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Introduction

Kidney cancer is a malignant cancer. According to a recent report, there were predicted to be ~14,890 cases of kidney cancer-associated mortalities in 2023(1). Renal cell carcinoma (RCC) originates from renal tubular epithelial cells and accounts for >90% of kidney cancer cases (2). Due to genetic heterogeneity, different genetic factors dominate in different cohorts of patients with RCC, resulting in difficulty when administering effective therapies (3). For example, different expression of CB2R could be used as a prognostic marker in RCC (4). Although patients with localized or regional kidney cancer show a good five-year survival rate, the 5-year survival rate of those with metastasis remains at ~15% (1). Additionally, ~20-30% patients with RCC already present with metastasis at the time of diagnosis (2). Therefore, it is of importance to understand the intrinsic mechanisms underlying RCC physiology for developing novel therapeutic strategies to treat RCC.

Inflammation is associated with cancer. In an inflammatory environment, normal cells can undergo malignant transformation, where aberrant events may occur, ultimately leading to the development of cancer (5). Therefore, inflammation may be a promising target for cancer therapy (6). Inhibition of tumor-associated macrophages, such as M2-like macrophages, has been reported to improve antitumor immune responses in solid cancer (7). Inter-α-trypsin inhibitor heavy chain 1 (ITIH1) is a gene located in chromosome 3p21.1 that encodes a preproprotein of the heavy chain of the inter-α-trypsin inhibitor complex, which is involved in inhibition of inflammatory diseases, such as sepsis (8). ITIH genes appear to serve a tumor suppressor role that frequently exhibit decreased expression in human cancers (9). Previous studies have reported that the reduced expression of ITIH1 can promote liver cancer progression, suggesting ITIH1 to be a prognostic or diagnostic indicator of this malignancy (10,11). In addition, Kopylov et al (12) reported that downregulation of ITIH1 expression was associated with the initial development of colorectal cancer. However, the role of ITIH1 in the initiation or progression of RCC remains unclear.

The hypo- or hyperactivation of certain growth or death signaling pathways can occur in cancer (13). Of these pathways, hyperactivation of the NF-κB signaling pathway is commonly observed in cancer (14). In gastrointestinal cancer, activation of NF-κB signaling has been reported to promote cancer initiation and development (14). In RCC, inhibition of NF-κB signaling has been reported to sensitize cancer cells to tyrosine kinase inhibitors (15). Furthermore, NF-κB signaling was considered a promising target for cancer therapy according to a report by Yu et al (16). However, it remains unclear if there is a relationship between ITIH1 and NF-κB signaling in RCC.

To investigate the role and mechanism of ITIH1 in RCC, the present study analyzed the effects of ITIH1 knockdown in RCC cell lines and measured the protein expression levels of downstream signaling molecules following ITIH1 knockdown. In addition, experiments after ITIH1 overexpression were performed to further investigate the signaling pathways regulated by ITIH1.

Materials and methods

Bioinformatics analysis

The mRNA expression profiles [transcripts per million (TPM)] of the ITIH1 gene in clear cell renal cancer tissues (n=110) and matched normal tissues (n=84) were obtained from The Cancer Genome Atlas (TCGA) database (https://ualcan.path.uab.edu/analysis.html) (17). The survival rate of patients with renal cancer was also extracted based on the clinical data in the TCGA database from Ualcan. Briefly, we input the name of gene ITIH1 to the box ‘Enter gene symbol’ on the web and selected the type of cancer ‘Kidney renal clear cell carcinoma’ in the box ‘TCGA dataset’. The ‘Explore’ button was pressed and the website (https://ualcan.path.uab.edu/cgi-bin/ualcan-res.pl) appeared. Then the ‘Expression’ button was clicked and the expression of ITIH1 gene was returned. ‘Survival’ was selected and the association of ITIH1 expression with patients' survival probability was returned. For survival analysis, the samples were grouped as below: High expression (with TPM values above upper quartile) and low/medium expression (with TPM values below upper quartile) (17).

