Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma

  • Authors:
    • Min Fan
    • Kai Wang
    • Xiaohui Wei
    • Hongwei Yao
    • Zhen Chen
    • Xiaozhou He
  • View Affiliations

  • Published online on: October 21, 2019     https://doi.org/10.3892/ijo.2019.4903
  • Pages: 1385-1395
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Abstract

Eukaryotic initiation factor 3c (eIF3C) is involved in the initiation of protein translation. Aberrant eIF3C expression has been reported in different types of human cancer. The present study aimed to assess the role of eIF3C in the malignant behavior of renal cell carcinoma in vitro and in vivo. eIF3C expression was assessed in 16 pairs of renal cell carcinoma (RCC) and matched distant normal tissues, and in RCC cell lines using immunohistochemistry. Subsequently, eIF3C was depleted using lentiviral short hairpin RNA and cell proliferation, cell cycle distribution and apoptosis of these eIF3C‑depleted cells were examined. Additionally, tumor cell xenograft assays in nude mice, Affymetrix microarrays and ingenuity pathway analyses were performed. eIF3C expression was upregulated in RCC tissues and cell lines. Depletion of eIF3C reduced tumor cell proliferation and arrested them at the G1 stage, thus promoting their apoptosis in vitro. Depletion of eIF3C also inhibited the formation and growth of tumor cell xenografts in nude mice. In addition, depletion of eIF3C altered the expression levels of 994 differentially expressed genes in RCC cells (516 genes were upregulated and 478 genes were downregulated). The expression levels of phosphorylated‑AKT, c‑JUN and NFKB inhibitor α were lower in the shorth hairpin RNA eIF3C‑transfected RCC cells compared with in the control group. In conclusion, the present study demonstrated that upregulated eIF3C expression contributed to the development and progression of RCC. Future studies should further evaluate whether eIF3C could be used as a potential strategy for RCC targeting therapy.

Introduction

There are >200,000 newly-diagnosed cases of kidney cancer each year globally according to a statistic calculated in 2013, with the highest incidence in North America and the lowest incidence in Asia and Africa (1). Histologically, kidney cancer can be divided into two common types: Renal cell carcinoma (RCC) and transitional cell carcinoma (2). RCC is the most common type of kidney cancer, accounting for 90-95% of cases in adults (3). The incidence and mortality of kidney cancer have also increased over the past decades at a rate of 2-3% per decade (3). Generally, RCC is asymptomatic, and is, therefore, frequently diagnosed at an advanced stage (4), and up to 30% of patients with RCC exhibit a metastatic tumor at diagnosis (3). To date, surgery remains the most effective treatment for RCC, whereas it is usually resistant to conventional chemoradiotherapy (5). Therefore, an improved understanding of the biology of RCC can help us develop novel therapeutic strategies and identify biomarkers for early detection and prediction of prognosis and treatment responses, leading to enhanced effectiveness of RCC control in clinical practice.

Previous studies have revealed that RCC development is associated with gene mutations in chromosome 3p, which activate oncogenes, such as c-Met, or inactivate tumor suppressor genes, such as VHL (6,7). However, alterations of various genes and gene pathways have also been associated with the development and progression of RCC (8-12). The present study of the gene alterations in RCC focused on eukaryotic initiation factors (eIFs). Aberrant expression levels of eIFs have been observed in several types of human cancer (13). eIFs regulate the initiation of protein translation in eukaryotic cells (14). For example, eIF3, the largest initiation factor, binds to the 40S ribosomal subunit, different initiation factors and mRNA to facilitate protein translation in cells (15,16). Overexpression or underexpression of a particular elF3 subunit is associated with the development and progression of a number of tumors, including lung cancer, breast cancer, hepatocellular cancer and intestinal cancer (17). During carcinogenesis and tumor progression, gene transcription and protein translation are usually upregulated in tumor cells (18). This has been confirmed in various previous studies of eukaryotic initiation factor 3c (eIF3C) in testicular seminoma (19), meningiomas (20), glioma (21,22), colorectal cancer (22), hepatocellular carcinoma (24,25) and breast cancer (26). Nevertheless, to the best of our knowledge, the role of eIF3C in RCC has not been assessed.

