A498 and 769-P cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. A498 cells were cultured in DMEM (Hyclone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA) and 100 mg/µl streptomycin (Sigma). 769-P cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were maintained at 37°C with 5% CO₂. Cells were mycoplasma free and authenticated by STR assay. Cells were seeded into 6-well plates and allowed to grow to 80% confluence prior to use in subsequent experiments.

The University of Alabama Cancer Database (UALCAN; ualcan.path.uab.edu/index.html) (21) was used to assess the expression of SEC61G in kidney cancer based on TCGA (https://cancergenome.nih.gov/) data. The Gene Expression Profiling Interactive Analysis (GEPIA; gepia.cancer-pku.cn/index.html) (22) was used to construct survival curves based on SEC61G gene expression data and clinical data in kidney cancer samples obtained from TCGA database, which were analyzed using the log-rank test. The cut-off value for overall survival (OS) was set to the median SEC61G gene expression value. The expression profile of SEC61G was clearly defined in human renal cancer using UALCAN and GEPIA.

A498 and 769-P cells were seeded into 6-well plates at a density of 4–8×10⁵ cell/well. At 2 h prior to transfection, culture medium was replaced with fresh complete medium (RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin). Subsequently, cells were transfected with 5 µl siRNAs against SEC61G (5′-CAUUGUUGGUGGCUGAAUATT-3′) or scrambled NC siRNA (5′-TTCTCCGAACGTGTCACGT-3′) at room temperature (~25°C) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation for 6 h, cells were supplemented with fresh complete culture medium and cultured for an additional 48 h. Then, cells were used for subsequent experiments. siRNAs were purchased from Oligobio Biotechnology Co., Ltd. (http://www.oligobio.com.cn/).

Total RNA was extracted from different transfected A498 and 769-P cells using an Ultrapure RNA kit (CoWin Biosciences) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using HiFiScript cDNA synthesis kits (CoWin Biosciences) following the manufacturer's protocol. Subsequently, with the SYBR Master Mixture (CoWin Biosciences), qPCR was performed to assess the effect of SEC61G knockdown on SEC61G mRNA gene expression levels. The following primers were used for qPCR (synthesized by Genewiz, Inc.): GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′; and Sec61G forward, 5′-TGGATCAGGTAATGCAGTTT-3′ and reverse, 5′-TCAGCCACCAACAATGATG-3′. The PCR reaction was as follows: Pre-incubation at 95°C for 10 min and incubation at 95°C for 30 sec, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min and a final dissociation stage (95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec). mRNA expression levels were quantified using the 2^−ΔΔCq method (23) and normalized to the internal reference gene GAPDH.

Total protein was isolated from A498 and 769-P cells using RIPA lysis buffer. The concentration of total protein was detected using the BCA method. Proteins (20 µg per lane) were separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). After blocking with TBS containing 0.1% Tween-20 containing 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies targeted against: SEC61G (Rabbit; 1:500; cat. no. 11147-2-AP; ProteinTech Group, Inc.), E-cadherin (Rabbit; 1:1,000; cat. no. 20874-1-AP; ProteinTech Group, Inc.), N-cadherin (Rabbit; 1:2,000; cat. no. 22018-1-AP; ProteinTech Group, Inc.), Bax (Rabbit; 1:2,000; cat. no. 50599-2-lg; ProteinTech Group, Inc.), GAPDH (Mouse; 1:5,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.), β-catenin (Rabbit; 1:1,000; cat. no. AF6266; Affinity Biosciences), BCL-2 (Rabbit; 1:1,000; cat. no. AF6139; Affinity Biosciences), Cleaved Caspase-3 (Rabbit; 1:1,000; cat. no. AF7022; Affinity Biosciences), AKT (Rabbit; 1:1,000; cat. no. AF6261; Affinity Biosciences), and p-AKT (Rabbit; 1:1,000; cat. no. AF0016; Affinity Biosciences) overnight at 4°C. Following primary antibody incubation, the membranes were incubated with corresponding HRP-conjugated anti-rabbit IgG (Goat; 1:5,000; cat. no. S0001; Affinity Biosciences) or anti-mouse IgG (Goat; 1:5,000; cat. no. S0002; Affinity Biosciences) secondary antibodies at room temperature for 2 h. Protein bands were visualized using an ECL detection kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression levels were semi-quantified using Quantity One Software (version 4.2.2; Bio-Rad Laboratories, Inc.) with GAPDH as the loading control.

A498 and 769-P cell proliferation was evaluated by performing a Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were harvested, resuspended in 100 µl medium (1.5×10⁴ cells/ml), seeded into 96-well plates (2,000 cells/well) and incubated at 37°C. At 24 h intervals, 10 µl CCK-8 reagent (Beijing TransGen Biotech Co., Ltd.) was added to each well and incubated at 37°C for an additional 1.5 h. Absorbance was measured at a wavelength of 450 nm using a microplate reader.

Flow cytometry was performed to determine A498 and 769-P cell apoptosis using the FITC-Annexin V Apoptosis Detection kit (BD Biosciences) according to the manufacturer's protocol. At 24 h post-transfection, cells (1–5×10⁵ cells/ml) were supplemented in serum-free medium and cultured for an additional 24 h. Subsequently, cells were harvested, resuspended, incubated with 5 µl FITC-conjugated Annexin V at room temperature for 5 min and then incubated with 10 µl PI in the dark at room temperature for 5 min. Cell apoptosis was analyzed using FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (version 7.6.1; FlowJo LLC). The percentage of total apoptotic events was defined as the sum of the apoptotic cells in the early stage (Annexin V positive/PI negative) and late stage (Annexin V positive/PI positive).

