In this study, the 30 BC patients and 30 normal samples were acquired between 2019 and 2021 from our hospital. In addition, four BC patients (T1, T2, T3, and T4) and four normal samples (N1, N2, N3, and N4) were chosen to analyze the protein level of CNN1. On the basis of The Cancer Genome Atlas (https://gdc-portal.nci.nih.gov/) database and the median expression value of CNN1, BC patients were fallen into high and low expression groups. The log-rank test was utilized to determine the statistical significance. The research involving human tissue was approved by the Medical Ethics Committee of The Second Hospital of Tianjin Medical University.

Human BC cell lines EJ, 5637, UMUC-3, T24, and J82 and normal control urothelial epithelial cell line SV-HUC were gained from the Chinese Academy of Medical Sciences Shanghai Cell Bank (Shanghai, China). These cells were routinely cultivated in Dulbecco’s minimum essential medium including 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 5% CO₂ incubator at 37°C.

pcDNA3.1-CNN1 and pcDNA3.1 (vector) were acquired from GenePharma Co. (Shanghai, China). When the cells were attaining 80% mixing, they were transfected into cells utilizing Lipofectamine 2000 reagents (Invitrogen, USA). After that 48 h, the transfection efficiency was determined by Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) measurements after 48 h.

Total RNA was extracted utilizing TRIzol (Invitrogen). For the reverse transcription of RNA, PrimeScript RT Reagent Kit (Takara, Kyoto, Japan) was applied. qPCR analysis was executed to detect SYBR Premix Ex Taq™ Kit (Takara) on the ABI 7300HT system. The expression level of CNN1 was normalized against glyceraldehyde-3-phosphate dehydrogenase and relatively quantified utilizing 2^-ΔΔCt method. The primers in this study were displayed in Table 1 .

The total protein was separated by radioimmunoprecipitation assay lysis buffer (Cw Biotech, Bejing, China) including proteinase suppressors. The protein concentration was assessed by bicinchoninic acid (Beijing ComWin Biotech Co., Ltd., Beijing, China) reagent. After that, 20 µg of protein samples was detected by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, followed by transfer to a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% fat-free milk for 1 h, the PVDF membranes were hatched with the antibodies at 4°C overnight. The blots were tested with ECL.

After 48 h of transfection, T24 and UMUC-3 cells were seeded into 96-well plates (1,000 cells per well). The proliferation of T24 and UMUC-3 cells was detected by CCK-8. One hundred microliters of cell suspension was taken, and 1 × 10³ cells per well were placed in 96-well plates. After that, 10 μl of CCK8 reagent was added into each well, followed by incubation for 1.5 h at 37°C incubator. The absorbance at 450 nm was detected by a microplate reader.

In vitro invasion and migration of T24 and UMUC-3 cells were tested by transwell inserts precoated with or without Matrigel. In brief, 200 μl of serum-free medium including approximately 1 × 10⁵ transfected cells was hatched into the upper chamber of each transwell. Besides, 500 μl of complete medium (including 10% FBS) was put onto the lower chamber and incubated for 24 h at 37°C. Then, the cells were fixed by 4% paraformaldehyde and dried with 0.1% crystal violet for 10 min each. Last, the number of cells was counted at selected randomly five fields utilizing a light microscope at magnification ×200.

When T24 and UMUC-3 cells were grown to the logarithmic growth phase, they were plated on six-well plates with 5 × 10⁵ cells per well. After 48 h of culture, the culture solution was collected, and the concentration of glucose and lactic acid as well as the level of ATP were determined by the biochemical automatic analyzer, respectively. Then, the cells were digested and counted after centrifugation. By subtracting the base values of glucose, lactic acid, and ATP in the culture fluid, the values of glucose production and lactic acid consumption as well as ATP level were calculated.

First, T24 cells were infected with the indicated lentiviral vector and CNN1 (5 × 10⁶ cells per mouse in 200-μl volume). Then, T24 vector and T24-CNN1 were subcutaneously into the left armpit of 6-week-old BALB/c nude mice. After 21 days, the mice were anaesthetized with 2%–3% isoflurane. Then, the mice were sacrificed. Then, the tumor was removed and weighed. All the animal assays were approved by the Animal Ethical and Welfare Committee of Tianjin Medical University.

Paraffin-embedded sections were firstly deparaffinized and then incubated with rabbit polyclonal anti-Ki-67 (#12075, Cell Signaling Technology) or rabbit polyclonal anti-CNN1 (#17819, Cell Signaling Technology) primary antibody at 4°C overnight. After being washed by TBST three times, the sections were then incubated with horseradish peroxidase–conjugated goat anti-mouse antibody (#4414, Cell Signaling Technology). The sections were washed by TBST three times and the signal was tested utilizing DAB Substrate Kit. Images were gained utilizing a microscopy.

The experimental data were analyzed by utilizing SPSS 22.0 statistical and GraphPad Prism 8.0 analysis software. The comparison of the two groups was carried out by Student’s t-test. One-way analysis of variance (ANOVA) and a post-hoc Dunnett’s test were utilized to analyze the mean comparison among more than two samples. P < 0.05 was regarded as statistically significant.

