All procedures were authorized by the Academic Ethics Committee of Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University (approval no. 2021-071) and were strictly implemented in accordance with the National Laboratory Animal Guide (27). Laboratory procedures were performed in such a manner as to reduce the pain and discomfort caused to mice. The need for approval for the use of primary human BMSCs was waived by the Ethics Committee of Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University.

The StarBase database (starbase.sysu.edu.cn/panCancer.ph) was used to determine PCYR1 expression in BC as follows: Select 'Gene Differential Expression', search 'PCYR1 (ENSG00000183010)', and select 'Bladder Urothelial Carcinoma'. The results were downloaded from the database.

Normal bladder epithelial cell SV-HUC-1 and BC cell lines T24, 5637, RT4, and TCCSUP (ATCC) were cultured in the RPMI-1640 medium (all from Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator (Eppendorf) supplied with 5% CO₂ air at 37°C. Cells in the logarithmic growth phase were collected and PCYR1 levels in cells were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting.

Human BMSCs (cat. no. 7500, ScienCell Research Laboratories, Inc.) were cultured and subcultured according to the manufacturer's instructions. After 5 subcultures, Exos in the BMSC supernatant culture medium were extracted by ultracentrifugation. BMSCs were cultured to 60% confluence, and the medium was replaced with Exo-free serum (EXO-FBS-50A-1, SBI). When the cell density reached 85%, the supernatant was centrifuged at 4°C at 2,000 g for 15 min. The collected supernatant was then centrifuged at 4°C at 16,500 g for 30 min, filtered with 0.22-µm filter membranes to remove particles >200 nm, and centrifuged twice at 4°C at 100,000 g for 70 min, and the Exos in the lower layer were collected, resuspended in 100 µl PBS, and stored at -80°C. The supernatant of BMSCs treated with GW4869, an inhibitor of exosome secretion (10 µM, cat. no. HY-19363, MedChemExpress), for 24 h was used as the negative control (NC) of Exos (28).

Cy3-si-PYCR1 (orange fluorescent label) was synthesized by Shanghai GenePharma Co., Ltd. The extracted Exos and Cy3-si-PYCR1 were added to the electroporation cuvettes (Bio-Rad Laboratories, Inc., Gene Pulser Xcell electroporation system) at the mass ratio of 5:1 (500 ng:100 ng) and were electroplated for 2 sec at 400 V and 125 µF. Subsequently, the mixture was centrifuged at 135,000 g and 4°C for 70 min to obtain Exo-si-PYCR1, resuspended in 100 µl PBS and stored at -80°C.

The morphological structure of Exos and Exo-si-PYCR1 was observed using a transmission electron microscope (TEM) (FEI). The particle size distribution and ζ potential of Exos and Exo-si-PYCR1 were measured using a Malvern Laser Particle Sizer (DLS, Zetasizer Nano ZS90, Malvern Instruments). The positive expression of surface marker proteins CD9, CD81, CD63, and TSG101 (Abcam) in Exos and Exo-si-PYCR1 was detected by western blotting. The content changes of free si-PYCR1 in Exos, free si-PYCR1, and Exo-si-PYCR1 after electroporation were determined by agarose gel electrophoresis to determine whether si-PYCR1 was loaded into Exos.