In a preliminary experiment, the eIF3C mRNA level was higher in RCC tissue than in adjacent normal tissues (Fig. S1). Therefore, the present study aimed to evaluate whether eIF3C could be used as a potential diagnostic marker or therapeutic target for RCC. To address this, eIF3C expression was assessed in RCC and normal kidney tissues, and the role of eIF3C in RCC malignant behavior was examined in vitro and in vivo.

Materials and methods

Patients and tissue collection

A total of 16 pairs of tumor and matched distant normal tissues were collected from patients with RCC (11 men and 5 women; median age at diagnosis, 54 years; age range, 37-74 years) who underwent radical resection between February 2016 and July 2016 at the Third Affiliated Hospital of Soochow University. Distant normal tissues were obtained >5 cm away from tumors to ensure their normality. All patients were histologically diagnosed with RCC and did not receive any pre-surgery chemotherapy or radiotherapy. The present study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University according to the principles of the Declaration of Helsinki. Written informed consent was provided by all participants prior to enrollment.

Immunohistochemistry

Tissues were fixed in 10% formalin at 4°C for 10 h. Fixed and paraffin-embedded tissue blocks were retrieved from the Pathology Department of The Third Affiliated Hospital of Soochow University and cut into 4-µm thick sections. For immunostaining of the eIF3C protein, the sections were deparaffinized in xylene and rehydrated in descending alcohol series. Antigens were repaired by heating the tissue sections at 100°C for 30 min in citrate (10 mmol/l; pH 6.0) (Beyotime). Then, the sections were immersed in a 0.3% hydrogen peroxide solution for 30 min to block endogenous peroxidase activity, rinsed in PBS for 5 min, blocked with 3% BSA (Beyotime Institute of Biotechnology) at room temperature for 30 min, and incubated with a primary antibody at 4°C overnight according to the manufacturer's instructions. Subsequently, the sections were incubated with the secondary antibody at 37°C for 30 min. The antigen-antibody complex was visualized after adding 3,3′-diaminobenzidine (DAB) for 2 min at room temperature. The primary antibody against eIF3C (1:1,000 dilution; cat. no. ab170841) was purchased from Abcam, and the secondary antibody and DAB were part of the MaxVision™ horseradish peroxidase (HRP)-Polymer anti-Mouse/Rabbit IHC kit (cat. no. 5010; Maxim Biotech, Inc.).

The immunostained sections were independently reviewed by two experienced pathologists who were blinded to the clinical parameters of the patients, and scores were evaluated from five randomly selected ×20 microscopic fields under a light microscope (Olympus Corporation), according to a previously described H-score method (25). The H-score = (% unstained tumor cells ×0) + (% weakly stained tumor cells ×1) + (% moderately stained tumor cells ×2) + (% strongly stained tumor cells ×3). The H-scores ranged between 0 (100% negative staining) and 300 (100% strong staining).

Cell lines and culture conditions

Four human RCC lines ACHN, 786-O, Caki-1 and A498, were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences in September 2017 and maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Biological Industries) and 1% penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. All cell lines were certified by Shanghai GeneChem Co., Ltd. for short tandem repeat analysis on October 31, 2017, as described in 2012 in American National Standards Institute Standard by the American Type Culture Collection Standards Development Organization (28) and in Capes-Davis et al (29). All cell lines were passaged <30 times.

Short hairpin RNA (shRNA) and cell transfection

Lentiviruses carrying eIF3C shRNA (cat. no. PSC2752) targeting the DNA sequence of 5′-GTC ACT AAA GGT CTG TTT A-3′, and negative control shRNA (cat. no. PSC3741) with a targeting sequence of 5′-TTC TCC GAA CGT GTC ACG T-3′ were obtained from Shanghai GeneChem Co., Ltd. Both eIF3C and negative control shRNA were designed and cloned into the GV115 vector (Shanghai GeneChem Co., Ltd.) double enzyme digested by AgeI/EcoRI. Reconstructed vectors with eIF3C-shRNA and the negative control shRNA were transformed into competent E. coli cells (Shanghai GeneChem Co., Ltd.) and transformed cells in serum-free LB solid medium (pH 7.0; Shanghai GeneChem Co., Ltd.) supplemented with ampicillin (0.1 mg/ml; Genebase) were cultured at 37°C. Positive colonies were selected by reverse transcription-quantitative PCR (RT-qPCR) and sequencing. To construct a stable eIF3C-depletion cell line, cells were seeded in 6-well plates at a density of 5×104 cells/well, grown overnight, and transfected with eIF3C-shRNA (8×108 TU/ml) (sheIF3C) or negative control shRNA (1×109 TU/ml) (shCtrl) lentivirus for 12 h. The culture medium was subsequently replaced with fresh complete RPMI-1640 medium, and cells were grown for an additional 72 h and subjected to fluorescence microscopy for the visualization of the green fluorescence protein (GFP). The efficiency of eIF3C depletion was evaluated through RT-qPCR and western blotting.

