DLX5 expression levels in kidney renal clear cell carcinoma (KIRC) and normal tissues were evaluated using the UALCAN (http://ualcan.path.uab.edu/index.html), TIMER (https://cistrome.shinyapps.io/timer/), and GEPIA (http://gepia2.cancer-pku.cn) databases.

RCC cell lines 786-O (CL-0010), A-498 (CL-0254) and Caki-1 (CL-0052), and HK-2 (CL-0109) cells were purchased from Procell (Wuhan, China). A-498 and HK-2 cells were cultured in Minimum Essential Medium (MEM, PM150410, Procell), while 786-O and Caki-1 cells were grown in RPMI-1640 (PM150110, Procell) and McCoy’s 5A media (PM150710, Procell), respectively, with 10% fetal bovine serum (FBS, 164210-50, Procell) and 1% penicillin/streptomycin (PB180120, Procell). Cells were authenticated by short tandem repeat (STR) profiling, tested negative for mycoplasma, and maintained in a 37 °C, 5% CO₂ incubator.

Two small interfering RNAs (siRNAs) targeting DLX5 (si-DLX5#1 and si-DLX5#2) and corresponding negative controls (si-NC) were synthesized by GenePharma (Shanghai, China). The sequences of si-DLX5#1, si-DLX5#2, and si-NC were 5^′-AGCUAUAGUCGGCAUAAGCUU-3^′, 5^′-GUGCAGCCAGCUCAAUCAA-3^′, and 5^′-UUCUCCGAACGUGUCACGU-3^′, respectively. Overexpression of c-Myc was achieved by transfecting pcDNA vector plasmids containing c-Myc (NM_005221.5) sequences (OE-c-Myc). The following transfections were performed: si-DLX5#1, si-DLX5#2, si-NC, si-NC+empty pcDNA vector plasmids (vector), si-DLX5#1+vector, and si-DLX5#1+OE-c-Myc into 786-O and Caki-1 cell lines using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), and the cells were then collected for subsequent analyses 48 hours later.

Total proteins were extracted using RIPA buffer (R0010, Solarbio, Beijing, China) and quantified with a BCA protein assay (ab102536, Abcam, Cambridge, UK). Proteins (20 µg) were separated by SDS-PAGE and transferred to PVDF membranes (EMD Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk (D8340, Solarbio) for 1 hour at room temperature, then incubated overnight at 4 °C with primary antibodies against DLX5 (1:1000, ab64827), gamma histone H2AX ($\gamma$H2AX) (1:1000, ab2893), c-Myc (1:20,000, ab152146), and $\beta$-actin (1:5000, ab8227) (all Abcam). Following this, membranes were incubated with horseradish peroxidase (HRP)-conjugated Anti-Rabbit IgG (1:50,000, ab288151, Abcam) for 2 hours and visualized using an ECL assay (P0018S, Beyotime, Shanghai, China).

Cells (3000 per well) were plated in 96-well plates and cultured overnight at 37 °C with 5% CO₂. After treatments, 10 µL of CCK-8 reagent (C0037, Beyotime) was added to each well, the plates were incubated for 2 hours at 37 °C, and optical density (450 nm) was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Cells (6 $\times$ 10⁵ per well) were plated in 6-well plates and incubated at 37 °C with 5% CO₂. After 12 hours, cells were exposed to 1 mL of 20 µM EdU solution (C0081, Beyotime) for 2 hours. Cells were then fixed, permeabilized, and treated with an anti-EdU Click solution (C0081, Beyotime). Nuclei were stained with Hoechst 33342 (C1025, Beyotime). Images were captured using fluorescence microscopy (IX71, Olympus, Tokyo, Japan), and the percentage of EdU-positive cells was calculated from five random fields.

Cells were stained with propidium iodide (PI) and fluoresceinisothiocyanate (FITC)-Annexin V (C1062S, Beyotime), and then washed with cold PBS (C0221A, Beyotime). Apoptosis was assessed using a FACScan flow cytometer (BD Biosciences, NJ, USA), and data was analyzed using BD CellQuest Pro software (version 5.1, BD Biosciences).