Cell culture

The A498, 786-O and ACHN RCC cell lines were purchased from Shanghai Fuheng Biotechnology Co., Ltd. and cultured in RPMI 1640 medium (Nanjing KeyGen Biotech Co., Ltd.) with 10% FBS (Gibco; Thermo Fisher Scientific) at 37˚C under an atmosphere with 5% CO2. HK-2 cells were purchased from Cellverse Bioscience Technology Co., Ltd. and cultured in DMEM (Nanjing KeyGen Biotech Co., Ltd.) with 10% FBS.

Reverse transcription-quantitative PCR

Total RNA was extracted from RCC cells (HK-2, A498, 786-O and ACHN) using the RNAeasy Kit (Beyotime Institute of Biotechnology). Total RNA (0.1-1.0 µg) was used as a template to transcribe the first strand of double-stranded complementary (c) DNA with BeyoRT III cDNA kit (cat. no. D7178M; Beyotime Institute of Biotechnology). The protocol for reverse transcription reaction is as follows: 25˚C, 10 min; 40˚C, 50 min; and 80˚C, 10 min. Subsequently, 1 µl the cDNA was used for the quantification of the expression of genes of interest using the BeyoFast SYBR Green qPCR Mix (Beyotime Institute of Biotechnology). The protocol is as follows: 95˚C, 5 min; 95˚C, 15 sec; 60˚C, 20 sec for 40 cycles. GAPDH was used as the internal reference control. The relative mRNA expression levels of the ITIH1 gene were calculated using the 2-IICq method (18). The primers used were as follows: ITIH1 forward (F), 5'-CTGCAGGGTTTCTACAGCCA-3' and reverse (R), 5'-CGCTCTCGGAGCAGTTTCTT-3'; and GAPDH F, 5'-GATGCTGGCGCTGAGTACG-3' and R, 5'-GTTCACACCCATGACGA-3'.

Synthesis of small interfering (si)RNA targeting ITIH1

SiRNAs targeting ITIH1 (siITIH1) or negative control (NC) were synthesized by Beijing Tsingke Biotech Co., Ltd. Briefly, siRNA (50 pmol) was mixed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 100 µl RPMI 1640 medium (Nanjing KeyGen Biotech Co., Ltd.) with 10% FBS and incubated for 30 min at room temperature. Subsequently, the mixture was delivered into the RCC cells (A498 and ACHN) and incubated at 37˚C for 4 h. The supernatant was then removed and substituted with fresh RPMI 1640 medium (Nanjing KeyGen Biotech Co., Ltd.) and incubated for 48 h at 37˚C before subsequent experimentation. The sequences of siITIH1 and NC used were as follows: si-#1 sense, 5'-UUAUGUCUCCGAUAAAUGCGU-3' and antisense, 5'-GCAUUUAUCGGAGACAUAAAG-3'; si-#2 sense, 5'-AACAUGAUGAGUAUUGAGGCA-3' and antisense, 5'-CCUCAAUACUCAUCAUGUUGA-3'; and NC sense, 5'-UUCUCCGTACGUGUCACGUTT-3' and antisense, 5'-ACGUGACACGUACGGAGAATT-3'.

Construction of recombinant plasmid overexpressing ITIH1

The coding sequence of the ITIH1 gene (accession no. NM_001166434.3) was chemically synthesized and subcloned into the pcDNA3.1 (General Biologicals Company) plasmid. The recombinant plasmid pcDNA-ITIH1 (~2 µg) was transferred into A498 and ACHN cells as aforementioned. The empty vector pcDNA3.1 was used as control.