RT-qPCR

Total cellular RNA was isolated from ACHN, 786-O, A498 and Caki-1 cell lines using TRIzol® reagent (Shanghai Pufei Biotechnology Co., Ltd.) and reverse transcribed into cDNA using the M-MLV cDNA kit (Promega Corporation) according to the manufacturer's instructions. The following steps were used: Step 1, adding 2 µg total RNA, 1 µl Oligo(dT) and RNase-Free H2O to 10 µl total volume, heating at 70°C for 5 sec; step 2, adding 4 µl 5X RT buffer, 2 µl 10 m MdNTPs, 0.4 µl Rnasin (40 U/µl), 1 µl M-MLV-RTase (200 U/µl) and 2.6 µl RNase-Free H2O, heating at 42°C for 1 h and at 70°C for 10 min. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression levels of eIF3C using a SYBR Master Mixture kit (cat. no. DRR041B) from Takara Biotechnology Co., Ltd. according to the manufacturer's instructions. The following thermocycling conditions were used: Stage 1, holding at 95°C for 30 sec, 1 cycle; stage 2, 2 steps PCR reacting at 95°C for 5 sec and at 60°C for 30 sec, respectively, 45 cycles; stage 3, dissociating at 95°C for 15 sec, at 55°C for 30 sec and at 95°C for 15 sec, respectively, 1 cycle. GAPDH was used as an internal control. The primers were designed as follows: Human eIF3C forward, 5′-AGA TGA GGA TGA GGA TGA GGA C-3′ and reverse, 5′-GGA ATC GGA AGA TGT GGA ACC-3′; and human GAPDH forward, 5′-TGA CTT CAA CAG CGA CAC CCA-3′ and reverse, 5′-CAC CCT GTT GCT GTA GCC AAA-3′. The relative expression level of eIF3C was calculated using the 2−ΔΔCq method (30). The experiments were performed in triplicate.

Celigo cell viability and proliferation assay

Following depletion of elF3c expression, tumor cells were subjected to a cell proliferation assay. Briefly, after 24 h gene transfection, ACHN cells were seeded into 96-well plates at a density of 2×103 cells/well and grown for up to 5 days. On each day, the green fluorescence emission was observed and recorded using a Celigo cytometer (Nexcelom Bioscience) to assess the proliferation of tumor cells. The data are presented as the mean ± SD (n=3).

The 96-well plates were subjected to an MTT assay using the MTT reagent (Genview) and formazan was dissolved by DMSO solution (Shiyi Corporation). An ELIASA microplate reader (Tecan Group, Ltd.) was used and the optical density value at 490 nm was used to estimate cell confluence. The data are presented as the mean ± SD (n=3).

Cell cycle distribution assay

ACHN cells were cultured in 6-cm cell culture dishes until they reached 80% confluence, and were then transfected for 5 days with a lentivirus targeting eIF3C or a negative control shRNA. Cells were then harvested, washed with ice-cold D-Hanks solution, and fixed with 75% ethyl alcohol at 4°C for at least 1 h. Subsequently, cells were washed with ice-cold D-Hanks solution and stained with 40X propidium iodide solution (2 mg/ml), 100X RNase (10 mg/ml) and 1X D-Hanks solution according to the manufacturer's instructions (Sigma-Aldrich; Merck KGaA). The cell cycle distributions were then analyzed using Guava easyCyte HT flow cytometry (EMD Millipore) and the results were analyzed by ModFit LT 4.0 software (Verity Software House, Inc.).

