All protocols involving human specimens were reviewed and approved by the Ethics Review Board at Xiantao First People’s Hospital (No. 2018H00109). Written informed consents were obtained from all participants. RCC tissues (CT) and adjacent noncancerous tissues (NT) were obtained from 10 RCC patients who underwent surgical resection at Xiantao First People’s Hospital. None of the 10 patients received any anti-RCC treatment before the surgery. The clinicopathological data of the patients were listed in Supplementary Table S1. Harvested tissue samples were immediately stored at −80°C till use.

Human normal renal proximal tubule epithelial cells (RPTEC), RCC cell lines A498 and 786-O were all purchased from ATCC, and cultured in DMEM/F12, EMEM and RPMI-1640 medium, respectively. A final concentration of 10% FBS (Gibco, Thermo Fisher Scientific) and antibiotics were added to all growth media. The human TUFT1 expression plasmid, designated as pTUFT1, was a kind gift from Dr. Kai Hu from Wuhan Institute of Virology, Chinese Academy of Sciences. TUFT1 shRNA (cat no: sc-61736-SH) and control shRNA (cat no: sc-108060) were both purchased from Santa Cruz Biotechnology.

RT-PCR was performed as previously described with modifications [18, 22]. In brief, total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse-transcribed into cDNA with FastQuant RT Kit (TIANGEN), according to the manufacturer’s instructions. TUFT1 mRNA level was then semi-quantified using a SYBR Green-based RT-PCR. GAPDH was used as an internal control. The primer pair used for TUFT1 detection were (forward) 5′-TCA​GAC​TGT​GTG​GCT​TTT​GAG-3′ and (reverse) 5′-GTC​AGC​ATT​GTT​GCT​CCG​AAG-3′. The primer pair used for GAPDH detection were (forward) 5′- GCC​AAG​GTC​ATC​CAT​GAC​AAC​TTT​GG-3′ and (reverse) 5′- GCC​TGC​TTC​ACC​ACC​TTC​TTG​ATG​TC-3′. The relative expression of TUFT1 was calculated using the 2^−ΔΔCt method.

All transfections were conducted using Lipo8000™ Transfection Reagent (Beyotime) according to the manufacturer’s instructions. In brief, plasmids were first diluted in Opti-MEM (Thermo Fisher Scientific) and then mixed with Lipo8000™ Transfection Reagent. Afterwards, the prepared the mixture was directly added into cell culture and cultured for 48 h. For signaling pathway inhibitor treatment, transfection mixture was removed 4 h post transfection and fresh medium supplemented with inhibitors were added and cells were cultured in the presence of inhibitor for another 48 h. The following signaling pathway inhibitors were used in the current study: SP600125 (JNK inhibitor; 5 μM), LY294002 (PI3K inhibitor; 25 μM) and PD98059 (MEK inhibitor; 50 μM). All inhibitors were purchased from Selleck and used as per the manufacturer′s instructions.

Western blot was performed as previously described with modifications [20, 23]. In brief, cells were first lysed using lysis buffer (150 mM NaCl, 1% NP-40 and 50 mM Tris pH 8.0) supplemented with protease inhibitor cocktail (Beyotime), and then cleared cell lysates were mixed with SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol) and boiled for 10 min. Prepared samples were then separated by a 12% SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was subsequently blocked with 5% non-fat milk for 1 h at room temperature. After three washes with PBST, membrane was then sequentially incubated with primary antibodies and HRP-conjugated secondary antibodies overnight at 4°C and 1 h at room temperature, respectively. Following extensive washes, membrane was developed with ECL substrate (Beyotime), according to the manufacturer’s instructions. The following primary antibodies were used: rabbit anti-human TUFT1 antibody (1:1,000 dilution; Cat no: 23385-1-AP; Proteintech), mouse anti-human E-cadherin antibody (1:1,000 dilution; Cat no: 60335-1-Ig; Proteintech), mouse anti-human N-cadherin antibody (1:1,000 dilution; Cat no: 66219-1-Ig; Proteintech), rabbit anti-human Snail (1:1,000 dilution; Cat no: 13099-1-AP; Proteintech), mouse anti-human AKT antibody (1:1,000 dilution; Cat no: 60203-2-Ig; Proteintech), rabbit anti-human phospho-AKT (Thr308) (p-AKT (Thr308), 1:1,000 dilution; Cat no: 9275; Cell Signaling Technology), mouse anti-human phospho-AKT (Ser473) (p-AKT (Ser473), 1:1,000 dilution; Cat no: 66444-1-Ig; Proteintech) and mouse anti-human GAPDH (1:2,000 dilution; Cat no: A00227; Boster). The following secondary antibodies were used: HRP-conjugated goat anti-mouse IgG (H + L) (1:30,000 dilution; Cat no: BA1050; Boster) and HRP-conjugated goat anti-rabbit IgG (H + L) (1:30,000 dilution; Cat no: BA1054; Boster). Western blot band intensity was semiquantified by ImageJ (version 1.53c; National Institutes of Health, Bethesda, MD, United States) and relative protein expression was calculated using GAPDH as the reference.