Western blotting assay

The total proteins were extracted from A498 cells treated with siITIH1 or NC for 48 h using the Nuclear and Cytoplasmic Protein Extract kit (Beyotime Institute of Biotechnology) and quantified on the UV Spectrophotometer (Hangzhou Lifereal Biotechnology Co., Ltd.). Total protein (~10 µg) was separated by 15% SDS-PAGE (Shanghai Yeasen Biotechnology Co., Ltd.), transferred onto PVDF membranes (Shanghai Yeasen Biotechnology Co., Ltd.) and blocked with 5% non-fat milk for 1 h at room temperature. Subsequently, the PVDF membranes were incubated with primary antibodies overnight at 4˚C, washed with TBST containing 0.5% Tween 20 (Tanon Science and Technology Co., Ltd.) three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After the PVDF membranes were washed with PBST three times, they were visualized with an enhanced ECL chemiluminescent kit (Shanghai Yeasen Biotechnology Co., Ltd.) and analyzed by ImageJ software (version 1.51j8; National Institutes of Health). The following antibodies were used in the present study: ITIH1 (1:2,000; cat. no. ab233032; Abcam), phosphorylated (p-) NF-κB (1:1,000; cat. no. ab194729; Abcam), NF-κB (1:1,000; cat. no. AG3101; Beyotime Institute of Biotechnology), IκB (1:2,000; cat. no. AG2737; Beyotime Institute of Biotechnology), IKK (1:1,000; cat. no. AF0198; Beyotime Institute of Biotechnology), cyclinD1 (1:1,000; cat. no. AF0126; Beyotime Institute of Biotechnology), proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. AG8029; Beyotime Institute of Biotechnology), GAPDH (1:2,000; cat. no. ab9485; Abcam), α-SMA (1:3,000; cat. no. AF1507; Beyotime Institute of Biotechnology) and goat anti-rabbit HRP-conjugated IGG antibodies (1:20,000; cat. no. ab6721; Abcam).

Cell Counting Kit-8 assay

RCC cells (A498 and ACHN) were seeded into 96-well plates (3-5x103 cells/well) and transfected with siITIH1 or NC. After the cells were cultured for 24, 48, 72 and 96 h at 37˚C,10% CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and incubated for 1 h at 37˚C. The absorbance value at 450 nm of each well was measured using a microplate-reader (Hangzhou Allsheng Instruments Co., Ltd.). For rescue experiments, the NF-κB signaling pathway inhibitor JSH-23 (cat. no. HY-13982; MedChemExpress) was used for 48 h at 37˚C after cells were treated with siITIH1 at 10 µM for 24 h.

Cell migration and invasion assays

RCC cells (A498 and ACHN) were seeded into Transwell inserts (8-µm pore size; Wuxi NEST Biotechnology Co., Ltd.) at 2-3x104 cells/well, placed into 24-well plates and transfected with siITIH1 or NC. In the upper chamber, culture medium without serum was added and culture medium with 10% fetal bovine serum was added to the bottom of the inserts. After incubating for 48 h at 37˚C, the inserts were removed, the cells on the upper surface were scraped away whereas cells on the lower surface were fixed using 4% formaldehyde for 10 min at room temperature and stained with 0.1% crystal violet dye (Beyotime Institute of Biotechnology) for 10 min at room temperature. After washing with PBS three times, the inserts were imaged using a light microscope and the number of positively stained cells at three fields of view were manually calculated as a percentage of total cells.

For the cell invasion assay, the Transwell inserts were pre-treated with 20 µl Matrigel (1 mg/ml per chamber; Corning, Inc.) overnight at 4˚C before being treated as aforementioned.

Apoptosis assay using flow cytometry

RCC cells (A498 and ACHN) were seeded into 6-well plates (1x104 cells/well) and transfected with siITIH1 or NC. After incubation for 48 h at 37˚C, the cells were collected at 250 x g for 5 min at room temperature and a total of 6x104 cells were stained with 100 µl dye buffer containing 5 µl Annexin V/FITC-A reagent (Shanghai Yeasen Biotechnology Co., Ltd.) for 15 min at room temperature in the dark. Stained cells were washed with cold PBS before the positively stained cells were detected using a flow cytometer equipment (FACSCelesta; BD Biosciences) and analyzed by Flowjo V10.7.1 (BD Biosciences).

