Rab14 Suppression Mediated by MiR-320a Inhibits Cell Proliferation, Migration and Invasion in Breast Cancer

Abstract

We found that microRNA-320a (miR-320a) was an attractive prognostic biomarker in breast cancer (BC) previously, whereas its regulatory mechanism in BC was not well understood. Our aim was to identify miR-320a target gene, examine the clinical relationship between miR-320a and its target, and further explore the functions of its target in BC. In this study, miR-320a downstream target gene was determined in HEK-293T cells by dual luciferase reporter assay. Then western blotting and immunohistochemistry were used to assess miR-320a target gene expression in fresh frozen (n=19, breast cancer and matched non-malignant adjacent tissue samples) and formalin-fixed paraffin-embedded (FFPE) (n=130, invasive BC tissues, the same panel detected for miR-320a expression previously) breast tissues, respectively. The results suggested that miR-320a could significantly suppressed Rab14 3'-untranslated region luciferase-reporter activity, and thus Rab14 was first identified as miR-320a target in BC. In 19 matched breast tissues, 12 (63%) breast cancer tissues showed high expression of Rab14 compared with the corresponding normal tissues. Rab14 immunoreactivity was mainly detected in the cytoplasm, 77/130 (59.2%) showed high expression. Furthermore, Rab14 expression was found to be inversely correlated with miR-320a expression in fresh-frozen breast tissues as well as in FFPE invasive breast cancer samples. In addition, Rab14 expression levels were positively related to tumor size (P = 0.034), lymph node metastasis (P < 0.001), distant metastasis (P = 0.001), histological grade (P = 0.035) and clinical tumor lymph-node metastasis stage (P = 0.001). Patients with higher Rab14 expression showed shorter overall survival time. Moreover, silencing of Rab14 could suppress proliferation, migration and invasion in breast cancer cell lines. Collectively, our results indicate that miR-320a could target Rab14 and that they could interact biologically in BC.

Keywords: Rab14; breast cancer; invasion.; miR-320a; migration; proliferation.

Figure 1

Rab14 is a target of miR-320a. a, Schematic of the two predicted binding sequences between miR-320a and Rab14 3'UTR at nucleotide 597-603 and 2262-2269, the pluc-Rab14-3'UTR-reporter constructs containing the wild-type (wt) or mutated (mut) sequences (underlined). b, Luciferase assay in HEK-293T cells. miR-320a suppressed the activity of Rab14-3'UTR-wt, whereas the joint mutation sequences (mut1&2) abolished the suppression in HEK-293T cells. c, Rab14 expression level by western blot in a series of breast epithelial cell lines. d, Rab14 protein level measured by western blot after transfection with pre-mir-320a or anti-miR-320a for 48 hours, respectively. e, Histogram figure about the densitometric analysis of Rab14 protein expression. f, RT-qPCR for Rab14 expression after transfection with pre-miR-320a or anti-miR-320a for 24 hours, GAPDH was used as an endogenous control. All experiments were repeated three times. *P < 0.05, **P < 0.01.

Figure 2

Rab14 and miR-320a might interact biologically. a, miR-320a expression in 19 paired fresh breast tissues by RT-qPCR. b, Rab14 expression in 19 sets of fresh BC and the adjacent non-tumor tissues assessed by western blot. c, Spearman correlation analysis between miR-320a and Rab14 in fresh frozen breast tissues (r = -0.69, P < 0.01). d, Representative case of Rab14 staining intensity. e, Rab14 and miR-320a expression level in 130 FFPE invasive BC tissues. Rab14 was immunostained with anti-Rab14 antibody, and the consecutive sections were detected for miR-320a with locked nucleic acid probes. f, Representative image of Rab-14 and miR-320a expression. g, Spearman correlation analysis between miR-320a and Rab14 in 130 FFPE invasive BC samples (r = -0.48, P < 0.001). Images were taken with a power of ×200.

Figure 3

Kaplan-Meier survival curves in 130 invasive BC patients with different Rab14 expressions. a, Survival analysis of patients stratified by LN status or categorized according to clinical stage. b, Overall survival rate of all patients with high or low Rab14 expression.

Figure 4

knockdown of Rab14 led to reduced viability, migration and invasion in breast cancer cells. a, Validation of Rab14 suppression by siRab14 using western blot. b, histogram related to densitometric analysis of Rab14 protein expression level. c, RT-qPCR for Rab14 expression after transfection of siRab14 for 24 hours. d, Effects of siRab14 on the proliferation of MDA-MB-231 and BT-549 cells. e, Transwell cell migration and invasion assay of MDA-MB-231 and BT-549 cells transfected with siRab14 or negative control. Cells were counted microscopically (×200). All experiments were repeated three times. *P < 0.05, **P < 0.01.