Cells were irradiated in ambient air with total doses of 0, 2, 4, 6, and 8 Gray (Gy) using a JL Shepherd Mark 1–68 137Cs irradiator. Mice with subcutaneous tumors were irradiated with 10 Gy using an XRad 320 irradiator (Precision X-Ray, 250 kV/s, 15 mA) following anesthesia with isoflurane (R510-22, RWD LIFE SCIENCE, Shenzhen, China).

Cells (800 per well) were plated in 6-well plates and incubated at 37 °C for 14 days. After fixation with 4% paraformaldehyde (P0099, Beyotime), cells were stained with 0.1% crystal violet (C0121, Beyotime) for 30 minutes. Colonies with 50 or more cells were counted manually and photographed. Plating efficiency (PE) was calculated by dividing the number of colonies in the control group by the number of cells plated. The survival fraction was determined by dividing the number of colonies by the product of the number of cells plated and the PE.

Cells were plated on glass coverslips and cultured overnight. After irradiation with 4 Gy, cells were collected at 0, 1, 4, and 8 hours. For immunofluorescence, cells were fixed with 4% paraformaldehyde (P0099, Beyotime) for 15 minutes, washed with PBS (C0221A, Beyotime), and permeabilized with 0.5% Triton X-100 (P0096, Beyotime) for 20 minutes. Following overnight incubation with anti-$\gamma$H2AX antibody (1:5000, ab11174, Abcam) at 4 °C, cells were washed and incubated with Goat anti-rabbit IgG-AlexaFluor 488 (1:1000, ab150077, Abcam) for 1 hour at 37 °C. Cells were mounted with DAPI-containing medium (S2110, Solarbio) and visualized by fluorescence microscopy.

Four-week-old BALB/c nude mice (Vital River, Beijing, China) were housed in a temperature-controlled specific pathogen free (SPF) facility with a 12-hour light-dark cycle. Mice were randomly assigned to four groups (n = 6 each): sh-NC, sh-DLX5#1, 10 Gy, and sh-DLX5#1+10 Gy. Each group had six mice throughout the study. The left flank of mice in the sh-NC and sh-DLX5#1 groups were subcutaneously injected with 2 $\times$ 10⁶ 786-O cells transfected with sh-NC or sh-DLX5#1, respectively. The 10 Gy group received 2 $\times$ 10⁶ 786-O cells and was irradiated with 10 Gy when tumors became measurable. Mice in the sh-DLX5#1+10 Gy group were injected with 2 $\times$ 10⁶ 786-O cells transfected with sh-DLX5#1 and then irradiated with 10 Gy when tumors were measurable. Tumor volumes were measured weekly using the formula: 1/2 $\times$ length $\times$ width². After four weeks, mice were euthanized with isoflurane (R510-22, RWD LIFE SCIENCE), and tumors were excised, weighed, and stored at –80 °C. All procedures were approved by the Animal Research Ethics Committee of Zhejiang Huitong Test & Evaluation Technology Group Co., Ltd (Approval No. HT-2023-LWFB-0018) .

Tumor samples were fixed in 4% formaldehyde (P0099, Beyotime), dehydrated, and embedded in paraffin (YA0012, Solarbio). Antigen retrieval was done with 10 mM sodium citrate buffer (pH 6.0, P0083, Beyotime) at 94 °C for 15 minutes. Sections were blocked with 1% bovine serum albumin (BSA) (ST023, Beyotime), then incubated with primary antibodies against DLX5 (1:100, ab64827, Abcam), c-Myc (1:100, ab32072, Abcam), and Ki-67 (1:100, ab15580, Abcam). Biotinylated secondary antibodies (1:2000, ab64256, Abcam) were used, and sections were counterstained with hematoxylin (C0105S, Beyotime). The samples were imaged with a light microscope (Olympus).

Data are presented as mean $\pm$ standard deviation (SD). Normality and variance homogeneity tests confirmed a normal distribution. For comparisons between two groups, an unpaired Student’s t-test was used. For comparisons among three or more groups, one-way ANOVA with Dunnett’s post hoc test was applied. Post hoc analyses were conducted with the Bonferroni method in SPSS 26.0 (IBM Corp., Chicago, IL, USA). Statistical significance was defined as p 0.05.

