on the proliferation and invasion of 786-O and A498 cells. (A) 786-O and A498 cells were cotransfected with miR-663 mimics and pcDNA3.1-PAK4 or pcDNA3.1 empty plasmid, and the protein expression level of PAK4 was detected at 72 h following transfection using western blot analysis. The proliferation of (B) 786-O and (C) A498 cells was evaluated by Cell Counting Kit-8 assays. The invasion of (D) 786-O and A498 cells following cotransfection with miR-663 mimics and pcDNA3.1-PAK4 or pcDNA3.1 was evaluated by cell invasion assays (magnification, ×200). *P<0.05 vs. miR-NC; #P<0.05 vs. miR-663 mimics+pcDNA3.1-PAK4. miR-663, microRNA-663; PAK4, p21 activated kinase 4; miR-NC, negative control microRNA; OD, optical density.

Discussion

Accumulating evidence has demonstrated that miRNAs are key gene regulators and are abnormally expressed in ccRCC (20,44,45). The dysregulation of miRNAs has been demonstrated to affect the initiation and progression of ccRCC (46,47). Therefore, identification of ccRCC-associated miRNAs may facilitate the determination of promising therapeutic targets for anti-cancer treatment. In the present study, the expression levels of miR-663 in ccRCC tissues and cell lines were significantly decreased compared with matched normal renal tissues and a normal renal cell line, respectively. In addition, miR-663 inhibited the proliferation and invasion of ccRCC cells. PAK4 was identified as a direct target gene of miR-663 in ccRCC cells. PAK4 expression was upregulated in ccRCC tissues, and its expression level was inversely correlated with miR-663 expression level. Furthermore, restoration of PAK4 expression was able to significantly inhibit the effects of miR-663 overexpression on ccRCC cells. miR-663 may serve as a novel type of tumor suppressor by directly targeting PAK4 in ccRCC.

miR-663 has been previously identified to be upregulated in multiple types of human cancer. miR-663 is overexpressed in nasopharyngeal carcinoma tissues, cell lines and serum (25,26,48). The high expression of miR-663 was correlated with grade, lymph node metastasis and clinical stage of nasopharyngeal carcinoma (25,26). An increase in miR-663 expression correlated with poor five-year survival rate and poor relapse-free survival of patients with nasopharyngeal carcinoma (25). miR-663 was highly expressed in lung cancer (27) and castration-resistant prostate (28) cancer. However, miR-663 expression is downregulated in papillary thyroid carcinoma tissues and cell lines (29). Decreased miR-663 expression was associated with age and tumor size of papillary thyroid carcinoma (29). Downregulation of miR-663 was observed in pancreatic cancer (30), glioblastoma (31) and gastric cancer (31). The previous findings suggested that the expression pattern of miR-663 is tissue specific. Therefore, this miRNA may be a promising biomarker for the detection and prediction of prognosis of patients with these specific human cancer types.

miR-663 serves oncogenic roles in numerous types of human cancer. miR-663 overexpression was suggested to promote cell proliferation in nasopharyngeal carcinoma by inducing cell cycle arrest at the G1 stage in vitro and increasing tumor growth in vivo (26,48). Liu et al (27) and Fiori et al (49) demonstrated that miR-663 inhibition suppressed cell proliferation and induced apoptosis in lung cancer. Jiao et al (28) identified that miR-663 overexpression attenuated dihydrotestosterone-induced upregulation of prostate-specific antigen expression, and promoted castration-resistant prostate cancer cell proliferation, invasion and neuroendocrine differentiation. Nevertheless, miR-663 serves as a tumor suppressor in papillary thyroid carcinoma by inhibiting cell migration and invasion (29). Zang et al (30) demonstrated that restoration of miR-663 expression restricted the growth and metastasis of pancreatic cancer in vitro and in vivo. Pan et al (31) demonstrated that ectopic miR-663 expression suppressed the growth and metastasis of glioblastoma cells. Collectively, the previous findings suggested that the biological roles of miR-663 are tissue specific, and that miR-663 may be investigated as a therapeutic target for antineoplastic therapy.

Numerous targets of miR-663 have been previously identified, including cyclin dependent kinase inhibitor 2A (26), cyclin dependent kinase inhibitor 1A (48) in nasopharyngeal carcinoma, p53 upregulated modulator of apoptosis (49), BTG anti-proliferation factor 2 (49) in lung cancer, transforming growth factor β1 (29) in papillary thyroid carcinoma, and elongation factor 1-α 2 (30) in pancreatic cancer. PAK4, a member of the serine/threonine protein kinase family, was identified as a direct target of miR-663 in ccRCC. In ccRCC, an increased expression level of PAK4 correlated with high Fuhrman grade and tumor necrosis (40). Patients with ccRCC with increased expression levels of PAK4, exhibited poor overall survival and recurrence-free survival compared with patients with decreased expression levels of PAK4 (40). Deregulated PAK4 expression was implicated in tumor oncogenesis and development, and PAK4 was described to regulate a multitude of biological processes, including ccRCC occurrence (38,41,42). Therefore, miR-663-based therapy targeting PAK4 expression may represent an effective therapeutic strategy for patients with ccRCC.

In the present study, miR-663 served tumor suppressive roles in ccRCC partly by targeting PAK4. The present study provided better understanding of the formation and progression of ccRCC, contributing to the investigation of novel therapeutic targets for treating patients with ccRCC in the near future. However, in the present study, a miR-663 inhibitor was not tested to knockdown endogenous miR-663 expression, and the effects of miR-663 knockdown in ccRCC cells was not examined. In addition, the regulatory roles of miR-663 on ccRCC cell apoptosis were not tested. These limitations of the present study require further investigation in future studies.

Acknowledgements

Not applicable.

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

ZT designed the present study. YL and DJ performed the functional experiments. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Research Ethics Committee of The China-Japan Union Hospital (Changchun, China), and was performed in accordance with the Declaration of Helsinki and the guidelines of the Research Ethics Committee of The China-Japan Union Hospital. Written informed consent was obtained from all patients for the use of their clinical tissues.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References