To investigate the correlation between R-spondin 2 (RSPO2) expression and clinicopathological characteristics in breast cancer，and evaluate its impact on the malignant biological behaviors of breast cancer cell line SKBR-3 in vitro.

RSPO2 expression profiles from 974 breast cancer tissue samples and 113 adjacent normal tissue samples were downloaded from the Cancer Genome Atlas （TCGA） database to analyze difference of RSPO2 expression between cancerous and adjacent normal tissues. Samples were divided into high and low RSPO2 expression groups using the median mRNA expression level as the cutoff value，and the clinicopathological characteristics of patients was compared between two groups. Prognostic implications of RSPO2 in breast cancer patients were evaluated using Kaplan-Meier survival analysis，with survival curves compared via the log-rank test. Functional enrichment and pathway analyses were conducted through Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG）. The mRNA and protein expression levels of RSPO2 in breast cancer cells were determined using RT-PCR and Western blot. SKBR-3 cell lines with RSPO2 knockdown （RSPO2-sh） and negative control （RSPO2-NC） were established via lentiviral transduction. Cellular proliferation，migration，invasion，and apoptosis were evaluated using CCK-8，wound healing，Transwell，and flow cytometry assays，respectively. Expression levels of β-catenin，Cyclin D1，and C-myc were measured by RT-PCR and Western blot. Statistical analyses were performed using the independent sample t test for comparison of quantitative data between two groups，one-way ANOVA for multi-group comparisons，and the LSD test for post-hoc pairwise comparisons.

In both unpaired samples，RSPO2 expression in cancer tissues was significantly higher than that in adjacent non-cancerous tissues ［0.046 （0.022，0.146） vs 0.027 （0.016，0.068），Z=2.751，P=0.006］. In paired samples，RSPO2 expression in cancer tissues was significantly higher than that in corresponding adjacent non-cancerous tissues ［0.022 （0.000，0.076） vs 0.000 （0.000，0.042），Z=-2.200，P=0.028］ breast cancer tissues. Its expression level was significantly correlated with patients' ER status and PR status （P<0.001）. Patients in the high RSPO2 expression group showed significantly lower OS compared with those in the low expression group （HR=1.50，95% CI：1.05-2.14，P=0.024）. GO and KEGG analyses indicated its potential involvement in multiple pathways related to breast carcinogenesis. Results from RT-PCR and Western blot analyses demonstrated differential expression levels of RSPO2 across the T47D，HCC1937，SKBR-3，and JIMT-1 breast cancer cell lines （Western blot： 1.143±0.182，0.980±0.092，2.416±0.059，0.516±0.214，F=46.900，P<0.001； RT-PCR： 1.094±0.075，0.961±0.102，2.634±0.131，0.822±0.221，F=104.912，P<0.001）. Functional assays revealed that compared with the control group （RSPO2-NC），SKBR-3 cells in the RSPO2 knockdown group （RSPO2-sh） exhibited significantly decreased proliferation （0.356±0.037 vs 0.242±0.033，t=4.670，P=0.009），invasion （84.789±8.301 vs 47.203±4.400，t=29.110，P<0.001），and migration capabilities （98.878±6.560 vs 53.670±6.250，t=21.610，P<0.001），and significantly increased apoptosis （18.687±0.460 vs 23.787±0.710，t=11.550，P<0.001）. The expression levels of β-catenin，Cyclin D1，and C-myc were significantly decreased in the RSPO2-sh group compared with the control group （2.266±0.102 vs 1.493±0.150，t=3.540，P<0.05； 1.176±0.051 vs 0.653±0.080，t=8.050，P< 0.001；1.885±0.050 vs 0.860±0.033，t=18.530，P<0.001）.

RSPO2 is highly expressed in breast cancer. High RSPO2 expression is significantly associated with an unfavorable prognosis. RSPO2 may influence the growth and metastatic capabilities of SKBR-3 cells by regulating the Wnt/β-catenin signaling pathway.