Pan-cancer analysis revealed a significant increase in DLX5 expression across various cancer types, including KIRC (Fig. 1A). Specifically, DLX5 was markedly upregulated in KIRC samples compared to normal kidney tissue samples (Fig. 1B,C). Additionally, high levels of DLX5 were validated in RCC cell lines (Fig. 1D). Notably, DLX5 expression was higher in 786-O and Caki-1 cells compared to A-498 cells, leading to the selection of 786-O and Caki-1 for further experiments. These findings indicate that DLX5 is highly expressed in RCC.

Fig. 1.

Elevated expression of DLX5 in renal cell carcinoma (RCC). (A) Pan-cancer analysis demonstrated increased DLX5 expression in kidney renal clear cell carcinoma (KIRC). *p 0.05, **p 0.01, and ***p 0.001 vs. Normal. (B) The Gene Expression Profiling Interactive Analysis (GEPIA) analysis showed heightened DLX5 expression in KIRC. ***p 0.001 vs. Normal. (C) Alignable Tight Genomic Clusters (ATGC) database results confirmed the upregulation of DLX5 in KIRC tumor samples compared to normal samples. *p 0.05 vs. Normal. (D) DLX5 levels in RCC cell lines. ***p 0.001 vs. HK-2. n = 3.

Given the high DLX5 expression in RCC cells, we used two siRNAs targeting DLX5 (si-DLX5#1 and si-DLX5#2) to lower DLX5 levels in 786-O and Caki-1 cells (Fig. 2A). DLX5 silencing significantly reduced cell proliferation, as shown by CCK-8 (Fig. 2B) and EdU assays (Fig. 2C and Supplementary Fig. 1), and increased apoptosis, as indicated by flow cytometry (Fig. 2D). Additionally, DLX5 knockdown enhanced p53 protein expression (Fig. 2E), suggesting DLX5’s role in regulating p53-mediated apoptosis and RCC cell proliferation. Thus, DLX5 downregulation impairs RCC cell growth and promotes apoptosis.

Fig. 2.

Silencing of distal-less homeobox 5 (DLX5) represses cell proliferation and enhances apoptosis in RCC cells. (A) DLX5 levels were decreased in 786-O and Caki-1 cells using si-DLX5#1 and si-DLX5#2, with $\beta$-actin as the normalization control. (B,C) Proliferation was assessed by cell counting kit-8 (CCK-8) (B) and, 5-Ethynyl-2^′-deoxyuridine (EdU) incorporation assay (C) . Scale bars = 100 µm. (D) Apoptosis was evaluated by flow cytometry. (E) p53 protein expression was measured by Western blot, normalized to $\beta$-actin. **p 0.01, and ***p 0.001 vs. small interfering RNA-negative control (si-NC). n = 3.

The impact of DLX5 on radioresistance was assessed in 786-O and Caki-1 cells. Following transfection with si-DLX5#1 and si-DLX5#2, both cell lines exhibited a dose-dependent reduction in survival rates (Fig. 3A). Notably, the survival rates of 786-O and Caki-1 cells were significantly diminished following 8 Gy of radiation (RI). Additionally, cell viability was markedly reduced at a total dose of 4 Gy (p 0.01), with further significant decreases observed upon DLX5 silencing (Fig. 3B). These findings indicate that DLX5 knockdown effectively impairs the radioresistance of RCC cells.

Fig. 3.

Interference with DLX5 inhibits radioresistance in RCC cells. (A) Colony formation assays assessed survival fractions of 786-O and Caki-1 cells transfected with si-NC, si-DLX5#1, and si-DLX5#2 following irradiation at 0, 2, 4, 6, and 8 Gray (Gy). **p 0.01 vs. si-NC. (B) Cell viability was measured by CCK-8 assay after 4 Gy irradiation and 96 hours of culture. **p 0.01, and ***p 0.001. n = 3.

