Long noncoding RNA RP11-70C1.3 confers chemoresistance of breast cancer cells through miR-6736-3p/NRP-1 axis

Abstract

Chemoresistance remains a major obstacle for improving the clinical outcome of patients with breast cancer. Recently, long noncoding RNAs (lncRNAs) have been implicated in breast cancer chemoresistance. However, the function and underlying mechanism are still largely unknown. Using lncRNA microarray, we identified 122 upregulated and 475 downregulated lncRNAs that might be related to the breast cancer chemoresistance. Among them, RP11-70C1.3 was one of the most highly expressed lncRNAs. In breast cancer patients, high RP11-70C1.3 expression predicted poor prognosis. Knockdown of RP11-70C1.3 inhibited the multidrug resistance of breast cancer cells in vitro and in vivo. Further investigations revealed that RP11-70C1.3 functioned as a competing endogenous RNA (ceRNA) for miR-6736-3p to increase NRP-1 expression. Notably, the rescue experiments showed that both miR-6736-3p inhibitor and NRP-1 overexpression could partly reverse the suppressive influence of RP11-70C1.3 knockdown on breast cancer chemoresistance. In conclusion, our study indicated that lncRNA RP11-70C1.3 regulated NRP-1 expression by sponging miR-6736-3p to confer chemoresistance of breast cancer cells. RP11-70C1.3 might be a potential therapeutic target in enhancing the clinical efficacy of chemotherapy in breast cancer.

FIGURE 1

LncRNA RP11-70C1.3 is ectopically overexpressed in the chemoresistant breast cancer tissues and associated with poor overall survival. (A) Heat map of top 10 upregulated and 10 downregulated lncRNAs in three chemotherapy-resistant and three chemotherapy-sensitive breast cancer tissues. (B) Relative RP11-70C1.3 expression in 32 chemotherapy-resistant tissues and 28 chemotherapy-sensitive breast cancer tissues, as detected by qRT-PCR. (C) Relative RP11-70C1.3 expression in five human breast cancer cell lines and a normal breast epithelial cell line MCF-10A, as detected by qRT-PCR. (D) Kaplan–Meier survival curves for breast cancer patients with high and low expression of RP11-70C1.3. **p < 0.01.

FIGURE 2

RP11-70C1.3 knockdown suppresses the chemoresistance of breast cancer cells in vitro. (A) Resistance index of MB231/ADM and MCF-7/PTX cells for ADM, PTX, DDP, CPM, 5-Fu, VCR, and MTX, as detected by CCK-8 assay. (B) Relative RP11-70C1.3 expression in MB231/ADM and MCF-7/PTX cells and their parental cells, as detected by qRT-PCR. (C) Relative RP11-70C1.3 expression in MB231/ADM and MCF-7/PTX cells after RP11-70C1.3 knockdown, as detected by qRT-PCR. (D) and (E) Resistance index of MB231/ADM (D) and MCF-7/PTX (E) cells for ADM, PTX, DDP, CPM, 5-Fu, VCR, and MTX in MB231/ADM and MCF-7/PTX cells after RP11-70C1.3 knockdown, as detected by CCK-8 assay. (F) RP11-70C1.3 knockdown promoted apoptosis of MB231/ADM and MCF-7/PTX cells compared with si-NC, as detected by flow cytometry assay. (G) RP11-70C1.3 knockdown reduced the capability of colony formation in MB231/ADM and MCF-7/PTX cells, as detected by colony formation assay. *p < 0.05; **p < 0.01.

FIGURE 3

RP11-70C1.3 knockdown inhibits breast cancer chemoresistance in vivo. Stably expressing sh-RP11-70C1.3 or sh-NC MB231/ADM and MCF-7/PTX cells were subcutaneously injected into the right flank area of nude mice. After 7 days, mice were treated with or without 5 mg/kg ADM or 20 mg/kg PTX through intraperitoneal injections twice a week until the end of the study. Five weeks later, the mice were killed and the tumor weight was recorded. (A) Representative pictures of tumor tissues. (B) and (C) Tumor volume from MB231/ADM (B) and MCF-7/PTX (C) cells. (D) and (E) Tumor weight from MB231/ADM (D) and MCF-7/PTX (E) cells. (F) Ki-67 immunohistochemical staining analysis. (G) and (H) Statistical analysis of Ki-67 positive cells from MB231/ADM (G) and MCF-7/PTX (H) cells. **p < 0.01.