A498 and 769-P cell migration and invasion were assessed using 24-well Transwell chambers. To assess cell invasion, 100 µl Matrigel was added to the upper chamber and incubated for 2 h. Subsequently, 500 µl serum-free medium was plated into a 24-well Transwell chamber for 30 min. Then, 100 µl cell suspension (5×10⁴ cells) was plated into the upper chamber, and 600 µl complete medium containing 10% FBS was plated into the lower chamber. Following incubation at 37°C for 24 h, cells on the upper surface of the filter membrane were removed using cotton swabs. Invading cells were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% Giemsa at room temperature for 5 min. Stained cells were quantified in five randomly selected fields of view per filter under a fluorescence microscope (CKX 51; Olympus Corporation). To assess cell migration, the aforementioned protocol was performed, but the upper chamber was not precoated with Matrigel.

Cells were seeded (5×10⁵ cells/well) into 6-well plates containing RPMI-1640 with 10% FBS to form a confluent cell monolayer. The following day, an artificial wound was created using a sterile 200-µl pipette tip. Following washing with PBS, cells were cultured for 24 h. Wounds were observed at 0 and 24 h using an inverted fluorescence microscope (CKX51; Olympus Corporation). The wound closure area of the migrating monolayer of cells was quantified using ImageJ software (version 1.49; National Institutes of Health).

All experiments were repeated three times. Statistical analyses were performed using SPSS software (version 18.0; SPSS, Inc.). Comparisons between groups were analyzed using the unpaired Student's t-test. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether SEC61G served as a novel and efficient candidate gene for predicting kidney cancer, the expression profile of SEC61G was obtained using the UALCAN web-tool based on TCGA database. The results demonstrated that SEC61G expression was significantly increased in human kidney tumor tissues compared with healthy tissues (P<0.001; Fig. 1A). Subsequently, the association between increased expression levels of SEC61G and the survival of patients with kidney cancer was investigated. Kaplan-Meier curve analysis revealed that high SEC61G expression levels were significantly associated with poor OS in patients with kidney cancer compared with low SEC61G expression levels (Fig. 1B). The results indicated that SEC61G might serve as a biomarker for kidney cancer.

Tumor progression is a complex process, which is characterized by tumor cell proliferation, migration, invasion, metastasis, colony formation and adhesion (24,25). Therefore, the present study investigated the effects of SEC61G on cell proliferation, migration, invasion and survival.

To determine the role of SEC61G in human kidney cancer cell proliferation, A498 and 769-P cells were transfected with SEC61G siRNA. Following transfection with SEC61G siRNA, SEC61G expression levels were dramatically decreased at the mRNA and protein levels in A498 (Fig. 2A) and 769-P (Fig. 2B) cells compared with the NC group. Subsequently, the present study examined whether SEC61G was essential for cell proliferation. The CCK-8 assay results demonstrated that SEC61G knockdown significantly inhibited A498 (Fig. 2C) and 769-P (Fig. 2D) cell proliferation compared with the NC group.

The PI3K/AKT signaling pathway promotes cell proliferation in the majority of cell types, including kidney cancer cells (26,27). In addition, it has been reported that the PI3K/AKT signaling pathway is activated in ~50% of RCC cases (28–30). Therefore, the effect of SEC61G knockdown on the activation status of AKT in A498 and 769-P cells was assessed. The results revealed that AKT phosphorylation was significantly decreased by SEC61G knockdown in both kidney cancer cell lines compared with the NC group (Fig. 2E and F). The results indicated that SEC61G affected human kidney cancer cell proliferation via the PI3K/AKT signaling pathway.

Subsequently, the effect of SEC61G on cell migration was investigated. Compared with the NC group, SEC61G knockdown significantly decreased A498 (Fig. 3A) and 769-P (Fig. 3B) cell migration. The effect of SEC61G on cell migration was further assessed by performing wound healing assays. Consistent with the previous findings, SEC61G knockdown significantly decreased the area of wound healing compared with the NC group (Fig. 3C and D). Therefore, the results suggested that SEC61G knockdown inhibited kidney cancer cell migration compared with the NC group.

To evaluate the effect of SEC61G on kidney cancer cell invasion, a cell invasion assay was performed. The results demonstrated that SEC61G knockdown significantly inhibited A498 (Fig. 4A) and 769-P (Fig. 4B) cell invasion compared with the NC group.

Several transcriptional factors are involved in cancer cell invasion and migration, including E-cadherin, N-cadherin and β-catenin (31,32). It has been reported that during cell invasion and migration, E-cadherin is downregulated, whereas N-cadherin and β-catenin are upregulated (33). Therefore, the expression levels of E-cadherin, N-cadherin and β-catenin were assessed following SEC61G knockdown in A498 and 769-P cells. Compared with the NC group, SEC61G knockdown significantly increased E-cadherin expression levels, and significantly decreased N-cadherin and β-catenin expression levels (Fig. 4C and D). The aforementioned results indicated that SEC61G enhanced kidney cancer cell invasion.

To further explore the role of SEC61G in kidney cancer cell apoptosis, Annexin V/PI staining was performed. Compared with the NC group, SEC61G knockdown significantly increased A498 and 769-P cell apoptosis (Fig. 5A and B). In addition, the expression levels of the apoptosis-related proteins BCL-2, Bax and Cleaved caspase-3 were also determined. The western blotting results demonstrated that compared with the NC group, SEC61G knockdown significantly downregulated the expression of the antiapoptotic protein BCL-2 and significantly upregulated the expression of the proapoptotic protein Bax (Fig. 5C and D). In addition, the protein expression levels of the apoptosis-related marker Cleaved caspase-3 were also significantly increased in SEC61G-knockdown A498 and 769-P cells compared with the NC group. Collectively, the results suggested that SEC61G promoted human kidney cancer cell survival.