The cell proliferation was assessed using a MTT cell proliferation and cytotoxicity assay kit (Sangon Biotech), according to the manufacturer’s instructions. In brief, cell culture medium was removed, and cells were treated with 1:1 diluted MTT solution in fresh serum-free medium for 3 h at 37°C. Following incubation, MTT solvent was added into each well and incubated on an orbital shaker for 15 min. Finally, the samples were read at OD590 nm with a microplate reader.

Cell migration was determined by wound healing assay, as previously described with modifications [24, 25]. In brief, cells pre-seeded at 50–60% confluency were first transfected with vector, pTUFT1, control shRNA or TUFT1 shRNA, and when cells formed a monolayer, a “wound” was introduced by scratching the cell monolayer with a sterile 200 µl tip. After washes with PBS to remove non-attached cells, the cell monolayer was then incubated in fresh medium and cultured for 24 h. The cell migration was measured by the “wound healing” rate. For cells receiving inhibitor treatment, inhibitors or mock controls were introduced into the culture right after the PBS washes.

All data were expressed as mean ± standard deviation (SD), and all statistical analyses were performed with Prism 8 (GraphPad). Either a Mann-Whitney test or a Kruskal-Wallis test with Dunn’s multiple comparisons test were used, and a p value less than 0.05 was considered statistically significant.

TUFT1 expression was previously reported to be elevated in HCC cancer tissue (CT) comparing to adjacent noncancerous tissue (NT) [20]. To check if a similar trend could be observed in RCC, we collected 10 pairs of CT and NT samples from RCC patients who underwent surgical resection and measured the TUFT1 mRNA and protein levels. To rule out possible drug interference, none of the patients had received any anti-RCC treatment before surgery. Our data showed similar results to what was reported in HCC (Figures 1A,B,D and Supplementary Figure S1). Namely, both the mRNA and protein levels of TUFT1 was significantly increased in CT comparing to adjacent NT.

We next further investigated whether such elevation in TUFT1 expression could also be observed in RCC cell lines. TUFT1 mRNA level in one normal renal proximal tubule epithelial cells (RPTEC) and 2 RCC cell lines A498 and 786-O were measured by RT-PCR. Consistently, TUFT1 mRNA was significantly increased in both RCC cell lines (Figure 2A). Similar results were observed when the TUFT1 protein level was determined by Western blot (Figures 2B,C). Taken together, our data here indicate that TUFT1 expression is upregulated in RCC.

To investigate the impact of TUFT1 expression on RCC cell growth and migration, TUFT1 expression was either upregulated or downregulated in A498 cells, and then cell proliferation and migration were determined. As shown in Figure 3A and Supplementary Figure S2, transfection of pTUFT1 considerably increased TUFT1 protein expression in A498 cells. In accordance with the increased TUFT1 expression, the proliferation rate of the A498 cells with TUFT1 overexpression showed significantly enhanced cell growth (Figure 3C) and migration (Figures 3E,G), comparing to vector-transfected cells. By contrast, cells with TUFT1 shRNA transfection showed downregulated TUFT1 expression (Figure 3B). Accordingly, cells with decreased TUFT1 showed slower proliferation rate (Figure 3D) as well as slower migration (Figures 3F,H). Together, these data indicate that TUFT1 enhances RCC cell growth and migration.