To examine DLX5’s impact on DNA damage, we analyzed 786-O and Caki-1 cells. DLX5 downregulation significantly increased $\gamma$H2AX levels (Fig. 4A and Supplementary Fig. 2). After 4 Gy irradiation for 8 hours, DLX5 silencing further elevated $\gamma$H2AX expression (Fig. 4B), suggesting that DLX5 knockdown exacerbates DNA damage in RCC cells.

Fig. 4.

Interference with DLX5 increases DNA damage in RCC cells. (A) The relative gamma histone H2AX ($\gamma$H2AX) levels were detected by immunofluorescence (IF) in 786-O and Caki-1 cells. Scale bars = 50 µm. (B) The relative protein expression of $\gamma$H2AX was measured by Western blot in both cell lines. Data are normalized to $\beta$-actin. *p 0.05, **p 0.01, and ***p 0.001 vs. si-NC. n = 3.

c-Myc is known to interact with and modulate the promoters of DNA double-strand break (DSB) repair genes, thereby enhancing radioresistance in tumor cells [9, 10]. Additionally, DLX5 has been shown to directly bind to the c-Myc promoter in lung tumor cells [17], suggesting that DLX5 may influence radioresistance via the regulation of c-Myc. In this study, we found that DLX5 modulated c-Myc expression, as silencing DLX5 significantly reduced the 4 Gy-induced relative protein expression of c-Myc in 786-O cells (Fig. 5A). Further validation was performed by co-transfecting 786-O cells with an overexpression plasmid of c-Myc and si-DLX5#1. Results showed that overexpression of c-Myc significantly rescued the reduced c-Myc expression induced by si-DLX5#1, both with and without radiation treatment (Fig. 5B). Moreover, c-Myc overexpression in 786-O cells restored the decreased survival rate (Fig. 5C) and cell viability (Fig. 5D) caused by DLX5 silencing. Additionally, increased c-Myc expression markedly reduced the $\gamma$H2AX levels induced by si-DLX5#1 (Fig. 5E). Collectively, these findings demonstrate that DLX5 promotes radioresistance through modulation of c-Myc.

Fig. 5.

DLX5 enhances radioresistance through c-Myc. (A) c-Myc protein levels were measured in 786-O cells transfected with si-NC, si-DLX5#1, or si-DLX5#2, normalized to $\beta$-actin. (B) c-Myc expression was assessed in cells transfected with si-NC+vector, si-DLX5#1+vector, or si-DLX5#1+OE-c-Myc, with data normalized to $\beta$-actin. (C) Colony formation assays determined survival fractions in the same transfections. (D) Cell viability was assessed by the CCK-8 assay for the same conditions. (E) $\gamma$H2AX protein expression was analyzed by Western blot, normalized to $\beta$-actin. *p 0.05, **p 0.01, and ***p 0.001. n = 3.

Lastly, we evaluated the role of DLX5 in tumor progression and radioresistance in nude mice. DLX5 knockdown or irradiation with 10 Gy significantly reduced tumor volume and weight. This reduction was further pronounced when DLX5 silencing was combined with 10 Gy irradiation (Fig. 6A). Similar trends were observed in the expression levels of ki-67 and c-Myc, which were significantly decreased under these conditions (Fig. 6B). Additionally, both DLX5 silencing and 10 Gy irradiation led to a marked increase in $\gamma$H2AX protein levels, with the combination of DLX5 knockdown and 10 Gy irradiation resulting in the most substantial increase (Fig. 6C). These findings demonstrate that downregulation of DLX5 effectively suppresses radioresistance in vivo.

Fig. 6.

Silencing of DLX5 inhibits radioresistance in vivo. Nude mice were inoculated subcutaneously with 2 $\times$ 10⁶ 786-O cells transfected with sh-NC or sh-DLX5#1, and irradiated with 10 Gy when tumors became measurable. (A) Tumor volume and weight were monitored. (B) c-Myc and Ki-67 expression levels were assessed by immunohistochemistry (IHC). Scale bars = 200 µm. (C) $\gamma$H2AX protein levels were measured by Western blot, normalized to $\beta$-actin. *p 0.05, and ***p 0.001. n = 6.