Cell apoptosis assay

ACHN cells were cultured in 6-cm cell culture dishes until they reached 80% confluence, and were transfected for 5 days with a lentivirus targeting eIF3C or a negative control shRNA. Transfected cells were harvested, washed with ice-cold D-Hanks solution and fixed with 75% ethyl alcohol at 4°C for at least 1 h. Staining was performed using Annexin V-APC apoptosis detection kit (eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, the cells were collected by centrifugation at 265 × g at 4°C for 5 min, washed with D-Hanks solution and then mixed with 1X binding buffer. Subsequently, 200 µl cell suspension was thoroughly mixed with 10 µl Annexin V solution, followed by incubation in the dark at room temperature for 10-15 min. Cells were then subjected to Guava easyCyte HT flow cytometry (EMD Millipore) and the results were analyzed by ModFit LT 4.0 software (Verity Software House, Inc.).

Nude mouse tumor xenograft formation assay

The animal protocol was approved by the Institutional Animal Care and Use Committee of the Third Affiliated Hospital of Soochow University (Changzhou, China). Female BALB/c nude mice (n=14; 4 weeks old; average weight, 18.7±1.75 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were raised under specific pathogen-free conditions (23±3°C; relative humidity, 40-70%) under a 12 h light/dark cycle. All mice were adaptively fed with free access to water and standard mouse chow. Animals were randomly divided into two experimental groups and subcutaneously injected into the right forelimb armpit with 1×107 786-O cells (in ~200 µl PBS) following transfection with a lentivirus carrying eIF3C- or scrambled shRNA. Mice were regularly monitored for weight, health and xenograft size for 7 weeks. Subsequently, all mice were euthanized by injection of 2% sodium pentobarbital (150 mg/kg of body weight), and after complete coma, cervical dislocation was performed. The tumor volume was measured for the greatest longitudinal diameter (length) and the greatest transverse diameter (width) using a vernier caliper. The tumor volume was calculated using the modified ellipsoidal formula as follows: V=3.14/6 × L × W2, in which V represents the whole volume of the tumor cell xenograft (mm3), L indicates the length (mm), and W is the width (mm).

Microarray and ingenuity pathway analyses

Total cellular RNA was isolated from 786-O cells after transfection with a lentivirus carrying eIF3C-shRNA (n=3) or scrambled shRNA (n=3) using TRIzol® reagent (31). RNA quantity and quality were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and RNA 2100 (Agilent Technologies, Inc.), respectively, according to the manufacturers' instructions. The genome-wide effects of eIF3C depletion were evaluated by GeneChip PrimeView Human Affymetrix microarray (Affymetrix; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The resultant raw data and the differentially expressed genes (DEGs) in the eIF3C-shRNA-infected RCC cell lines were identified based on the criteria of an absolute fold change >1.3 and P<0.05. An ingenuity pathway analysis (Ingenuity Systems; Qiagen, Inc.) was performed to assess the functional and pathway annotations based on all the DEGs.

Western blotting

Total cellular protein from tissue samples and cell lines was extracted using an ice-cold RIPA (high) lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g at 4°C for 15 min. The protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg/µl) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). These membranes were blocked with 5% fat-free milk in TBS-0.5% Tween-20 (TBS-T) at room temperature for 1 h and then incubated with primary antibodies at room temperature for 2 h. The antibodies used in the present study were anti-c-JUN (1:200 dilution; cat. no. ab32137; Abcam), anti-NFKB inhibitor α (NFKBIA; 1:2,000 dilution; cat. no. ab7217; Abcam), anti-AKT (1:1,000 dilution; cat. no. 9272; Cell Signaling Technology, Inc.), anti-phosphorylated-(p-)AKT (1:1,000 dilution; cat. no. 13038; Cell Signaling Technology, Inc.), anti-caspase-3 (1:1,000 dilution; cat. no. 9662; Cell Signaling Technology, Inc.), anti-caspase-8 (1:1,000 dilution; cat. no. 4790; Cell Signaling Technology, Inc.), anti-caspase-9 (1:1,000 dilution; cat. no. ab2324; Abcam) and anti-GAPDH (1:2,000 dilution; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.). All aforementioned antibodies of caspases could detect both the cleaved and total caspases. Blots were washed three times in TBS-T and further incubated with a secondary antibody goat anti-mouse immunoglobulin G (IgG)-HRP (1:5,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or goat anti-rabbit IgG-HRP (1:5,000 dilution; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Immunoreactive protein bands were developed using the Pierce™ ECL Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.) and exposed to x-ray films. The protein bands were semi-quantified using ImageJ v1.37 software (National Institutes of Health).