T24 cells (2×10⁵) were seeded in the confocal Petri dishes and used for the control group, si-PYCR1 group, and Exo-si-PYCR1 group. The control cells were not treated, cells in the si-PYCR1 group were transfected with free Cy3-si-PYCR1 (100 ng) using Lipofectamine^® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.), and cells in the Exo-si-PYCR1 group were transfected with Exo-si-PYCR1 with Cy3-si-PYCR1 (100 ng). After 24 h of transfection, cells were incubated with immunostaining fixative (cat. no. P0098, Beyotime Institute of Biotechnology) for 10 min and washed with immunostaining detergent (cat. no. P0106, Beyotime Institute of Biotechnology) for 5 min x 4 times. Then, to the confocal Petri dishes, 200 µl green-fluorescent probe was added (Actin-Tracker Green, cat. no. C1033, Beyotime Institute of Biotechnology) diluted with immunofluorescence staining secondary antibody diluent (1:200, cat. no. P0108, Beyotime Institute of Biotechnology) and incubated at 37°C for 30 min. After staining, the cells were washed with detergent for 5 min x 4 times, incubated with DAPI working solution for 10 min, and washed with PBS 4 times; the uptake rate (%) of Cy3-si-PYCR1 by T24 cells was observed under a laser confocal microscope (magnification, ×200, Leica GmbH).

T24 cells and RT4 cells were seeded in 6-well plates, cultured until 80% confluence, and divided into the following groups based on different treatments: i) blank group, T24 cells or RT4 cells normally cultured for 48 h; ii) si-PYCR1 group and si-NC group, T24 cells transfected with the si-PYCR1 or the NC, respectively; iii) oe-PYCR1 group and oe-NC group, RT4 cells transfected with the oe-PYCR1 or the NC, respectively; iv) oe-PYCR1 + Vehicle group and oe-PYCR1 + CL-387785 group, RT4 cells treated with EGFR inhibitor CL-387785 (1 µM, cat. no. HY-10325, MedChemExpress) or solvent (0.1% DMSO) for 2 h and transfected with oe-PYCR1. The dosage and method of use of CL-387785 were determined according prior to the study (data not shown); v) Exo group, T24 cells were cultured with Exos (100 ng/well) for 48 h; and vi) Exo-si-NC group and Exo-si-PYCR1 group, T24 cells were cultured with Exo-si-NC or Exo-si-PYCR1 (containing si-NC or si-PYCR1 100 ng/well) for 48 h. si-PYCR1, oe-PYCR1, and corresponding NCs used in transfection were synthesized by Shanghai GenePharma CO., Ltd. The sequences of si-RNAs were: si-PYCR1 forward, 5′-UGC UAU CAA CGC UGU GG-3′ and reverse, 5′-CCA CAG CGU UGA UAG CA-3′; and si-NC forward, 5′-AAU UCU CCG AAC GUG UAC GU-3′ reverse, 5′-ACG UAC ACG UUC GGA GAA UU-3′. pcDNA3.1 was used as the vector of oe-PYCR1, and oe-NC corresponded to the empty plasmid. After 48 h of transfection, PYCR1 levels were determined by RT-qPCR and western blotting.

Cell viability was detected using an MTT assay. Briefly, 2,000 T24 or RT4 cells were seeded in 96-well plates. After 48 h of culture, cells were incubated with 20 µl MTT solution (0.5%, cat. no. M1025, Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 4 h, and then MTT solution was removed. Next, 100 µl DMSO was added to cells, and the plates were shaken at a constant speed for 10 min to fully dissolve the formazan crystals. The optical density value at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.).

After 48 h of transfection, the cells in each group were collected and made into cell suspensions. The invasive ability of BC cells was assessed using Transwell chambers (cat. no. 3413, Corning, Inc.). Matrigel matrix adhesive was spread (60 µl, Corning) on the bottom of the apical chamber, 100 µl cell suspension was added to the chamber, with a cell count of approximately 1×10⁵, and 700 µl RPMI-1640 medium comprising 10% fetal bovine serum was added to the basolateral chamber. Following 48 h of incubation, the non-invasive cells in the apical chamber of the Transwell chamber were wiped off with a cotton swab, cells were fixed with 4% paraformaldehyde at 37°C for 30 min, and stained with 0.1% crystal violet at 37°C for 10 min. The number of cells passing through the membrane pores was observed under a microscope (200×, Olympus Corporation) in three random fields for each well; the number of invasive cells in the different groups was counted.

