AUP1 transcriptionally activated by KDM5B reprograms lipid metabolism to promote the malignant progression of cervical cancer

Abstract

Cervical cancer is one of the reproductive malignancies threatening women's lives worldwide. In the present study, it was aimed to explore the role and mechanism of ancient ubiquitous protein 1 (AUP1) in cervical cancer. Through bioinformatics analysis, AUP1 expression in cervical cancer tissues and the correlation between AUP1 and the prognosis of patients were analyzed. AUP1 expression in several cervical cancer cell lines was detected. Following the co‑transfection of short hairpin RNA specific to AUP1 with or without lysine demethylase 5B (KDM5B) overexpression plasmids in SiHa cells, the proliferation and apoptosis of SiHa cells were detected. Additionally, wound healing and Transwell assays were used to detect SiHa cell migration and invasion. Cellular lipid droplets level was detected using the Oil red O staining. Meantime, the levels of triglyceride, cholesterol, oxygen consumption rates and expression of lipid metabolism‑related proteins were detected to assess the lipid metabolism in SiHa cells. Then, the luciferase reporter assay and ChIP assay were used to verify the binding between KDM5B and AUP1. Finally, the effects of AUP1 and KDM5B on the growth and lipid metabolism in SiHa tumor‑bearing mice were measured. AUP1 was significantly upregulated in cervical cancer tissues and cells. AUP1 interference inhibited the malignant biological behaviors and lipid metabolism reprogramming of SiHa cells, which was blocked by KDM5B overexpression. Moreover, KDM5B could transcriptionally activate AUP1 and upregulate AUP1 expression. Furthermore, AUP1 knockdown transcriptionally regulated by KDM5B limited the tumor growth and suppressed the lipid metabolism reprogramming in vivo. Collectively, AUP1 could be transcriptionally activated by KDM5B to reprogram lipid metabolism, thereby promoting the progression of cervical cancer. These findings reveal possible therapeutic strategies in targeting metabolic pathways.

Keywords: ancient ubiquitous protein 1; cervical cancer; lipid metabolism reprogramming; lysine demethylase 5B; proliferation.

Figure 1

AUP1 expression is significantly upregulated in cervical cancer tissues and cells. (A) AUP1 expression in normal cervical tissues (Normal; n=3) and tissues of patients with primary CESC (n=305) was analyzed using UALCAN database. ^***P<0.001 vs. Normal group. (B) The association between AUP1 expression and the prognosis of patients with cervical cancer was analyzed using Kaplan-Meier Plotter database. (C) Reverse transcription-quantitative PCR and (D) western immunoblot analysis were used to analyze AUP1 expression in four cervical cancer cell lines (SiHa, Caski, HeLa and C33A cells) and the human immortalized cervical epithelial cell line (H8 cells). ^**P<0.01 and ^***P<0.001 vs. H8 group. AUP1, ancient ubiquitous protein 1; CESC, cervical squamous cell carcinoma.

Figure 2

AUP1 interference inhibits the proliferation of cervical cancer cells. (A) Reverse transcription-quantitative PCR and (B) western immunoblot analysis were used to examine AUP1 expression in SiHa cells after transfection with sh-AUP1-1/2. (C) Cell Counting Kit-8 assay measured the viability of SiHa cells. (D and E) Cell proliferation was assessed by colony formation assay. (F and G) EdU staining detected the proliferation of SiHa cells. ^***P<0.001 vs. sh-NC group. AUP1, ancient ubiquitous protein 1; EdU, 5-ethynyl-2′-deoxyuridine; sh-, short hairpin; NC, negative control.

Figure 3

AUP1 interference facilitates the apoptosis of cervical cancer cells. (A and B) SiHa cell apoptosis was detected using flow cytometric analysis. (C) Western immunoblot analysis measured Ki67, PCNA, Bcl-2 and Bax expression in SiHa cells. ^**P<0.01 and ^***P<0.001 vs. sh-NC group. AUP1, ancient ubiquitous protein 1; PCNA, proliferating cell nuclear antigen; sh-, short hairpin; NC, negative control.

Figure 4

AUP1 interference inhibits the migration and invasion of cervical cancer cells. (A) Wound healing assay was used to detect the migration of SiHa cells. (B) The invasion of SiHa cells was detected by Transwell assay. (C) Western immunoblot analysis was used to identify MMP2 and MMP9 expression in SiHa cells. ^***P<0.001 vs. sh-NC group. AUP1, ancient ubiquitous protein 1; MMP, matrix metalloproteinase; sh-, short hairpin; NC, negative control.

Figure 5

AUP1 interference inhibits the lipid metabolism reprogramming of cervical cancer cells. (A) Oil red O staining was used to detect lipid droplets in SiHa cells. The contents of (B) TG and (C) CHOL in SiHa cells were analyzed using the corresponding kits. (D) OCR in SiHa cells was detected using the Seahorse XF Cell Mito Stress Test Kit. (E) The expression of proteins related to lipid metabolism was detected using western immunoblot analysis. ^**P<0.01 and ^***P<0.001 vs. sh-NC group. AUP1, ancient ubiquitous protein 1; TG, triglyceride; CHOL, cholesterol; OCR, oxygen consumption rates; sh-, short hairpin; NC, negative control; CPT1A, carnitine palmitoyltransferase IA; STARD5, StAR-related lipid transfer domain containing 5; MGLL, monoglyceride lipase.