Statistical analysis

All data are presented as the mean ± SD of at least three repeated experiments, and the difference between groups was analyzed using Student's t-test by GraphPad Prism v6.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

eIF3C expression is upregulated in RCC tissues

The present study analyzed the expression levels of eIF3C in 16 paired RCC and distant normal tissues using immunohistochemistry. In almost all cases, eIF3C staining was stronger in the RCC tissue compared with in the paired distant normal tissue (Fig. 1A). Subsequently, the immunostaining data was quantified using the H-score. The H-score was significantly higher in the RCC tissue compared with in the normal tissues (Fig. 1B), indicating that eIF3C may contribute to the development or progression of RCC.

RCC cell proliferation is reduced and cell cycle distribution is altered following eIF3C depletion

The present study assessed the mRNA expression levels of eIF3C in four different RCC cell lines using RT-qPCR. eIF3C expression was high in all four tested cell lines (Fig. 2F). Among them, eIF3C mRNA exhibited the highest expression levels in 786-O cells and the lowest expression levels in ACHN cells. Therefore, ACHN and 786-O cell lines were selected for subsequent experiments in which eIF3C was depleted (Fig. 2A), and alterations in the tumor cell malignant phenotypes were explored. Fig. 2G-I shows that transfection with a lentivirus carrying eIF3C-shRNA resulted in significantly decreased eIF3C mRNA and protein expression compared with transfection with lentivirus carrying shCtrl.

Subsequently, the proliferation capacity of these tumor cells was assessed using cell proliferation and viability assays. The present study revealed that depletion of eIF3C significantly reduced the viability and proliferation rate of ACHN and 786-O cells compared with the control group (Fig. 2B-E). Additionally, the depletion of eIF3C remarkably altered the cell cycle distribution. There were 42.64±0.81, 53.70±1.28 and 3.67±0.60% of tumor cells in G1, S and G2 phases, respectively, following transfection with shCtrl, whereas there were 49.64±0.77, 45.96±0.46 and 4.40±0.41% of tumor cells in G1, S and G2 phases, respectively, after transfection with eIF3C-shRNA (Fig. 3A and B). The number of G1-phase cells was significantly higher in the sheIF3C group compared with in the shCtrl group (P<0.001). However, the numbers of S-phase cells were lower in the sheIF3C group compared with in the shCtrl group (P<0.001).

Depletion of eIF3C triggers apoptosis of RCC cells

To assess the underlying mechanism of the reduced cell viability following the depletion of eIF3C, an apoptosis assay by flow cytometry as performed. The proportion of apoptotic cells in eIF3C-depleted ACHN cells was ~2-fold higher compared with in the cells transfected with scrambled shRNA (7.57±0.38 vs. 3.73±0.16%, P<0.01; Fig. 3C and D). Additionally, the expression levels of caspase-3, caspase-8 and caspase-9 were detected, and it was ascertained that the depletion of eIF3C could induce their upregulation with the exception of caspase-8 (Fig. 3E and F).

Effects of reduced eIF3C expression on the formation and growth of nude mouse xenograft

The in vitro findings were further confirmed using a nude mouse tumor cell xenograft assay. At the beginning of the experiment, the average weights of the sheIF3C and shCtrl mice were 18.5±2.32 and 18.9±0.99 g, respectively. At the end of the experiment, the average body weights of the sheIF3C and shCtrl mice were 22.9±2.30 and 23.0±0.96 g, respectively (data not shown). The average tumor volume of the sheIF3C group was 106.72 mm3, whereas it was 491.61 mm3 in the shCtrl group (Fig. 4). Additionally, the average tumor xenograft weight in the sheIF3C group was significantly lower compared with the shCtrl group (0.100±0.10 vs. 0.45±0.132 g; P<0.05; Fig. 4).