The migratory ability of BC cells was assessed using a wound-healing assay. After 48 h of transfection, T24 or RT4 cells were seeded at 5×10⁴ cells/well in 96-well plates and cultured in an. Once a confluent monolayer had formed, a scratch was created using a 200-µl sterile pipette gun head by vertically marking a line under an inverted microscope (Olympus Corporation) and imaged at 0 and 24 h. The cell migration rate (%) was calculated by measuring the width of the wound.

The levels of aerobic glycolysis-related indicators, glucose uptake, lactate production, and ATP production were determined using the colorimetric method. The glucose uptake colorimetric assay kit (cat. no. 36503, AAT Bioquest), lactate detection kit (cat. no. KTB1100-1, Abbkine Scientific Co., Ltd.), and ATP detection kit (cat. no. YT6278, Beijing YITA Biotechnology) were used according to the instructions of the kits.

T24 cells in the exponential phase were collected and total protein was extract using weak RIPA lysate (P0013D, Beyotime, Shanghai, China), and protein concentration was determined using BCA reagent kit (P0012, Beyotime). The 200 µg of total protein was taken and 1 µg PYCR1 antibody (cat. no. ab102601, Abcam) was added and incubated overnight at 4°C. Then, 10 µl pre-treated protein A (cat. no. YJ101, Epizyme Biomedical Technology) agarose bead was added to the solution and incubated for 2 h at 4°C to allow coupling of the antibody with protein A agarose bead. Following immunoprecipitation, the mixture was centrifuged for 3 min at 4°C and 200 × g to sink the agarose bead to the bottom of the tube. The supernatant was removed carefully, and the agarose bead was washed 3 times with 1 ml lysis buffer before examining the binding of PYCR1 and EGFR protein using western blotting. IgG antibody (cat. no. ab6715, Abcam) was used as a negative control, and protein lysis buffer without immunoprecipitation was used as a positive control (Input).

The 6-week-old male BALB/c nude mice (Cavens Laboratory Animal) were used to establish the xenograft tumor model. T24 cells have a high tumor formation rate in nude mice. T24 cells in the logarithmic growth phase were detached into a single-cell suspension and subcutaneously injected into the armpits of mice (2×10⁶ cells per mouse). Drug intervention was performed when the subcutaneous tumor grew to ~100 mm³ (tumor volume=long × width² × 1/2). The mice with tumor formations were randomly assigned to one of four groups (n=6 per group): NC group, BMSC-Exo group, BMSC-Exo-si-NC group, and BMSC-Exo-si-PYCR group, in which mice were administered 200 µl normal saline, BMSC-Exo, BMSC-Exo-si-NC, and BMSC-Exo-si-PYCR1, respectively, by tail vein injection on days 0, 3, 6, and 9, respectively. The physical conditions and behavior of mice were observed daily during the medication period. The weight of mice was measured on days 0, 2, 4, 6, 8, and 12, respectively. The volume of the xenografted tumor was measured once a week. The tumor volume was measured for the last time in the 5th week (the 35th day) (maximum tumor long diameter: 15.0 mm). Then, mice were euthanized by intraperitoneal injection of 1% pentobarbital sodium (200 mg/kg). The tumors were removed for observation and imaging, weight measurement, and immunohistochemical staining.

BC cells or tumor tissues of nude mice were collected. Total RNA was extracted using the TRIzol^® reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the RNA RT kit according to the manufacturer's protocol (cat. no. 4387406; Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the ABI Prism 7300 system using the following amplification temperature protocol: Pre-denaturation, 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The relative gene expression was analyzed using the 2^−ΔΔCq method (29), with β-Actin as the internal reference control. The primers were synthesized by Sangon Biotech Co., Ltd. The sequences were as follows: PYCR1:F: 5′-GGC TGC CCA CAA GAT AAT GGC-3′; R: 5′-CAA TGG AGC TGA TGG TGA CGC-3′ and β-actin: F: 5′-AAT GAG CTG CGT GTG GCT-3′; R: 5′-TAG CAC AGC CTG GAT AGC AA-3′.