Statistical analysis

Data were analyzed using SPSS software (version 16.0; SPSS, Inc.) and were presented as the mean ± standard deviation (n=3). Differences between two groups were analyzed using the unpaired Student's t-test, whilst differences among >2 groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. Kaplan-Meier survival curve analysis was used to analyze survival data followed by log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical significance of ITIH1 in renal cancer

According to the data obtained from the TCGA database, the protein expression level of ITIH1 in primary RCC tissues (n=110) was significantly higher compared with that in the normal tissues (n=84) (Fig. 1A). The expression levels of ITIH1 in female (n=30) and male (n=80) patients with RCC was significantly higher compared with that in normal tissues (n=84) (Fig. 1B). Moreover, patients with high expression levels of ITIH1 (n=133) exhibited a significantly decreased 5-year survival rate compared with those with low/medium expression levels of ITIH1 (n=398; Fig. 1C). However, the mRNA and protein expression levels of ITIH1 in the ACHN, A498 and 786-O RCC cell lines were significantly lower compared with that in the control HK-2 cells (Fig. 2A-C). Therefore, the role of ITIH1 in renal cancer requires further investigation.

Figure 1 Role of ITIH1 in RCC based on The Cancer Genome Atlas data. (A) ITIH1 expression was significantly higher in RCC tissues (n=110) compared with the normal tissues (n=84). (B) ITIH1 expression was significantly higher in both female (n=30) and male (n=80) patients with RCC compared with that in normal controls. (C) Patients with RCC with high ITIH1 expression levels (n=133) showed a significantly poorer survival probability compared with those with low/medium ITIH1 expression levels (n=398) for 4,000 days. *P<0.05. ITIH1, inter-α-trypsin inhibitor heavy chain 1; RCC, renal cell carcinoma.

Figure 2 Expression levels of ITIH1 in renal cell carcinoma cell lines. (A) mRNA expression levels of ITIH1 in 786-O, A498 and ACHN cell lines was significantly lower compared with that in HK-2 cells. (B and C) Protein expression levels of ITIH1 in 786-O, A498 and ACHN cell lines was significantly lower compared with that in HK-2 cells as observed by western blotting. *P<0.05 vs. HK-2 cells. ITIH1, inter-α-trypsin inhibitor heavy chain 1; RCC, renal cell carcinoma.

ITIH1 contributes to the inhibition of renal cancer cell proliferation

The mRNA and protein expression levels of ITIH1 in A498 and ACHN cells were found to be significantly decreased following transfection with siRNAs targeting ITIH1 (Fig. 3A-C). The knockdown efficiency of si-#1 in both cell lines was >70% at mRNA level. On protein level, si-#1 was also more effective at decreasing the protein expression levels of ITIH1 compared with si-#2. Therefore, subsequent transfection experiments used si-#1 (siITIH1). Cells transfected with siITIH1 exhibited significantly increased proliferative capabilities compared with those by NC cells (Fig. 3D). In 786-O cells, knockdown of ITIH1 expression also significantly increased proliferation (Fig. S1A and B). By contrast, overexpression of ITIH1 significantly decreased the proliferation of A498, ACHN and 786-O cells (Fig. S2A and B). Therefore, it could be suggested that ITIH1 negatively regulated proliferation of RCC cells.

Figure 3 Knockdown of ITIH1 promotes cell proliferation in renal cell carcinoma cells. (A) Knockdown efficiency of si-#1 was higher compared with that of si-#2 in A498 and ACHN cells as shown by reverse transcriptase-quantitative PCR. (B) Knockdown efficiency of si-#1 was higher compared that of si-#2 in A498 and ACHN cell lines as shown by western blotting, (C) which was semi-quantified. (D) A498 and ACHN proliferation was increased significantly in the siITIH1-treated group compared with that in the NC group. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control.

