Evaluation of MiR-1908-3p as a novel serum biomarker for breast cancer and analysis its oncogenic function and target genes

Abstract

Background: Breast cancer is one of the most common tumors for women globally. Various miRNAs have been reported to play a crucial role in breast cancer, however the clinical significance of miR-1908-3p in breast cancer remains unclear. The present study aimed to explore the role of miR-1908-3p in breast cancer.

Methods: The expression of miR-1908-3p was detected in 50 pairs of breast cancer tissues and adjacent normal tissues, 60 breast cancer patient serum and 60 healthy volunteer serum. The functional roles of miR-1908-3p in breast cancer cells such as proliferation, migration and invasion were evaluated using CCK8, SRB, wound healing and transwell chambers. In addition, bioinformatics tools were used to identify potential targets of miR-1908-3p.

Results: The results showed that the expression of miR-1908-3p were increased in breast cancer tissues and serum compared with normal breast tissues and serum of healthy volunteers respectively. Furthermore, the young breast cancer patients and HER2-positive patients had a higher level of tissues' miR-1908-3p than elder breast cancer patients and HER2-negative patients, respectively. The young breast cancer patients had a higher level of serum miR-1908-3p than elder breast cancer patients, ROC analysis suggested that miR-1908-3p had the potential as a promising serum diagnostic biomarker of breast cancer. Up-regulation of miR-1908-3p promoted the cells proliferation, migration and invasion while knockdown of miR-1908-3p inhibited these processes in breast cancer cell MCF-7 and MDA-MB-231. The potential target genes of miR-1908-3p in breast cancer included ID4, LTBP4, GPM6B, RGMA, EFCAB1, ALX4, OSR1 and PPARA. Higher expression of these eight genes correlated with a better prognosis for breast cancer patients.

Conclusions: These results suggest that miR-1908-3p may exert its oncogenic functions via suppression of these eight genes in breast cancer.

Keywords: Breast cancer; Invasion; Migration; Proliferation; miR-1908-3p.

Fig. 1

MiR-1908-3p is highly expressed in breast cancer tissues and breast cancer cells. a The miR-1908-3p expression level in breast cancer tissues and adjacent normal breast tissues were compared using TCGA. b The miR-1908-3p expression level in 50 pairs of fresh breast cancer tissue and adjacent normal tissue was determined by RT-qPCR. Quantification of miR-1908-3p expression were calculated with the 2-∆Ct method. c The expression of miR-1908-3p in two breast cancer cell lines and the non-transformed mammary epithelial MCF-10A was determined by RT-qPCR. Relative quantification of miR-1908-3p expression were calculated with the 2-∆∆Ct method. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 2

Serum miR-1908-3p level is increased in breast cancer patients compared with healthy donors. a The serum miR-1908-3p level in 60 breast cancer patients and 60 healthy donors were determined by RT-qPCR. Quantification of miR-1908-3p expression were calculated with the 2-∆Ct method. b High levels of serum miR-1908-3p as a diagnostic marker in patients with breast cancer based on 60 breast cancer patients and 60 healthy donors.***, p < 0.001

Fig. 3

miR-1908-3p promotes MCF-7 cell proliferation, migration and invasion. a The expression of miR-1908-3p in MCF-7 cell were affected by transfection of miR-1908-3p mimics or inhibitor. b & c CCK8 and SRB assay were used to evaluated the proliferation of MCF-7 cells following transfection with miR-1908-3p mimics or inhibitor. d The migration ability of MCF-7 cells with miR-1908-3p mimics or inhibitor transfection. e The invasion ability of MCF-7 cells with miR-1908-3p mimics or inhibitor transfection. Relative quantification of miR-1908-3p expression were calculated with the 2-∆∆Ct method. ***, p < 0.001

Fig. 4

miR-1908-3p promotes MDA-MB-231 cell proliferation, migration and invasion. a The expression of miR-1908-3p in MDA-MB-231 cell were affected by transfection of miR-1908-3p mimics or inhibitor. b & c CCK8 and SRB assay were used to evaluated the proliferation of MDA-MB-231 cells following transfection with miR-1908-3p mimics or inhibitor. d The migration ability of MDA-MB-231 cells with miR-1908-3p mimics or inhibitor transfection. e The invasion ability of MDA-MB-231 cells with miR-1908-3p mimics or inhibitor transfection. Relative quantification of miR-1908-3p expression were calculated with the 2-∆∆Ct method. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 5

Gene ontology terms and KEGG pathway enriched by the potential target genes of miR-1908-3p. a The predicted target genes of miR-1908-3p. b The GO Term molecular function enriched by the potential target genes of miR-1908-3p. c The GO Term cellular component enriched by the potential target genes of miR-1908-3p. d The GO Term biological process enriched by the potential target genes of miR-1908-3p. e The KEGG pathways enriched by the potential target genes of miR-1908-3p

Fig. 6

Identification of candidate miR-1908-3p targeted genes. a The DEGs between breast cancer samples and normal breast samples from GSE33447 database. b The intersection of miR-1908-3p target genes and down-regulated DEGs. c-o The expression levels of ID4, LTBP4, CCNB1IP1, GPM6B, RGMA, BEGAIN, EFCAB1, ALX4, TRIOBP, OSR1, ANO4, PPARA and ZDHHC15 obtained from the GEPIA database. *, p < 0.05

Fig. 7

Prognostic analysis of miR-1908-3p target genes. a-i The prognostic analysis of hsa-miR-1908, ID4, LTBP4, GPM6B, RGMA, EFCAB1, ALX4, OSR1 and PPARA obtained from the Kaplan-Meier Plotter website