Effects of reduced eIF3C expression on gene expression in vitro and in vivo

To explore the potential molecular events following eIF3C depletion, a microarray analysis was conducted to identify DEGs in eIF3C-depleted RCC 786-O cells. A total of 994 DEGs were identified, including 516 upregulated and 478 downregulated genes (Fig. 5A). The ingenuity pathway analysis revealed that eIF3C-regulated genes were mainly involved in the pathways of 'cell growth and proliferation', 'cell death and survival', 'cancer', 'organismal injury and abnormalities', 'cell cycle', 'cellular development' and 'cellular movement' (Fig. 5B). Common tumor-associated genes, such as AKT, c-JUN and NFKBIA, whose expression levels changed significantly, were selected. Western blotting results revealed that all of them could be regulated by eIF3C (Fig. 5C and D).

Discussion

RCC is usually diagnosed at advanced stages of the disease and is insensitive to chemoradiotherapy (5). Therefore, identification of novel gene alterations and targets involved in this disease could help medical oncologists effectively control RCC in clinical practice. The present study first assessed eIF3C expression at the protein level in RCC and normal kidney tissues and then investigated the effects of eIF3C depletion on the regulation of malignant behaviors of RCC cells in vitro and in nude mice. The data revealed that eIF3C protein expression was significantly higher in RCC tissues and cell lines compared with their corresponding controls. The depletion of eIF3C reduced tumor cell proliferation, increased the proportion of G1-phase tumor cells, and enhanced tumor cell apoptosis compared with the controls. Furthermore, depletion of eIF3C also inhibited the formation and growth of tumor cell xenograft in nude mice. At the gene level, depletion of eIF3C resulted in 516 upregulated and 478 downregulated genes. These genes were involved in cell proliferation, survival and cancer-related pathways, including the AKT, c-JUN and NFKBIA signaling pathways. These findings were confirmed through western blotting. Collectively, the data demonstrated that eIF3C exerted oncogenic effects on the development and progression of RCC.

The proteins in the eIF family mainly function as initiators of protein translation from their corresponding mRNAs in eukaryotic cells (32). To date, ~11 eIF members have been reported to be involved in this process (16). One of these protein family members, eIF3, has 13 different subunits (eIF3a to eIF3 m) (16) that can bind to the 40S ribosome through the facilitation of methionyl-tRNA and mRNA binding for protein translation (33). Previous studies have demonstrated that the eIF3C subunit of eIF3 is a crucial binding partner for eIF1 and eIF5 (34,35), and this subunit serves an essential role in the selection of the translational start codon (36). During the development and progression of cancer, protein synthesis is frequently upregulated, and overexpression of eIF3C has been observed in various types of cancer (19-26). However, to the best of our knowledge, no studies have investigated the role of eIF3C in RCC. The present study demonstrated that eIF3C expression was upregulated in RCC tissues and cell lines. Zang et al (37) have reported that overexpression of eIF3b in tumors is associated with an aggressive tumor phenotype and poor prognosis in patients with RCC. However, as the present study had a small sample size, future studies with large tissue sample sizes can further confirm the upregulation of eIF3C and determine whether such upregulation is associated with the prognosis or even the treatment response in RCC.

The data revealed that depletion of eIF3C inhibited the malignant behavior in RCC, including a decrease in tumor cell proliferation, increased apoptosis and altered cell cycle progression. This corroborates the important role of eIF3C in the development and progression of RCC. Indeed, a previous study has demonstrated that eIF3C is able to induce exosome secretion and promote angiogenesis and tumorigenesis in human hepatocellular carcinoma (25). In breast cancer, eIF3C targets the mTOR signaling pathway to inhibit cell proliferation and induce apoptosis (26). Therefore, targeting eIF3C could be a potential therapeutic approach for cancer treatment (13). Furthermore, the expression of other eIF3 subunits could also be altered in RCC. For example, aberrant eIF3e expression is essential for embryonic development and cell proliferation (38), whereas aberrant eIF3a expression occurs in non-small cell lung cancer, which is associated with p27 expression and poor patient prognosis (39). Additionally, eIF3b is able to activate the β-catenin signaling pathway, leading to accelerated progression of esophageal squamous cell carcinoma (40). Zhu et al (41) have reported that eIF3 h targets the transforming growth factor-β and mitogen-activated protein kinase signaling pathways in patients with hepatocellular carcinoma. Qi et al (42) have reported that overexpression of eIF3i can activate the synthesis of prostaglandin-endoperoxide synthase 2 and β-catenin in colorectal cancer. Therefore, it is necessary to further investigate the functions of eIF3C in the development and progression of RCC.

