SQLE Knockdown inhibits bladder cancer progression by regulating the PTEN/AKT/GSK3β signaling pathway through P53

Abstract

Bladder cancer (BCa) is one of the most common malignancies worldwide. However, the lack of accurate and effective targeted drugs has become a major problem in current clinical treatment of BCa. Studies have demonstrated that squalene epoxidase (SQLE), as a key rate-limiting enzyme in cholesterol biosynthesis, is involved in cancer development. In this study, our analysis of The Cancer Genome Atlas, The Genotype-Tissue Expression, and Gene Expression Omnibus databases showed that SQLE expression was significantly higher in cancer tissues than it was in adjacent normal tissues, and BCa tissues with a high SQLE expression displayed a poor prognosis. We then confirmed this result in qRT-PCR and immunohistochemical staining experiments, and our vitro studies demonstrated that SQLE knockdown inhibited tumor cell proliferation and metastasis through the PTEN/AKT/GSK3β signaling pathway. By means of rescue experiments, we proved that that P53 is a key molecule in SQLE-mediated regulation of the PTEN/AKT/GSK3β signaling pathway. Simultaneously, we verified the above findings through a tumorigenesis experiment in nude mice. In conclusion, our study shows that SQLE promotes BCa growth through the P53/PTEN/AKT/GSK3β axis, which may serve as a therapeutic biological target for BCa.

Keywords: Apoptosis; Bladder cancer; Cell cycle; P53; PTEN/AKT/GSK3β signaling pathway; Proliferation; SQLE.

Fig. 1

SQLE is highly expressed in BCa tissues and SQLE expression is associated with various clinical characters. (A) The mRNA expression levels of SQLE in 33 types of tumor tissues and adjacent normal tissues from the TIMER database; (B) The differential expression of SQLE between unpaired BCa tissues and adjacent normal tissues. The nucleus is blue and the brownish-yellow stain is positive; the darker the color, the higher the level of gene expression; (C) The differential mRNA expression of SQLE between bladder tumor tissue and adjacent normal tissue in BCa; (D) Representative immunohistochemistry of SQLE in BCa tissues and adjacent tissues. The nucleus is blue and the brownish-yellow stain is positive; the darker the color, the higher the level of gene expression; (E) The landscape of SQLE-related clinicopathological features of BCa in TCGA database; (F) The landscape of SQLE-related clinicopathological features of BCa in the GEO database; (G–J) The correlation between the expression of SQLE and some clinicopathological features of BCa. (*p < 0.05, **p < 0.01, *** p < 0.001)

Fig. 2

Comprehensive diagnostic and prognostic analyses of SQLE in BCa (A, B) The ROC curve indicated the high-expression specificity of SQLE in tumor and normal tissues in TCGA and GSE188715 databases; (C, D) Kaplan–Meier analysis of SQLE expression in TCGA and GSE13507 databases. The significance of the prognostic value was tested using a log-rank test; (E) Univariate and multivariate analyses of prognostic parameters in TCGA database overall survival (OS)

Fig. 3

Alteration of SQLE and the association between SQLE expression and immune cell infiltration in BCa. (A) Alteration frequency of SQLE in BCa and pan-cancer. (B) The mutation sites, types of mutation, and alteration frequency of SQLE somatic mutation in BCa. (C) The correlation between tumor mutation burden and expression of SQLE. (D) The stromal, immune, and ESTIMATE scores were different between low-expression SQLE and high-expression SQLE. (E) SQLE expression was positively associated with CD4 + Th2 T cells, plasma cells, CD8 + T cells, and macrophage infiltration in BCa and negatively correlated with CD4 + T cells, myeloid-derived suppressor cells, and NK cells. (F) Gene expression after ICB treatment and in responders and non-responders in different tumor models. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