ITIH1 negatively regulates cell migration and invasion in renal cancer cells

Significantly increased cell migratory ability was observed in siITIH1-transfected A498 and ACHN cells compared with that in NC cells (Fig. 4A and B). The number of successfully invading cells in the siITIH1 group was also found to be significantly higher compared with that in the NC group in A498, ACHN and 786-O cells (Figs. 4C, D, S1C and D). However, the number of invading cells was significantly reduced in A498, ACHN and 786-O cells following the overexpression of ITIH1 compared with the vector group (Fig. S2C and D). These findings suggest that ITIH1 can suppress migration and invasion by renal cancer cells.

Figure 4 Cell migration and invasion abilities are increased after ITIH1 knockdown. (A) Transwell migration assay images of A498 and ACHN cells transfected with siITIH1. (B) Cell migration was significantly increased in siITHI1 cells compared with that in NC cells. (C) Transwell invasion assay images of A498 and ACHN cells transfected with siITIH1 through Matrigel-covered inserts. (D) Cell invasion was significantly increased in siITIH1 cells compared with that in NC cells. Magnification, x100. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control.

ITIH1 enhances the apoptosis of renal cancer cells

The percentage of apoptotic cells of A498 and ACHN was found to be significantly reduced when ITIH1 expression was knocked down compared with that in NC cells (Fig. 5A and B). The percentage of apoptotic cells in A498 cells in the NC group was 32.07%, whereas it was 7.08% in the siITIH1 group. In ACHN cells, the percentage of apoptotic cells in the NC group was 15.81%, whilst it was 3.20% in the siITIH1 group. Therefore, these results suggest that ITIH1 can promote apoptosis in renal cancer cells.

Figure 5 Apoptosis is suppressed after ITIH1 knockdown in renal cell carcinoma cells. (A) The apoptosis rate was measured by flow cytometry. (B) The apoptosis rate in the siITIH1 group was significantly lower compared with that in the NC group in A498 and ACHN cells. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control.

ITIH1 regulates the NF-κB signaling pathway

NF-κB signaling was activated when ITIH1 expression was knocked down in A498 cells (Fig. 6A and B). Specifically, the phosphorylation level of NF-κB was observed to be significantly increased in the siITIH1 group compared with that in the NC group (Fig. 6C). Furthermore, the protein expression level of IκB was downregulated. By contrast, the protein expression level of IKK, the negative regulator of IκB (19), was upregulated. Additionally, the protein expression levels of proliferation markers cyclin D1 and PCNA, in addition to those of the migration marker α-smooth muscle actin (α-SMA), were significantly increased in the siITIH1 group compared with those in the NC group (Fig. 6A and B).

Figure 6 NF-κB pathway is activated by ITIH1 knockdown. (A) Western blotting analysis of the knockdown of siITHI1 on the expression of components in the NF-κB pathway, showing that the protein levels of (B) piNF-κB, IKK, Cyclin D1, PCNA and α-SMA were significantly increased whereas those of IκB were significantly decreased compared with NC cells. (C) A significant increase in the phosphorylation of NF-κB was demonstrated in siITIH1 cells compared with that in NC cells. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control; pi, phosphorylated; PCNA, proliferating cell nuclear antigen; α-SMA, α-smooth muscle actin.

Treatment of cells with JSH-23, an inhibitor of NF-κB signaling, in addition to siITIH1 transfection, significantly decreased A498 and ACHN cell invasion ability compared with that in the siITIH1 transfection-only group (Fig. 7A and B). In addition, A498 and ACHN cell proliferation was also significantly inhibited when JSH-23 was used in combination with siITIH1 compared with that in the siITIH1 transfection-only group (Fig. 7C).