Cells were collected and lysed using RIPA lysis buffer (cat. no. P0013B, Beyotime Institute of Biotechnology) to obtain total proteins from cells or tumor tissues of nude mice. Protein concentration was determined using a BCA kit. Equal amounts of proteins protein were loaded on SDS-gels, resolved using 10% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h and incubated overnight with the following antibodies: PYCR1 (1:1,000, cat. no. ab102601; Abcam), GLUT1 (1 µg/ml, cat. no. ab115730, Abcam), hexokinase 1 (HK1; 1:1,000, cat. no. ab154839, Abcam), lactic dehydrogenase A (LDHA; 1:5.000, ab52488, Abcam), EGFR (1:10.000, ab52894, Abcam), p-EGFR (1:1,000, ab40815, Abcam), PI3K (1:1,000, ab32089, Abcam), p-PI3K (0.5 µg/ml, ab278545, Abcam), AKT (1:500, ab8805, Abcam), p-AKT (1:1,000, ab38449, Abcam), and β-Actin (1:1,000, ab8227, Abcam). After incubation with the primary antibodies, the membranes were probed with horseradish peroxidaseconjugated secondary antibody (1:3,000, ab6721, Abcam) for 1 h. Finally, Signals were visualized using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.).

Tumor tissues were fixed with 4% paraformaldehyde at 4°C for 24 h and cut into 5-µm thick paraffin-embedded sections. As previously reported (30), the sections were routinely dewaxed and hydrated, and subjected to antigen retrieval using sodium citrate buffer (pH=6.0) in a microwave oven at 100°C for 10 min and incubated with 3% H₂O₂ at room temperature for 10 min to block the activity of endogenous peroxidase. After washing with PBS, the sections were incubated with the primary antibody against Ki-67 (1:250, cat. no. ab92742, Abcam) in the dark at 4°C overnight. After PBS washes, the sections were incubated at room temperature with the secondary antibody goat anti-rabbit IgG H&L HRP (1:500, cat. no. ab97051, Abcam) for 30 min at 37°C, washed with PBS, counterstained with hematoxylin for 30 sec at 37°C, differentiated with 0.1% HCl, rinsed with tap water to return to blue for 5 min. Subsequently, the sections were dehydrated with a gradient of alcohol solutions, cleared with xylene, and sealed with neutral gum. After air drying, the sections were observed and imaged under a light microscope (×200, Olympus Corporation).

The safety of Exo-si-PYCR1 was investigated by observing the weight changes of mice in each group during the drug intervention period (days 0-12). After mice were euthanized by intraperitoneal injection of 1% pentobarbital sodium (200 mg/kg), the major organs (heart, liver, spleen, lung, and kidney) of mice were collected for pathological HE staining to further investigate the effects of Exo-si-PYCR1 on these organs.

GraphPad Prism version 8.01 (GraphPad Software Inc.) was used to perform the statistical analysis. A Shapiro-Wilk test was used to evaluate the distribution of data. In vitro experiments were performed three times in each group, and 6 nude mice were included in each group for the in vivo experiments. The normally distributed data are presented as the mean ± SD. An independent samples Student's t-test was used for comparisons between 2 groups and a one-way ANOVA followed by Tukey's post hoc test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference. a statistically significant difference.