SQLE knockdown inhibited T24 and 5637 cell proliferation, migration, and viability. (A, B) The qRT-PCR assay detected shRNAs transfection efficiency in two BCa cell lines T24 and 5637; (C) Western blot assay detected the sh2 efficiency of transfection for protein level in two BCa cell lines T24 and 5637; (D, E) The CCK-8 assay was used to evaluate the proliferation of shSQLE-treated tumor cells and NC-treated tumor cells from 0 to 96 h; (F) Representative images of the colony-forming assay; (G, H) Immunofluorescence results showed that the expression of KI67 was decreased in BCa; (I, J) The wound-healing assay demonstrated that the capacity of migration in two BCa cell lines T24 and 5637 (K–M) The migration and invasion of T24 and 5637 cell lines were inhibited by SQLE knockdown as measured by a transwell assay. Data represent the mean ± standard deviation (SD) of three independent experiments. (The magnification under the microscope is shown as marked in the diagram; *p < 0.05, **p < 0.01, ***p < 0.001 versus the vector group, ns, not significant)

Fig. 5

SQLE Knockdown inhibited T24 and 5637 cell cycle and promoted cell apoptosis. (A) Flow cytometry was used to detect the proportion of G1, S, and G2/M phase cell population in T24 and 5637; (B) Flow cytometry was used to detect the early apoptosis and late apoptosis cell population in T24 and 5637; (C, D) The western blot assay was used to evaluate the expression of cell cycle and cell apoptosis related genes; (E–G) Representative images of JC-1 immunofluorescence and the ratio of red to green fluorescence showed a decrease in the mitochondrial membrane potential. Data are presented herein as the from three independent experiments. Data represent the mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 versus the vector group, ns, not significant)

Fig. 6

Knockdown of SQLE affects the P53 signaling pathway and PTEN/AKT/GSK3β signaling pathway. (A) The top 147 genes associated with SQLE were constructed with a PPI network (23 genes were not found in the STRING platform); (B) KEGG enrichment analysis of genes in PPI network; (C) GO enrichment analysis of genes in PPI network; (D) Protein expression levels of P53, PTEN, AKT, P-AKT, GSK3β, and P-GSK3β in T24 and 5637 cells; (E–G) Representative images of P53, PTEN, and P-AKT immunostaining. Data represent the mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001 versus the vector group, ns, not significant)

Fig. 7

SQLE increases tumor cell proliferation and inhibits cell apoptosis of Bca by regulating the PTEN/AKT/GSK3β signaling pathway through P53. (A) Protein expression levels of P53, PTEN, AKT, P-AKT, GSK3β, P-GSK3β, Bcl2, and Cyclin D1 in T24 and 5637 cells; (B, C) The CCK-8 assay was used to assess the proliferation of shSQLE-treated tumor cells, NC-treated tumor cells, negative controls, and cells treated with shSQLE + PFT β from 0 to 96 h; (D) Representative images of the colony-forming assay; (E, F) Representative images of KI67 immunostaining showed that overexpression of P53 increased the proliferation of T24 and 5637 cells after SQLE knockdown; (G–I) Flow cytometry detected the early apoptosis and late apoptosis cell population in T24 and 5637. Data represent the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant)

Fig. 8

SQLE increases tumor cell migration and invasion by regulating the PTEN/AKT/GSK3β signaling pathway through P53. (A–C) The wound-healing assay demonstrated the capacity of migration in two BCa cells T24 and 5637; (D, E) The migration of T24 and 5637 cells were rescued by upregulation of P53 as measured by a transwell assay; Data represent the mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant)

Fig. 9

SQLE increases tumor cell proliferation, migration, and invasion by regulating the PTEN/AKT/GSK3β signaling pathway through P53 in vivo. (A–B) Representative image of T24 xenograft tumors under different treatments; (C) Tumor weight in each group; (D) Tumor volume in each group; (E) Expression levels of P53, PTEN, P-AKT, P-GSK3β, cyclin D1, and Bcl2 of xenograft tumors in each group as determined by the western blot assay; (F) Expression of P-AKT, PTEN, P53, Cyclin D1 and Bcl2 in xenograft tumors was analyzed by immunohistochemical staining and TUNEL staining assays. The nucleus is blue and the brownish-yellow stain is positive; the darker the color, the higher the level of gene expression. Data represent the mean ± SD of three independent experiments. (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant)

Fig. 10

This schematic diagram explains the molecular mechanisms underlying the pro-BCa effects of SQLE. Briefly, SQLE regulates the expression of downstream PTEN/AKT/GSK3β through P53 molecules, thereby promoting BCa proliferation, migration, and invasion and inhibiting apoptosis