The protocol of the research project had been approved by the Ethics Committee of the Ruijin Hospital, Shanghai Jiaotong University (Shanghai, China) and it conformed to the provisions of the Declaration of Helsinki. The patients' informed consents were all obtained.

All formalin-fixed paraffin-embedded RCC tissue specimens and normal renal tissue of RCC patients who underwent radical nephrectomy or nephron-sparing surgery were obtained from the Department of Urology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine (Shanghai, China) between 2009 and 2015. The diagnosis of RCC was made by at least two contrast-enhanced imaging studies [ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)], and confirmed by US-assisted fine-needle biopsy. None of patients had received chemotherapy or radiotherapy before the operation. Follow up was performed from the date of tumor resection until May 2015 or until patient mortality. All clinicopathological parameters were collected from the medical records of the hospital.

Specimens were stained at 4°C overnight with LDHA polyclonal antibody (cat. no. SC-27231; dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), E-cadherin polyclonal antibody (cat. no. 3195; dilution, 1:200), N-cadherin polyclonal antibody (cat. no. 13116; dilution, 1:200), vimentin polyclonal antibody (cat. no. 5741; dilution, 1:200) and Snail polyclonal antibody (cat. no. 3879; dilution, 1:200) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). A biotinylated secondary antibody (cat. no. 10K06A; dilution, 1:100; ZSGB-BIO; OriGene Technologies, Inc.) was used to detect the primary antibody at 37°C for 40 min. A score was calculated via multiplying the extent of staining (1, 0–5%; 2, 5–25%; 3, 26–50%; 4, 50–75%; 5, 75–100%) by the staining intensity (0, negative; 1, weak; 2, medium; 3, strong). For the purpose of statistical analyses, tumors with a final staining score of >3 were considered as high expression.

Cells and tissues were lysed in lysis buffer containing protease inhibitor cocktail (50 mM Tris pH 7.5, 150 mM NaCl, 1% TritonX-100 and 5 mM ethylenediaminetetraacetic acid). Protein concentration was determined using a Bio-Rad protein assay system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Western blotting was performed following the routine protocol. Signals were detected on X-ray film using the ECL detection system (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The relative protein levels were calculated based on β-actin as the loading control.

Human RCC cell lines 786-O, OS-RC-2, Caki-1, A498, HK-2 and ACHN were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were preserved in our institute. All the cells were cultured in RPMI-1640 medium (HyClone Laboratories; GE Healthcare Life Sciences, Chalfont, UK) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin and maintained at 37°C with 5% CO₂. Transfection of specific siRNAs targeting LDHA (Shanghai GenePharma Co., Ltd., Shanghai, China) and negative control (NC)-siRNA into RCC cells was performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Following this, the interference efficiency was determined by western blotting. ACHN cells with stable knockdown of LDHA and their control line were established using the lentiviral transduction system (Santa Cruz Biotechnology, Inc.) (26).

RCC cells (1×10⁶) were transfected with siRNAs or cultured with 30 mM oxamate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and prepared for LDHA activity, lactate production and glucose utilization assay. LDHA activity and lactate production assay were performed using the Lactate Dehydrogenase Activity Assay kit (cat. no. MAK066) and Lactate Assay kit (cat. no. MAK064; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Glucose concentration in the media was measured using colorimetric glucose assay kit (BioVision, Milpitas, CA, USA) and normalized according to cell number (27).

The scratch migration assay was performed in 24-well plate, and cells were transfected with control or LDHA siRNA, or cultured with 30 mM oxamate. Cells were scratched using a tip of sterile 200 µl pipette in each well following 24 h. The plates were washed twice with PBS, and then incubated at 37°C in 5% CO₂. Wound width was monitored at various time points and quantified by the ratio of gap distance at 24 or 48 h to that at 0 h. The experiment was conducted in triplicate. The migration and invasion assay were both performed using Transwell (Corning Incorporated, Corning, NY, USA) in 24-well plate. Filters coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were used for invasion assay. At 48 h following RNA interference, cell suspension (1×10⁵ cells in 200 µl serum-free medium) was plated into the upper chamber, while the lower chamber was filled with 500 µl RPMI (HyClone Laboratories; GE Healthcare Life Sciences) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). Following incubation at 37°C for 24 h, tumor cells adhere to the lower surface of the membrane were fixed with methanol and stained with crystal violet. The staining cells were photographed and counted using Image J software.

All procedures for animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80-23, reversed 1996) and according to the Ethical Guidelines for animal experiments of the Ruijin Hospital, Shanghai Jiaotong University (Shanghai, China). The orthotopic renal xenograft model was established in previous research (26). RCC cells were suspended in 20 µl RPMI-1640/Matrigel (1:1; R&D Systems, Inc., Minneapolis, MN, USA) and inoculated into the right subrenal capsule of kidneys of nude mice. At 7 weeks, the two group of ten mice were sacrificed by cervical dislocation and the lungs were removed and embedded in paraffin for evaluating the frequency of metastasis, serial sections from lung were stained with hematoxylin and eosin (H&E) and then screened independently by two researchers who were blinded to the treatment.

All data from at least three independent experiments was presented as mean ± standard deviation and statistically analyzed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). Statistical analysis was performed by Student's t-test. The rate and constituent ratio were compared by the Chi-square test. Kaplan-Meier survival curve was used to analyze patient's overall survival (OS) rates and disease-free survival (DFS) rates, and the differences were compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To verify the expression of LDHA in RCC, the authors examined the level of LDHA in 43 RCC and corresponding non-cancerous renal (CNR) tissues using immunohistochemical staining and western blotting. Subsequent analyses revealed that LDHA was upregulated in 55.8% (24/43) of RCC patients compared with those of CNR tissues (P=0.016; Fig. 1A and B). In addition, western blotting implied that LDHA expression level was enhanced in 786-O and ACHN cells compared to OS-RC-2, Caki-1, A498 and HK-2 cells (Fig. 1C). Therefore, 786-O and ACHN cells were employed in the subsequent experiments. The above observations suggested that LDHA was upregulated in RCC.