Figure 6

KDM5B could transcriptionally activate AUP1 and upregulate AUP1 expression in cervical cancer cells. (A) The putative KDM5B-binding site on the AUP1 promoter was predicated by the HumanTFDB database. (B) RT-qPCR and (C) western immunoblot analysis were used to measure KDM5B expression in H8 cells and SiHa cells. ^***P<0.001 vs. H8 group. (D) RT-qPCR and (E) western immunoblot analysis analyzed KDM5B expression in SiHa cells after transfection with oe-KDM5B or sh-KDM5B-1/2. ^***P<0.001 vs. oe-NC group; ^#P<0.05 and ^###P<0.001 vs. sh-NC group. The binding of KDM5B on AUP1 promoter site was verified using (F) luciferase reporter assay and (G) chromatin immunoprecipitation assay. ^***P<0.001 vs. oe-NC group or IgG group. (H) RT-qPCR and (I) western immunoblot analysis were used to analyze KDM5B expression in SiHa cells after transfection with oe-KDM5B or sh-KDM5B. ^***P<0.001 vs. oe-NC group; ^#P<0.05 and ^##P<0.01 vs. sh-NC group. KDM5B, lysine demethylase 5B; AUP1, ancient ubiquitous protein 1; RT-qPCR, reverse transcription-quantitative PCR; sh-, short hairpin; NC, negative control; oe-overexpressing; WT, wild-type; MUT, mutant.

Figure 7

KDM5B overexpression blocks the inhibitory effects of AUP1 silencing on the proliferation and apoptosis of cervical cancer cells. (A) Cell Counting Kit-8 assay was used to measure the viability of SiHa cells. (B and C) Cell proliferation was detected by colony formation assay. (D and E) EdU staining was used to detect the proliferation of SiHa cells. (F and G) SiHa cell apoptosis was detected using flow cytometric analysis. (H) Western immunoblot analysis was used to measure Ki67, PCNA, Bcl-2 and Bax expression in SiHa cells. ^***P<0.001 vs. control group; ^#P<0.05 and ^###P<0.001 vs. sh-AUP1 + oe-NC group. KDM5B, lysine demethylase 5B; AUP1, ancient ubiquitous protein 1; dU, 5-ethynyl-2′-deoxyuridine; PCNA, proliferating cell nuclear antigen; sh-, short hairpin; NC, negative control; oe-overexpressing.

Figure 8

KDM5B overexpression blocks the inhibitory effects of AUP1 silencing on the migration and invasion of cervical cancer cells. (A) Wound healing assay was used to detect the migration of SiHa cells. (B) The invasion of SiHa cells was identified using Transwell assay. (C) Western immunoblot analysis was used to evaluate MMP2 and MMP9 expression in SiHa cells. ^***P<0.001 vs. control group; ^##P<0.01 and ^###P<0.001 vs. sh-AUP1 + oe-NC group. KDM5B, lysine demethylase 5B; AUP1, ancient ubiquitous protein 1; MMP, matrix metalloproteinase; sh-, short hairpin; NC, negative control; oe-overexpressing.

Figure 9

KDM5B overexpression abolishes the impacts of AUP1 silencing on the lipid metabolism reprogramming of cervical cancer cells. (A) Oil red O staining was used to detect lipid droplets in SiHa cells. The contents of (B) TG and (C) CHOL in SiHa cells were analyzed using the corresponding kits. (D) OCR level in SiHa cells was detected using the Seahorse XF Cell Mito Stress Test Kit. (E) The expression of proteins related to lipid metabolism was detected using western immunoblot analysis. ^***P<0.001 vs. control group; ^#P<0.05 and ^###P<0.001 vs. sh-AUP1 + oe-NC group. KDM5B, lysine demethylase 5B; AUP1, ancient ubiquitous protein 1; TG, triglyceride; CHOL, cholesterol; OCR, oxygen consumption rates; sh-, short hairpin; NC, negative control; oe-overexpressing.

Figure 10

AUP1 knockdown transcriptionally regulated by KDM5B interferes with the tumor growth in vivo. (A) The images of SiHa tumor-bearing mice and the dissected tumor. (B) The tumor volumes were recorded every two days from the 6th day after injection. (C) The expression of AUP1 and KDM5B was detected using western immunoblot analysis. (D) Ki67 expression in tumor tissues was detected by immunofluorescence staining. (E) Apoptosis was detected using TUNEL staining. ^***P<0.001 vs. control group; ^#P<0.05 and ^###P<0.001 vs. sh-AUP1 + oe-NC group. AUP1, ancient ubiquitous protein 1; KDM5B, lysine demethylase 5B; sh-, short hairpin; NC, negative control; oe-overexpressing.

Figure 11

AUP1 knockdown transcriptionally regulated by KDM5B suppresses the lipid metabolism reprogramming in vivo. (A) Oil red O staining was used to detect the lipid accumulation in tumor tissues. (B) The expression of proteins related to lipid metabolism in tumor tissues was detected using western immunoblot analysis. ^***P<0.001 vs. control group; ^#P<0.05 and ^###P<0.001 vs. sh-AUP1 + oe-NC group. AUP1, ancient ubiquitous protein 1; KDM5B, lysine demethylase 5B; sh-, short hairpin; NC, negative control; oe-overexpressing; CPT1A, carnitine palmitoyltransferase IA; STARD5, StAR-related lipid transfer domain containing 5; MGLL, monoglyceride lipase.