FIGURE 4

RP11-70C1.3 is a sponge of miR-6736-3p. (A) and (B) DIANA-LncBase analysis revealed that 16 candidate miRNAs could interact with RP11-70C1.3. qRT-PCR was used to determine their expression levels in MB231/ADM (A) and MCF-7/PTX (B) cells after RP11-70C1.3 knockdown. (C) Putative binding site of miR-6736-3p for RP11-70C1.3. (D) and (E) Luciferase reporter assay results showed that miR-6736-3p mimics reduced the luciferase activity of wt-RP11-70C1.3 but not that of the mut-RP11-70C1.3 vector in MB231/ADM (D) and MCF-7/PTX (E) cells. (F) Relative miR-6736-3p expression in 32 chemotherapy-resistant tissues and 28 chemotherapy-sensitive breast cancer tissues, as detected by qRT-PCR. (G) Relative miR-6736-3p expression in MB231/ADM and MCF-7/PTX cells and their parental cells, as detected by qRT-PCR. (H) Pearson correlation analysis demonstrated that miR-6736-3p negatively correlated with RP11-70C1.3 expression in breast cancer tissues. ns p > 0.05; * p < 0.05; ** p < 0.01.

FIGURE 5

miR-6736-3p directly targets NRP-1 and represses its expression. (A) Predicted binding sites of miR-6736-3p for the 3’UTR of NRP-1, as analyzed by TargetScan v7.2. (B) and (C) Luciferase reporter assay results showed that miR-6736-3p mimics reduced the luciferase activity of NRP-1 wt-3’UTR but not that of the NRP-1 mut-3’UTR vector in MB231/ADM (B) and MCF-7/PTX (C) cells. (D) Biotinylated miR-6736-3p and miR-NC were transfected into MB231/ADM and MCF-7/PTX cells. RNA pull-down assay results showed that NRP-1 mRNA was highly enriched in Bio-miR-6736-3p group. (E) Effects of miR-6736-3p overexpression on the mRNA levels of NRP-1 in MB231/ADM and MCF-7/PTX cells determined by qRT-PCR. (F) Effects of miR-6736-3p overexpression on the protein levels of NRP-1 in MB231/ADM and MCF-7/PTX cells were determined by Western blot. (G) Quantitative analysis of Figure 5F. (H) Relative NRP-1 mRNA expression in MB231/ADM and MCF-7/PTX cells and their parental cells, as detected by qRT-PCR. (I) and (J) Relative NRP-1 mRNA and protein expression in 32 chemotherapy-resistant tissues and 28 chemotherapy-sensitive breast cancer tissues, as detected by qRT-PCR and Western blot. (K) Pearson correlation analysis demonstrated that miR-6736-3p negatively correlated with NRP-1 mRNA expression in breast cancer tissues. ** p < 0.01.

FIGURE 6

RP11-70C1.3 promotes breast cancer chemoresistance through regulating miR-6736-3p/NRP-1 axis. (A) and (B) miR-6736-3p inhibitors reversed the suppression of RP11-70C1.3 knockdown on NRP-1 mRNA (A) and protein (B) level in both MB231/ADM and MCF-7/PTX cells. (C) Pearson correlation analysis demonstrated that RP11-70C1.3 positively correlated with NRP-1 mRNA expression in breast cancer tissues. (D) and (E) qRT-PCR and Western blot were used to validate the upregulation of NRP-1 in mRNA (D) and protein (F) levels in MB231/ADM and MCF-7/PTX cells after transfected with pcDNA3.1-NRP-1 or its negative control pcDNA3.1. (F) and (G) CCK-8 assay results showed that transfection of miR-6736-3p inhibitor or pcDNA3.1-NRP-1 attenuated the chemoresistance inhibition induced by RP11-70C1.3 knockdown in MB231/ADM (F) and MCF-7/PTX (G) cells. (H) The apoptosis rate induced by RP11-70C1.3 knockdown in MB231/ADM and MCF-7/PTX cells was obviously decreased after transfection with miR-6736-3p inhibitor or pcDNA3.1-NRP-1. (I) The effects of RP11-70C1.3 knockdown on the capability of colony formation in MB231/ADM and MCF-7/PTX cells were reversed after miR-6736-3p silence or NRP-1 overexpression. * p < 0.05; ** p < 0.01.