The expression and prognostic associations of RGS10 in breast cancer

(A) RGS10 mRNA levels in 31 normal human tissues. Data were derived from the Genotype-Tissue Expression database.

(B) RGS10 mRNA levels in cell lines representing 21 human cancers. Data were derived from the Cancer Cell Line Encyclopedia database.

(C) qRT-PCR showing RGS10 mRNA levels in freshly resected breast cancer tissues (n = 20) and matched adjacent normal breast tissues

(D–E) Survival analyses showing disease-free survival (DFS) (D) and overall survival (OS) (E) in patients with breast cancer stratified by high versus low RGS10 mRNA levels. Data were derived from the Kaplan–Meier plotter database.

(F) Representative images showing immunohistochemical staining of RGS10 protein expression in breast cancer tissues or normal tissues (n = 133) (magnification: 200× and 400×).

(G–H) Kaplan–Meier analysis showing DFS (G) and OS (H) in patients with breast cancer stratified by presence versus absence of RGS10 protein in breast cancer tissues (n = 133).

Clinicopathological characteristics of 20 patients

Correlations between RGS10 expression and clinicopathological characteristics

Univariate and multivariate Cox regression analyses of clinicopathological risk factors for disease-free survival

Univariate and multivariate Cox regression analyses of clinicopathological risk factors for overall survival

RGS10 silencing increases the proliferation and migration of breast cancer cells in vitro

(A) Western blotting showing RGS10 protein levels in molecularly distinct breast cancer cell lines.

(B) Western blotting showing RGS10 protein levels in SKBR3 cells transfected with three independent shRNA constructs, shRNA-RGS10-161, shRNA-RGS10-321, and shRNA-RGS10-506, and shRNA-NC.

(C) CCK-8 assay showing the viability of SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC.

(D–F) Colony formation (D) and transwell migration/invasion (E, F) assays in SKBR3 cells transfected with shRNA-RGS10-161, shRNA-RGS10-506, or shRNA-NC.

Protein–protein network interaction network and GO functional- and KEGG pathway-enrichment analysis of genes co-expressed with RGS10

(A) Volcano plot showing differentially expressed genes between SKBR3 cells transfected with shRNA-RGS10 or shRNA-NC.

(B–E) KEGG pathway analysis and GO enrichment analysis of differentially expressed genes showing the ten most enriched terms. BP: biological processes; MF: molecular function; CC: cellular compartment.

(F) LCN2, E-cadherin, and vimentin protein levels in SKBR3 cells transfected with shRNA-RGS10 or shRNA-NC.

(G) Schematics of predicted miR-539-5p binding sites between wild-type and mutant RGS10 sequences in the 3ʹ-untranslated regions.

(H) Relative luciferase activities detected after cotransfection of wild-type or mutant luciferase reporter plasmids and an miR-539-mimic.

miR-539-5p regulates the migration, invasion, proliferation, and EMT of breast cancer cells

(A) qPCR showing the transfection efficiency of the miR-539-5p mimic. *** P < 0.001, one-way ANOVA

(B–C) qRT-PCR and Western blotting showing RGS10 mRNA and protein levels in SKBR3 cells transfected with the miR-539-5p mimic, negative control (NC), or wild type (WT). *** P < 0.001, one-way ANOVA.

(D) CCK-8 assay showing the viability of SKBR3 cells transfected with the miR-539-5p mimic, NC, or WT. *** P < 0.001, one-way ANOVA.

(E–G) Colony formation (E) and transwell migration/invasion (F, G) assays in SKBR3 cells transfected with the miR-539-5p mimic, NC, or WT. * P < 0.05, ** P < 0.01, Student’s t test.

(H) Western blotting showing protein levels of LCN2 and biomarkers of EMT in SKBR3 cells transfected with the miR-539-5p mimic or NC.

(I) Immunofluorescence staining showing E-cadherin, vimentin, and snail protein expression in SKBR3 cells transfected with the miR-539-5p mimic or NC. Scale bar: 50 µm.

Gene sets enriched in phenotype ‘High’

miR-539-5p inhibitor suppresses breast cancer cell proliferation and invasion

(A) qPCR showing transfection efficiency of the miR-539-5p inhibitor after 48 h. *** P < 0.001, one-way ANOVA

(B–C) qRT-PCR and Western blotting showing RGS10 mRNA and protein levels in MDA-MB-231 cells transfected with the miR-539-5p inhibitor, negative control (NC), or wild type (WT). *** P < 0.001, one-way ANOVA.

(D) CCK-8 assay showing the viability of MDA-MB-231 cells transfected with the miR-539-5p inhibitor, NC, or WT. *** P < 0.001, one-way ANOVA.

(E–G) Colony formation (E) and transwell migration/invasion (F, G) assays in MDA-MB-231 cells transfected with the miR-539-5p inhibitor, NC, or WT. * P < 0.05, ** P < 0.01, Student’s t test.

(H) Western blotting showing protein levels of LCN2 and biomarkers of EMT in MDA-MB-231 cells transfected with the miR-539-5p inhibitor or NC.

(I) Immunofluorescence staining showing E-cadherin, vimentin, and snail protein expression in MDA-MB-231 cells transfected with the miR-539-5p inhibitor or NC. Scale bar: 50 µm.

RGS10 inhibits breast cancer growth by targeting LCN2 in vivo

(A) Size of tumors derived from RGS10-depleted SKBR3 cells, negative control (NC) and wild type(WT).

(B) Volume of tumors derived from RGS10-depleted SKBR3 cells, NC and WT.

(C) Hematoxylin and eosin staining of tumors derived from RGS10-depleted SKBR3 cells and NC.

(D) Immunohistochemical staining showing LCN2, E-cadherin, snail, and vimentin protein expression in tumors derived from RGS10-depleted SKBR3 cells and NC.