In the present study, it was also observed that depletion of eIF3C was able to suppress different signaling pathways, including the Akt, c-JUN and NFKBIA singaling pathways. This indicated that eIF3C promoted RCC malignant phenotypes in vitro through these signaling pathways. Several eIFs family members have been found to participate in these pathways indicated above in various tumors, such as lung cancer, breast cancer, hepatocellular cancer and intestinal cancer (17). A previous study has linked eIF1, eIF5 and eIF6 to the PI3K/Akt/mTOR signaling pathway in colorectal cancer (43), whereas Zang et al (37) has demonstrated that depletion of eIF3b suppresses the Akt pathway network in RCC cells, including decreased expression of integrin/focal adhesion kinase/Akt, Akt/mTOR/hypoxia-inducible factor/vascular endothelial growth factor, Akt/mTOR/NF-κB, Akt/Bcl-2/Bax and Akt/glycogen synthase kinase-3β pathway genes. Furthermore, Chen et al (44) have demonstrated that eIF4b may integrate the signals from the Pim and PI3K/Akt/mTOR signaling pathways in Abl-expressing leukemic cells. Phosphorylated eIF2 exhibits translation-dependent control of the activation of NF-κB (45). In order to review what occurred in tumors, the aforementioned signaling pathways were listed in Table SI. However, there was insufficient data demonstrating eIF3C-related gene regulation. The present study demonstrated that there were a total of 994 differentially expressed genes in RCC cells following eIF3C depletion. Future studies could characterize some of these genes in RCC cells.

Overall, the findings provided initial evidence regarding the role of eIF3C in the development and progression of RCC. However, the underlying molecular mechanisms require further investigation.

Supplementary Data

Abbreviations:

eIFs

eukaryotic initiation factors

eIF3C

eukaryotic initiation factor 3c

RCC

renal cell carcinoma

DAB

3,3′-diaminobenzidine

GFP

green fluorescence protein

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

Acknowledgments

Not applicable.

Funding

The present study was financially supported in part by grants from the Natural Science Foundation of Jiangsu Province (grant no. BK20151180), Applied Basic Research of Changzhou City (grant no. CJ20159014) and Major Science and Technology Project of Changzhou Health Bureau (grant no. ZD201405).

Availability of data and materials

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

Authors' contributions

XH and ZC conceived and designed the project. KW, HY and XW performed the experiments and acquired the data. MF analyzed the data. KW and MF participated in writing the article. All authors read and approved the final version of this manuscript.

Ethics approval and consent to participate

Written informed consent was obtained from every participant enrolled in the present study. The current study was approved by the Ethics Committee of the Third Affliated Hospital of Soochow University.

Patient consent for publication

All patients in the present study provided consent for their data to be published.

Competing interests

The authors declare that they have no competing interests.

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December-2019
Volume 55 Issue 6

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Online ISSN:1791-2423

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Spandidos Publications style
Fan M, Wang K, Wei X, Yao H, Chen Z and He X: Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma. Int J Oncol 55: 1385-1395, 2019
APA
Fan, M., Wang, K., Wei, X., Yao, H., Chen, Z., & He, X. (2019). Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma. International Journal of Oncology, 55, 1385-1395. https://doi.org/10.3892/ijo.2019.4903
MLA
Fan, M., Wang, K., Wei, X., Yao, H., Chen, Z., He, X."Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma". International Journal of Oncology 55.6 (2019): 1385-1395.
Chicago
Fan, M., Wang, K., Wei, X., Yao, H., Chen, Z., He, X."Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma". International Journal of Oncology 55, no. 6 (2019): 1385-1395. https://doi.org/10.3892/ijo.2019.4903