EMT is a biological process allowing immobilized polarized epithelial cells transdifferentiate into motile mesenchymal cells, which exhibit enhanced invasiveness and resistance to apoptosis [26, 27]. The activation of an EMT process is considered the critical mechanism mediating cancer metastasis [28]. The involvement of TUFT1 in the regulation of EMT in RCC cells were subsequently investigated by determining the expression of EMT-related markers (E-cadherin, N-cadherin and Snail). E-cadherin is an important adhesion molecule maintaining the epithelial phenotype while loss of E-cadherin leads to enhanced cell mobilization and activation of several EMT transcription factors [29]. Decrease of E-cadherin is considered the EMT hallmark. N-cadherin, in contrast, is mainly expressed in non-epithelial cells and serves as an indicator of ongoing EMT [30, 31]. Snail is one of the essential EMT-inducing transcription factors that facilitates a mesenchymal phenotype [32]. Our data showed that in A498 cells with TUFT1 overexpression, the expression of E-cadherin was decreased while the expression of N-cadherin and Snail was increased, indicating that TUFT1 promoted EMT of RCC cells (Figures 4A,C). To further confirm that TUFT1 is involved in EMT of RCC cells, a downregulation of TUFT1 in A498 cells was performed by TUFT1 shRNA. By contrast to TUFT1 overexpression, elevation of E-cadherin expression while decrease of N-cadherin and Snail were observed in TUFT1 knockdown cells (Figures 4B,D). In accordance with the cell line data, E-cadherin expression was decreased in primary RCC tumor tissue than in adjacent noncancerous tissue (Figures 1C,E). Furthermore, a negative correlation between TUFT1 and E-cadherin expression was detected in the CT but not NT clinical samples (Figures 1F,G). Together, our data demonstrate that TUFT1 promotes EMT progression of RCC cells.

We next explored possible signaling pathways underlying TUFT1-mediated proliferation, migration and EMT progression of RCC cells. Canonical pathways involved in cancer progression (JNK, PI3K, and AKT pathways) were investigated using signaling pathway inhibition assay [33–35]. The effects of signaling pathway inhibitors on RCC cell proliferation was first determined. As shown in Figure 5A, the overexpression of TUFT1, in consistent with our previous data, promoted cell proliferation, and the addition of inhibitor solvent, or JNK and MEK inhibitors did not induce any apparent impact on the cell growth. Of note, the enhancement of RCC cell proliferation by TUFT1 overexpression was significantly suppressed by the introduction of PI3K inhibitor into the system, indicating PI3K pathway was involved in TUFT1-mediated RCC cell proliferation. Further experiments were performed to confirm whether this pathway was also involved in TUFT1-mediated migration and EMT progression of RCC cells. Similar to the proliferation assay, the migration assay showed that TUFT1 overexpression promoted RCC cell migration while the PI3K inhibitor counteracted at such enhancement (Figures 5B,C). Western blot results showed that TUFT1 overexpression did not increase the expression of AKT, a serine/threonine-specific protein kinase playing an important role in PI3K pathway but increased the phosphorylation of AKT (p-AKT) (Figure 5D). In consistent, the elevation of p-AKT, N-cadherin and Snail as well as the decrease of E-cadherin induced by TUFT1 overexpression were suppressed by the addition of PI3K pathway inhibitor (Figure 5D). These data here indicate that TUFT1 exerts oncogenic effects on RCC cells through PI3K/AKT signaling pathway.

Taken together, our current study has revealed that TUFT1 expression is increased in RCC tissue and cell lines both on the mRNA and protein levels. TUFT1 elevation in RCC can promote cancer cell proliferation, migration and EMT progression, via the PI3K/AKT signaling pathway.