Figure 7 JSH-23 reverses the effect of ITIH1 in renal cell carcinoma cells. (A) Transwell assay images of A498 and ACHN cells transfected with siITIH1 and treated with JSH-23. (B) JSH-23 reversed the (B) increased invasive capacity and (C) increased proliferation in A498 and ACHN cells transfected with siITIH1. Magnification, x100. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control. Supplementary figure legends

Discussion

Renal cell carcinoma is a major subtype of kidney cancer (1). Treating RCC effectively remains a challenge at present, particularly for patients with distant metastasis (2). The survival and quality of life of patients with RCC has been improved in recent years due to medical improvements. However, there are currently few successful treatment options for patients with late-stage disease (20). An obstacle for the complete remission of such patients is that RCC is a highly heterogeneous cancer and the mechanisms underlying its occurrence remain unknown. Therefore, it is of importance to determine the mechanism driving the initiation and progression of RCC.

TCGA database has been a useful public resource for cancer research over the past decade (21). It provides data from patients with various types of cancer. In particular, the genetic information available in TGCA in each cancer, such as gene expression changes and alterations, have already been explored, making this database a valuable asset for drug development and gene therapy research. Based on the data from TCGA, the present study demonstrated that the expression level of ITIH1 was significantly higher in RCC tumor tissues compared with that in normal tissues. However, in RCC cell lines, ITIH1 exhibited lower expression levels compared with that in the HK-2 control cell line. This expression pattern was the opposite of that demonstrated by data obtained from the TCGA. This may be due to the selected cases of clinical specimens or the small sample size used. The ITIH protein family has been previously reported to be responsible for extracellular matrix stability, the expression of which was frequently lost in certain types of solid tumors, such as breast cancer (9). Additionally, Hamm et al (9) previously showed that ITIH1 expression was lost in kidney cancer. In a number of types of liver and colorectal cancers, ITIH1 was reported to serve a suppressor role in tumor progression and was associated with good clinical prognosis (11,12). In RCC cell lines, it was demonstrated that ITIH1 was significantly downregulated compared with HK-2 cells, which was consistent with the findings reported by Hamm et al (9). Furthermore, based on the TCGA database, patients with RCC with high ITIH1 expression exhibited a poorer survival rate. This is opposite to the role of ITIH1 in HCC (11). Therefore, the role of ITIH1 in the development of RCC requires further exploration.

ITIH1 knockdown in RCC cells was found to significantly increase the proliferation of cells compared with that in the NC group. Additionally, both the migratory and invasion capabilities of RCC cells were significantly increased by ITIH1 knockdown compared with those by NC cells. Tumors can be characterized by aggressive proliferation and expansion (22). Tumor cells will typically lose the contact inhibition feature exhibited by normal cells and proliferate without inhibitions as long as the energy supply is sufficient, which results in the formation of solid tumor spheres (23). In addition, cancer cells can secrete MMPs, which digest the extracellular matrix, causing the leakage of tumor cells (24). This allows cancer cells to invade the surrounding tissues to form new tumor foci (25). In clinical studies, drugs targeting cell proliferation or metastasis have been reported to be efficient in controlling the cancer progression. AKT inhibitors have been documented to potently inhibit cell proliferation and survival in cancer, such as breast cancer and ovarian cancer (26). Furthermore, Traditional Chinese Medicine has been found to effectively suppress cell invasion and migration in certain types of cancers, such as lung cancer and gastric cancer (27). In the present study, ITIH1 knockdown increased cell proliferation and invasion in vitro. Therefore, ITIH1 may serve an important role in the progression of RCC. Inducing apoptosis is a main aim of therapy targeting cancer cells (28). Cancer cells prevent apoptosis by changing the expression levels of critical molecules, such as caspase-3 in the caspase cascade response signaling pathway (22). The present study demonstrated that ITIH1 expression induced apoptosis in RCC cells, suggesting that ITIH1 is a critical molecule in mediating apoptosis of RCC cells.

