Figures & data
Figure 1. TDRG1 is prominently up-regulated in BC and knockdown of TDRG1 impedes malignant behaviors of BC progression in vitro
A．QRT-PCR analysis was utilized to evaluate the TDRG1 expression level in BC cell lines and normal epithelial cell line (MCF-10A). B. The efficiency of TDRG1 silencing was ensured by qRT-PCR analysis. C-D. CCK8 and CFSE assays were conducted to test cell proliferation ability after knockdown of TDRG1. E. Flow cytometry was utilized to assess apoptosis after knockdown of TDRG1. F-G. Transwell assays were implemented to assess the number of migrated and invaded cells after knockdown of TDRG1. H. Tube formation assay was implemented to detect angiogenesis after knockdown of TDRG1 in HUVECs. **P < .01.
Figure 2. TDRG1 exerts oncogenic properties in BC via negatively regulating miR-214-5p
A-B. Subcellular fractionation and FISH assay were conducted to determine the location of TDRG1. C. QRT-PCR analysis was implemented to investigate miRNA expression when TDRG1 was silenced. D. QRT-PCR analysis was performed to detect the expression profile of miR-214-5p in cell lines. E. The binding sites of miR-214-5p with TDRG1 were predicted. F. Wild and mutant TDRG1 sequences were sub-cloned into luciferase reporter. Luciferase activities were normalized to renilla luciferase. G. RNA pull down was performed to explore the mechanical relationship between miR-214-5p and TDRG1. H. The inhibiting efficiency of miR-214-5p inhibitor was evaluated by qRT-PCR. I-N. Rescue experiments were performed to evaluate the impact of miR-214-5p inhibitor on sh-TDRG1induced BC biological activities. **P < .01.
Figure 3. TDRG1 exerts positive effects on CLIC4 expression by negatively regulating miR-214-5p
A. QRT-PCR was utilized to evaluate the transfection efficiency of miR-214-5p mimics plasmid. B. MiRNA candidates’ expression was examined by qRT-PCR analysis after overexpressing miR-214-5p. C. QRT-PCR was performed to evaluate miR-214-5p expression in BC cell lines and normal one. D. Putative binding sites between miR-214-5p and CLIC4 and. E. Wild and mutant CLIC4 sequences were sub-cloned into luciferase reporter, and dual luciferase reporter was utilized to determine the relationship between CLIC4 and miR-214-5p. F. RIP assay was performed to verify the association of miR-214-5p, CLIC4, and TDRG1. G. The inhibiting efficiency of sh-CLIC4 plasmid was examined by qRT-PCR. H-L. Functional experiments were conducted to investigate CLIC4’s role in BC cellular activities. **P < .01.
Figure4. TDRG1 expedites cell proliferation and migration by regulating miR-214-5p/CLIC4 axis
A. QRT-PCR was utilized to determine CLIC4 expression after pcDNA3.1/CLIC4 plasmid transfection. B-C. CLIC4 mRNA expression and protein expression were detected, respectively, by QRT-PCR and western blot. D-I. Functional experiments were conducted to detect the effects of pcDNA3.1/CLIC4 on sh-TDRG1-mediated biological activities in rescue analysis. J. Xenograft tumors shrank after knockdown of TDRG1. K. Xenograft tumor growth curved within four weeks after inoculating MDA-MB-231 cells. L-M. Xenograft tumors were measured and compared in terms of volume and weight. **P < .01.