In order to probe the influences of CNN1 on BC, bioinformatics was used. First, the expression differences of CNN1 between BC and normal tissues were analyzed. The outcomes revealed that CNN1 was downregulated in BC tissues (n = 30) relative to normal tissues (n = 30; Figure 1A , P < 0.001). Then, Western blotting was utilized to test the relationship between CNN1 protein expression in BC patients and adjacent tissues. The outcomes indicated that the protein level of CNN1 was obviously lowly expressed in BC tissues ( Figure 1B , P < 0.001). Then, we investigated the correlation of CNN1 expression and prognostic power in patients with BC. The consequences indicated that patients with low expression of CNN1 had a worse overall survival relative to those with high expression of CNN1 ( Figure 1C , P = 0.0389). Then, the mRNA and protein expression of CNN1 in BC cell lines EJ, T24, 5637, UMUC-3, and J82 was detected. The human normal bladder epithelial cell line SV-HUC was used as a control. The results exhibited that the mRNA ( Figure 1D ) and protein ( Figure 1E ) levels of CNN1 were lowly expressed in both T24 and UMUC-3 cells. Thus, T24 and UMUC-3 cells were used in the subsequent analysis to overexpression CNN1. Altogether, these findings suggested that CNN1 was lowly expressed in BC tissues and cells; also, low expression of CNN1 could be considered as a prognostic factor in BC patients.

To probe the influences of CNN1 on BC cells, CNN1 expression was overexpressed in T24 and UMUC-3 cells by pcDNA3.1. The results of qRT-PCR and Western blotting demonstrated that pcDNA3.1 sequences visibly facilitated CNN1 expression in T24 and UMUC-3 cells, both in mRNA ( Figure 2A ) and protein levels ( Figure 2B ). Then, CCK8 and transwell were used to test the properties of BC cells. As illustrated in Figures 2C-E , overexpression of CNN1 repressed T24 and UMUC-3 cell proliferation, invasion and migration. Then, Western blotting was applied to further detect the impacts of CNN1 overexpression on the expression levels of glycolysis-related proteins GLUT1, PKM2, and LDHA. Figure 2F displayed that the protein levels of GLUT1, PKM2, and LDHA were all eased by CNN1 overexpression. Then, the kits were used to detect glucose absorption, lactate secretion, and ATP levels. ( Figures 2 G-I ) illustrated that, after overexpression of CNN1, the glucose consumption, lactate production, and ATP levels of T24 and UMUC-3 cells decreased ( Figures 2G-I ). Above all, overexpression of CNN1 eased the property of T24 and UMUC-3 cells.

It is believed that HIF-1α plays a significant role in the development of the BC (22). Besides, previous pieces of evidences have pointed out the association of CNN1 with HIF-1α in smooth muscle cell (21). For the purpose of exploring the mechanisms of CNN1 in BC, the impacts of CNN1 silence on HIF-1α pathway were determined. Western blotting was utilized to test the protein levels of HIF-1α and PDK1. As displayed in Figures 3A, B , overexpression of CNN1 suppressed the mRNA and protein levels of HIF-1α and PDK1 in T24 and UMUC-3 cells ( Figure 3 ). All the above findings suggested that overexpression of CNN1 suppressed HIF-1α signaling pathway in BC.

To further manifest the mechanism of CNN1 function on BC cells, CNN1 overexpression combined with HIF-1α was cotransfected into T24 and UMUC-3 cells. Western blotting outcomes showed that HIF-1α activation accelerated the suppressed role of CNN1 overexpression on the protein levels of HIF-1α and PDK1 in T24 and UMUC-3 cells ( Figure 4A ). CCK8 results indicated that overexpression of CNN1 suppressed the T24 and UMUC-3 cell proliferation, and HIF-1α activation receded the repressed role of CNN1 on the proliferation of T24 and UMUC-3 cells ( Figure 4B ). Transwell assay results revealed that HIF-1α activation weakened the inhibitory impact of CNN1 on the invasion and migration of BC cells ( Figures 4C, D ). Moreover, CNN1 overexpression suppressed the protein levels of GLUT1, PKM2, and LDHA, which were rescued by HIF-1α activation ( Figure 4E ). Furthermore, the decreased glucose consumption, lactate production, and ATP levels of T24 and UMUC-3 cells were promoted by HIF-1α activation ( Figures 4F-H ). Together, these results provide important insights into that HIF-1α activation enhanced the suppressive influence of CNN1 overexpression on cell proliferation, invasion, migration, and glycolysis in BC cells.

In order to research the relationship between CNN1 and BC in vivo, subcutaneous xenograft tumor model in nude mice was built. T24-pcDNA3.1-CNN1 (CNN1) or T24-pcDNA3.1 (vector) cells were injected into mice. Xenograft tumor model with CNN1 revealed overtly lowered in tumor volume and weight relative to mice vector xenograft tumor model ( Figures 5A-C ). Nude mouse tumor tissues were used for immunohistochemical experiments to detect the expression levels of Ki-67 and CNN1. Figure 5D illustrated that CNN1 decreased Ki-67 in BC nude mice. Collectively, the above findings indicated that CNN1 overexpression could impede BC cell growth in nude mice.