Apolipoprotein L3 inhibits breast cancer proliferation and modulates cell cycle via the P53 pathway

Figure 1. APOL3 Expression in normal and breast cancer tissue. A: Pan-cancer expression profile of APOL3; B-C: Reduced expression of APOL3 in normal mammary tissues and tumors in the GSE29431 (B) and GSE42568 (C) series; D: Reduced expression of APOL3 in normal mammary tissues and tumors from TCGA; E: Comparative analysis showing decreased APOL3 levels in tumors versus paired normal breast tissues from TCGA; F: Lower mRNA levels of APOL3 in the MCF10A cell line compared with MCF7, T47D, MDA-MB-231, MDA-MB-468, and BT549 cell lines; G: Diminished protein expression of APOL3 in the MCF10A cell line compared with MCF7, T47D, MDA-MB-231, MDA-MB-468, and BT549 cell lines; H: IHC staining for APOL3 in breast cancer and adjacent normal breast tissues. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 2. Prognostic Significance of APOL3 in Breast Cancer. A-C: Elevated APOL3 expression correlates with poor prognosis in OS (A), PFI (B), and DSS (C) of breast cancer patients; D: ROC curve for APOL3; E: Association between APOL3 expression and histological subtypes of breast cancer; F: Correlation of APOL3 expression with PR levels; G: Multivariate Cox proportional hazards regression analysis for APOL3. *: p < 0.05, ***: p < 0.001.

Figure 3. In Vitro Proliferative Effects of APOL3 Overexpression. A: qRT-PCR of APOL3 mRNA levels in MCF7 and MDA-MB-231 cells transfected with PEGFP-APOL3-MYC vector; B: Western blotting analysis of APOL3-MYC protein levels in MCF7 and MDA-MB-231 cells transfected with PEGFP-APOL3-MYC vector; C: CCK-8 assays monitoring cell growth rates in MCF7 and MDA-MB-231 cells transfected with PEGFP-APOL3-MYC vector; D: Evaluation and counting of colony formation; E: Flow cytometry of the cell cycle in MCF7 and MDA-MB-231 cells 24h after transfection with PEGFP-APOL3-MYC vector; F: Western blotting analysis of CDK6, Cyclin D1, and Cyclin A2 levels. *: p < 0.05, **: p < 0.01, ***: p < 0.001. ns: no significance.

Figure 4. Effect of APOL3 Knockdown on In Vitro Proliferation. A: qRT-PCR of APOL3 mRNA levels in MCF7 and MDA-MB-231 cells transfected with siRNA; B: Western blotting analysis of APOL3 protein levels in MCF7 and MDA-MB-231 cells transfected with siRNA; C: CCK-8 assays monitoring cell growth rates in MCF7 and MDA-MB-231 cells transfected with siRNA; D: Evaluation and quantification of colony formation; E: Flow cytometry of cell cycle in MCF7 and MDA-MB-231 cells 24h after siRNA transfection; F: Western blotting analysis of CDK6, Cyclin D1, and Cyclin A2 levels. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5. APOL3 Overexpression and Its effects on Tumor Growth In Vivo. A: Growth curves of tumors in nude mice inoculated with APOL3-overexpressing MCF7 cells; B: Representative images of nude mice after tumor formation; C: Macroscopic view of excised tumors; D: IHC staining of tumor tissue sections. E: IHC staining for cell cycle related proteins in tumor tissue sections. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 6. APOL3 Exerts Significant Effects via the P53 pathway. A: Mass spectrometry results; B: CO-IP confirming APOL3-YBX1 interaction; C: Immunofluorescence assay of APOL3 and YBX1. D: Western blotting analysis of YBX1 alterations following APOL3 knockdown; E: Verification of YBX1 knockdown efficiency by western blotting; F-G: Reversal of si-APOL3-induced cell proliferation by YBX1 knockdown, assessed by CCK8 assay (F) and colony formation counting (G); H: Western blotting analysis of CDK6, Cyclin D1, and Cyclin A2 levels; I: APOL3 knockdown diminishes P53 and P21 expression; J: Restoration of P53 and P21 levels upon concurrent knockdown of YBX1 and APOL3. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. ns: no significance.

Figure 7. APOL3 modulates cell cycle and proliferation of breast cancer through binding to YBX1 through P53 pathway.