PYCR1 expression is upregulated in various types of cancer and it is involved in the regulation of BC cell behaviors (31,32). Using the StarBase database, it was found that PYCR1 levels were upregulated in BC (Fig. 1A, P<0.05). Next, PYCR1 levels in SV-HUC-1 normal human bladder epithelial immortalized cells and four BC cells (T24, 5637, RT4, and TCCSUP) were determined by RT-qPCR and western blot assay. PYCR1 was found to be upregulated in the four BC cell lines, in which T24 cells exhibited the highest level and RT4 cells exhibited the lowest levels (Fig. 1B and C, all P<0.05). To investigate the effects of PYCR1 on BC cells, interference plasmids were transfected into T24 cells to knock down PYCR1, and overexpression plasmids were transfected into RT4 cells to increase PYCR1 levels. RT-qPCR and western blot assays showed that PYCR1 was successfully knocked down in T24 cells and overexpressed in RT4 cells (Fig. 1D and E, all P<0.001). MTT assay showed that T24 cell viability was decreased following PYCR1 knockdown and increased following PYCR1 overexpression (Fig. 1F, all P<0.001). Transwell wound-healing assays showed that PYCR1 knockdown reduced T24 cell invasion and migration, whereas PYCR1 overexpression stimulated RT4 cell invasion and migration (Fig. 1G and H, all P<0.001). These results confirmed that PYCR1 was highly expressed in BC where it facilitated BC cell proliferation, invasion, and migration.

Aerobic glycolysis is important in regulating tumor cell behaviors (33,34). It was speculated that PYCR1 may regulate aerobic glycolysis in BC. The levels of glucose uptake, lactate production, and ATP production in cells after 48 h of transfection were assessed. The levels of glucose uptake, lactate production, and ATP production in T24 cells with PYCR1 expression knocked down were lower, whereas they were increased in RT4 cells overexpressing PYCR1 (Fig. 2A-C, all P<0.001). Furthermore, the protein expression levels of aerobic glycolysis-related enzymes GLUT1, HK1, and LDHA were assessed by western blotting. PYCR1 knockdown reduced the protein expression levels of GLUT1, HK1, and LDHA, while PYCR1 overexpression increased the expression levels of these proteins (Fig. 2D, all P<0.001). These results suggested that PYCR1 promoted aerobic glycolysis in BC.

The EGFR/PI3K/AKT pathway is closely associated with BC cell proliferation, invasion, and metastasis (35) and is implicated in the regulation of aerobic glycolysis (17,18). It was speculated that PYCR1 regulated the EGFR/PI3K/AKT pathway. Western blot assay demonstrated that the EGFR/PI3K/AKT pathway was inhibited, and the p-EGFR/EGFR, p-PI3K/PI3K, and p-AKT/AKT levels were repressed in T24 cells following PYCR1 knockdown, whereas this pathway was activated in RT4 cells following PYCR1 overexpression (Fig. 3A, all P<0.001). Thus, PYCR1 activated the EGFR/PI3K/AKT pathway. To probe the mechanism by which PYCR1 regulated the EGFR/PI3K/AKT pathway, the protein-protein interactions between PYCR1 and EGFR were examined by Co-IP, which revealed that endogenous PYCR1 interacted with EGFR protein in T24 cells, suggesting that PYCR1 could interact with EGFR (Fig. 3B); that is, PYCR1 activated the PI3K/AKT pathway by binding to EGFR.

To confirm that PYCR1 regulated aerobic glycolysis and BC cell growth through the EGFR/PI3K/AKT pathway, RT4 cells overexpressing PYCR1 were treated with the pathway inhibitor CL-387785. Western blotting showed that the EGFR/PI3K/AKT pathway was inhibited by CL-387785, while PYCR1 protein expression was not altered by CL-387785 (Fig. 4A, all P<0.01). This result confirmed that EGFR was downstream of PYCR1. Glucose uptake, lactate production, and ATP production levels were also repressed by CL-387785 treatment (Fig. 4B-D, all P<0.001), and RT4 cell proliferation, invasion, and migration were also suppressed (Fig. 4E-G; all P<0.001). Overall, CL-387785 abrogated the promoting effects of PYCR1 overexpression on aerobic glycolysis and BC cell proliferation, invasion, and migration.

