Fig 1: High STARD4 expression is associated with shorter DMFS times in BRCA. (A) Kaplan-Meier survival curve analysis with a log rank test indicated that higher expression levels of STARD4 were significantly associated with shorter DMFS time in patients with BRCA. Association between STARD4 expression and DMFS time in patients with (B) ER negative, (C) PR negative, (D) HER2 positive, (E) ER positive, (F) PR positive and (G) HER2 negative BRCA. BRCA, breast cancer; DMFS, distant metastasis-free survival; ER, estrogen receptor; PR, progesterone receptor; STARD4, steroidogenic acute regulatory protein-related lipid transfer 4.
Fig 2: Knockdown of STARD4 suppresses breast cancer cell migration. (A and B) Transwell assay revealing that STARD4 knockdown significantly inhibited HCC1937 cell migration. Three replicates from an independent assay are presented. Magnification ×200. (C and D) Transwell assay revealing that STARD4 knockdown significantly inhibited MDA-MB-231 cell migration. Three replicates from an independent assay are presented. Magnification ×200. (E and F) Wound healing assay revealing that STARD4 knockdown significantly inhibited HCC1937 cell migration. Magnification ×100. (G and H) Wound healing assay revealing that STARD4 knockdown significantly inhibited MDA-MB-231 cell migration. Magnification ×100. Data are presented as the mean ± SD (n=3). ***P<0.001. CTRL, control; sh, short hairpin RNA; STARD4, steroidogenic acute regulatory protein-related lipid transfer 4.
Fig 3: MLT inhibits CRPC progression and reverses enzalutamide resistance. (A) Meta-analysis of the melatonin and T-CHO levels in the blood. (B) Viability of the LNCaP, C4-2, and 22RV1 cells treated with cholesterol (10 µM) and/or MLT (1 mM) was detected by CCK-8 assay. (C) The CES1 expression in C4-2-ENZR cells compared with that in C4-2 cells as determined by WB. Densitometry and statistical analysis. Representative images are shown. (D) TG and T-CHO contents were measured in C4-2-ENZR cells treated with DMSO/MLT (1 mM) and in C4-2-ENZR cells with stable CES1 overexpression. The graphs represent the means ± SEM. (E) Western blotting was used to detect the effect of MLT treatment (1 mM, 48 h) on the expression of the proteins encoded by steroid hormone metabolism-related genes (CYPA11A1 and STARD4) in enzalutamide-resistant cell lines (C4-2-ENZR and 22RV1 cells). Densitometry and statistical analysis. Representative images are shown. (F) LC-MS/MS was performed to evaluate the effect of MLT on the T (testosterone) and DHT (dihydrotestosterone) contents in C4-2-ENZR cells or in the cells with stable knockout of CES1 expression. The results represent the mean ± SEM of three independent experiments. (G) CCK-8 assay was used to evaluate the viability of enzalutamide-resistant cell lines (C4-2-ENZR and 22RV1 cells) treated with ENZ (20 µM) and/or MLT (1 mM). The data from a representative of three independent experiments are shown. *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations: ns, no significance; RR, risk ratio; SMD, standard mean difference
Fig 4: Knockdown of STARD4 suppresses BRCA proliferation. (A) Expression levels of STARD4 in 57 BRCA cell lines. (B) RNA and (C) protein levels of STARD4 in HCC1937 cells after transfection with shSTARD4. (D) RNA and (E) protein levels of STARD4 in MDA-MB-231 after transfection with shSTARD4. (F) Celigo cell counting and (G) CCK-8 assays demonstrated that knockdown of STARD4 suppressed HCC1937 cell proliferation. Magnification ×100. (H) Celigo cell counting and (I) CCK-8 assays demonstrated that knockdown of STARD4 suppressed MDA-MB-231 cell proliferation. Magnification ×100. Data are presented as the mean ± SD (n=8). **P<0.01 and ***P<0.001 vs. shCTRL. BRCA, breast cancer; CCK-8, Cell Counting Kit-8; CTRL, control; OD, optical density; sh, short hairpin RNA; STARD4, steroidogenic acute regulatory protein-related lipid transfer 4.
Fig 5: STARD4 expression is upregulated in BRCA samples. STARD4 expression was (A) negative, (B) weakly positive, (C) positive and (D) strongly positive in clinical BRCA samples as detected by IHC. Scale bar, 50 µm. (E) STARD4 protein levels were significantly upregulated in 121 BRCA samples compared with 49 adjacent normal tissue, and (F) upregulated in 49 paired BRCA samples. (G) STARD4 was upregulated in stage T1, T2, T3 and T4 BRCA samples compared with normal breast samples. (H) STARD4 expression was upregulated in basal and Her2-positive, but not in luminal BRCA samples compared with in normal breast tissues. For (E) statistical comparisons between groups of normalized data were performed using Mann-Whitney U test according to the test condition. For (F) statistical comparisons between groups of normalized data were performed using the Wilcoxon matched-pairs signed rank test according to the test condition. *P<0.05, ***P<0.001 vs. normal group. BRCA, breast cancer; IHC, immunohistochemistry; STARD4, steroidogenic acute regulatory protein-related lipid transfer 4.
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