NF-κB signaling is important for normal embryonic development and physiological activities, such as the inflammatory response (29). However, aberrant activation of NF-κB signaling has been reported in a number of diseases, including certain types of cancers. NF-κB signaling was found to be increased in cancers, such as lung cancer, where targeting the NF-κB pathway may serve as a promising method for therapy (16). Yan et al (30) previously reported that activation of NF-κB by S1P promoted RCC progression. In the present study, it was demonstrated that ITIH1 was a negative regulator of the NF-κB pathway, since this pathway was activated when ITIH1 expression was knocked down in RCC cells. The protein expression levels of proliferative markers Cyclin D1 and PCNA were also found to be upregulated after ITIH1 knockdown in the present study. In addition, the protein expression level of α-SMA was also increased. α-SMA is a gene that positively contributes tO cell migration processes (31). α-SMA was previously reported to accelerate the progression of liver and colorectal cancer by facilitating metastasis (32,33). Therefore, NF-κB signaling may induce the expression of Cyclin D1, PCNA and α-SMA, in turn promoting cell proliferation and metastasis. Treatment of RCC cells with JSH-23, an inhibitor of NF-κB signaling, in addition to siITIH1 transfection, decreased the proliferative and invasion ability of cells compared with the siITIH1 group alone. This further suggested that NF-κB signaling is an important pathway downstream of ITIH1. Akt signaling pathway is another important pathway during development and aberration could lead to cancer (34). The present study also detected the expression of critical members in Akt signaling such as Akt and PI3K. However, no significant alterations of these members occurred. It may be possible that the clinical specimens of the TCGA data are not of a large enough sample size. A larger cohort of samples encompassing more subtypes of RCC may support the findings of the present study. Therefore, the lack of clinical specimens, which could've been used to detect the expression pattern of ITIH1 and analyze the association of ITIH1 expression with clinicopathological factors, is a limitation of the present study. These data could be used to validate the conclusions reported in the present study. Further research is required to fully elucidate the role of ITIH1 expression in clinical specimens and the underlying mechanism of action of ITIH1 in RCC.

In conclusion, data from the present study suggest that ITIH1 can negatively regulate cell proliferation, migration and invasion by RCC cells. NF-κB signaling may be an important signaling pathway regulated by ITIH1 in RCC. The results of the present study may provide a potential avenue of treatment for patients with RCC.

Supplementary Material

Knockdown of ITIH1 promotes cell proliferation and invasion in 786-O cells. (a) Knockdown efficiency of si-#1 was higher compared with that of si-#2 in 786-O cells. (b) Proliferative ability of 786-O cells increased significantly in the siITIH1-transfected group compared with that in the NC group. (c) Transwell assay images demonstrated that (d) the number of invading cells was significantly higher in cells treated with siITIH1 compared with that by NC cells. Magnification, x100. *P<0.05 vs. NC. ITIH1, inter-α-trypsin inhibitor heavy chain 1; si, small interfering RNA; NC, negative control.

Overexpression of ITIH1 inhibits cell proliferation and invasion in RCC. (a) Transfection of RCC cells with oeITIH1 significantly increased the expression level of ITIH1 compared with that in the vector group. (b) Proliferative ability of cells decreased significantly in the oeITIH1-treated group compared with that in the vector group. (c) Transwell assay images demonstrated that (d) the number of invaded cells was significantly lower in the oeITIH1 group compared with that in the vector group. Magnification, x100. *P<0.05 vs. vector. ITIH1, inter-α-trypsin inhibitor heavy chain 1; oe, overexpression.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

SY and JG designed the study, supervised the experiments and reviewed the paper. GYu, JG and YY drafted the paper and performed the experiments. WH, WW and DH performed the data analysis and contributed to the draft of this paper. GYa and JW reviewed the draft and contributed to the data analysis. All authors read and approved the final version of the manuscript. JG and SQY confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors' information

Dr Shiquan Yang ORCID iD: 0009-0005-7416-5603.

References