MSC-shuttled Exos have a good biological affinity, low toxicity, and low immunogenicity and can be used as carriers to load si-RNA and other drugs to target cancer (20). BMSC-secreted Exos were isolated by differential centrifugation and loaded with si-PYCR1 by electroporation. TEM revealed that Exos were present, visible as the circular saccular body with a double-layer membrane structure and loading si-PYCR1 had no obvious effect on their morphology (Fig. 5A). The particle diameter of the Exos was 54.8±9.6 nm, and the particle size of Exo-si-PYCR1 after loading si-PYCR1 was not changed significantly (Fig. 5B). The potential of Exos was −8.67±0.21 mV, while that of Exo-si-PYCR1 was −14.32±0.18 mV, which indicated that the negatively charged si-PYCR1 was loaded into Exos (Fig. 5C, P<0.01). The Exo surface markers CD9, CD81, CD63, and TSG101 in Exos and Exo-si-PYCR1 were positively expressed (Fig. 5D). Agarose gel electrophoresis found that the levels of free si-PYCR1 in Exo-si-PYCR1 were lower, which indicated that si-PYCR1 was loaded into Exos (Fig. 5E). These results indicated that BMSC-shuttled Exos loaded with si-PYCR1 had been successfully obtained.

To evaluate the effects of Exo-si-PYCR1 on BC cells, the proportion of Cy3-labeled Exo-si-PYCR1 entering T24 cells was observed using a laser confocal microscope. The proportion of si-PYCR1 plasmid introduced into T24 cells increased relative to the control group, and the levels of Exo-si-PYCR1 entering the cells was higher in the si-PYCR1 group (Fig. 6A, all P<0.05). Exo-si-PYCR1 knocked down PYCR1 levels in T24 cells (Fig. 6B and C, all P<0.01). The glucose uptake, lactate production, and ATP production levels were suppressed after Exo-si-PYCR1 treatment, and the inhibitory effect of Exo-si-PYCR1 was more prominent than that of si-PYCR1 transfection (Fig. 6D and F, all P<0.05). In addition, Exo-si-PYCR1 inhibited T24 cell proliferation, invasion, and migration, and the inhibitory effect was stronger than that of si-PYCR1 (Fig. 6G-I, all P<0.05). Collectively, Exo-si-PYCR1 inhibited aerobic glycolysis and BC cell growth, and the inhibitory effect of Exo-si-PYCR1 was stronger than that of si-PYCR1.

To validate the inhibitory effects of Exo-si-PYCR1 on BC in vivo, T24 cells were injected into nude mice to establish xenograft tumor models of BC. When the volume of the xenograft tumors was ~100 mm³, BMSC-Exos, BMSC-Exo-si-NC, and BMSC-Exo-si-PYCR1 were injected into nude mice through the tail vein. After BMSC-Exo-si-PYCR1 treatment, the PYCR1 levels in tumor tissues were repressed (Fig. 7A and B, all P<0.001), and BMSC-Exo-si-PYCR1 inhibited the growth rate, volume, and weight of xenograft tumors in nude mice (Fig. 7C-E, P<0.001), and the number of Ki67-positive cells that marked the proliferation of cancer cells in tumor tissues was reduced (Fig. 7F, P< 0.001), indicating that BMSC-Exo-si-PYCR1 inhibited BC cell proliferation in vivo. After the injection of BMSC-Exo-si-PYCR1, any toxic effects of BMSC-Exo, BMSC-Exo-si-NC, and BMSC-Exo-si-PYCR1 were evaluated by observing the changes in the body weight of nude mice and observing any pathological changes in the primary organs (heart, liver, spleen, lung, and kidney) by HE staining. BMSC-Exo, BMSC-Exo-si-NC, and BMSC-Exo-si-PYCR1 had no notable effects on the body weight and the primary organs of nude mice (Fig. 7G-H). These results suggested that the use of BMSC-Exo-si-PYCR1 may be a feasible method for the management of BC.