In order to investigate the relationship between LDHA expression and clinicopathological characteristics of RCC, the clinical data of 43 RCC patients were retrospectively analyzed. Results showed that increased LDHA expression was significantly correlated with lymph node metastasis (χ²=4.449, P=0.025), distant metastasis (χ²=5.044, P=0.035), and tumor size (χ²=6.894, P=0.009; Table I). The OS rate and DFS rate between the two groups had significant differences, as evidenced by the Kaplan-Meier survival curve and the log-rank test (OS, χ²=3.923, P=0.0476; DFS, χ²=4.245, P=0.0394; Fig. 1D). All of the data indicated that LDHA might have an important role in the development of RCC.

Following this, the authors hypothesized about the functional role of LDHA in tumor cells. Western blotting revealed that LDHA protein levels were obviously decreased in 786-O and ACHN cells that transfected with LDHA-siRNA, compared with their control groups respectively (Fig. 2A). Then assays were performed to investigate the role of LDHA in migration of invasion of RCC cells. The Transwell assay indicated that downregulation of LDHA significantly decreased the number of 786-O and ACHN cells crossing the membrane compared to the control group, as presented in Fig. 2B (P<0.01 for 786-O and ACHN). Matrigel invasion assay showed that the number of LDHA-siRNA cells crossing Matrigel-coated membrane significantly decreased compared to the control group (P<0.01 for 786-O and ACHN, Fig. 2C). The same conclusions were reached following the scratch migration assay (P=0.0374 for 786-O and P=0.0246 for ACHN, Fig. 2D and E). These data illustrated that the low-expression of LDHA significantly suppresses the ability of migration and invasion of RCC cells.

To determine the function of LDHA on tumor metastasis in vivo, an orthotopic renal xenograft model was established in the present research. At 7 weeks, the mice were sacrificed and the pulmonary metastasis foci were counted. Of note, average metastatic nodules of lung were dramatically decreased in the LDHA-siRNA group, compared to NC siRNA groups (P<0.033; Table II). Hematoxylin and eosin (H&E) staining of serial sections of lung were used to identify metastasis nodules (Fig. 2F). These results suggested that LDHA is a critical factor inhibiting RCC metastasis in vivo.

Accumulating evidence has demonstrated that the Warburg effect played an important role in the development of tumor, and may have potential clinical applications (24). The influence of LDHA on the Warburg effect in RCC cells was also investigated. In the present study, the effect of LDHA on glucose utilization and lactate production was assessed. Significant decreasing in glucose utilization (P=0.0019 for 786-O cells and P=0.035 for ACHN cells) and lactate production (P<0.01 for 786-O cells and P=0.019 for ACHN cells) in LDHA-siRNA RCC cells were observed (Fig. 3A and B). These data indicated that LDHA could affect the Warburg effect, which influences the development of RCC.

Limited investigation attempted to elucidate that LDHA could promote EMT in muscle-invasive bladder cancer (25). In the current study, retrospective analyses showed that LDHA expression was related to tumor metastasis. Therefore, the authors ask whether LDHA expression was related to EMT process. To this end, the correlation between the expression of LDHA and EMT markers was tested. The results of IHC indicated that the level of reduction of E-cadherin (P=0.0498) and elevation of N-cadherin (P=0.0286) and vimentin (P=0.033) were dramatically correlated with the upregulation of LDHA (Fig. 4A). In addition, a negative correlation between LDHA expression and E-cadherin expression and a positive correlation between LDHA expression and N-cadherin were explored using Pearson's correlation coefficient (Fig. 4B). Western blotting of RCC tissues revealed that the downregulation of LDHA was negatively associated with epithelial phenotype and positively associated with mesenchymal phenotype, which was consistent with the IHC observations (Fig. 4C).

In order to substantiate the hypothesis that LDHA may inhibit EMT, we further tested the EMT makers in LDHA-siRNA cells and the control group, which yielded similar results. An increased level of E-cadherin was observed as a response to the treatment of LDHA-siRNA in RCC cells. On the contrary, N-cadherin and vimentin were attenuated in LDHA-siRNA cells compared to the control group (Fig. 4D). Furthermore, downregulation of LDHA suppressed the expression of EMT inducer Snail (Fig. 4C and D). These findings make clear that EMT process plays an important role in LDHA induced metastasis in RCC cells.

Considering the clinical application of target therapy, the authors treated the RCC cells with oxamate to investigate the contribution of LDHA inhibitor to LDHA activity and LDHA-driven EMT. The concentration of oxamate was 30 mM according to the authors' previous study. The results demonstrated that oxamate significantly decreased LDHA activity, glucose utilization, and lactate production of RCC cells compared with the control group (P<0.01; Fig. 5A). As illustrated in Fig. 5B, oxamate apparently inhibited the ability of migration and invasion of 786-O RCC cells by Transwell assays compared to the control group (P<0.01). The scratch migration assay, in line with Transwell assays confirmed that oxamate significantly suppressed the migration of 786-O RCC cells (P<0.05; Fig. 5C). The above observations increasingly support the notion that LDHA promote RCC metastasis by mechanisms orchestrated by EMT.