miR-6126 modulates GRP78 to suppress the Warburg effect and mitochondrial dynamics in triple-negative breast cancer

Abstract

Cancer cells often exhibit a metabolic shift towards aerobic glycolysis, known as the Warburg effect, leading to excessive energy production that facilitates tumorigenesis, including in breast cancer. Recently, non-coding RNAs, including microRNAs (miRNAs), have been identified as playing crucial roles in various human cancers. However, their roles in regulating metabolic reprogramming in breast cancer remain largely unexplored. Here, we identified the novel miRNA miR-6126, which is highly expressed in TNBC cells by using a miRNA microarray analysis. Overexpression of miR-6126 reduced the growth of TNBC cells and induced apoptosis by targeting GRP78 in vitro and in vivo. In addition, a luciferase reporter assay confirmed that GRP78 is a direct target of miR-6126. Elevated glucose metabolism, indicated by increased levels of LDHA and glucose transporter-1, was observed in TNBC following GRP78 overexpression. Treatment with miR-6126 mimics or GRP78 siRNA not only reduced LDHA and GLUT1 expression but also decreased glucose uptake and lactate release in TNBC cells. Moreover, miR-6126 impaired mitochondrial function by inducing mitochondrial fission through the downregulation of phospho-Drp1 (Ser616) and FIS1. Furthermore, we demonstrated that the expression of miR-6126 is negatively correlated with GRP78 in human tumor tissues. Our findings revealed that miR-6126 is implicated in tumorigenesis via the Warburg effect by targeting GRP78 and restoring mitochondrial function in TNBC.

Keywords: GRP78; Warburg effect; miR-6126; mitochondria dynamics; triple-negative breast cancer.

Figure 1

miR-6126 inhibits cell proliferation and induces apoptosis in triple-negative breast cancer (TNBC) cell lines. (A) MicroRNA expression in normal breast epithelial cells (MCF10A) and TNBC cell line (MDA-MB-231) was assessed by miRNA microarrays to discover that miR-6126 was downregulated. (B) Analysis results of miR-6126 by qRT-PCR. (C) MDA-MB-231 cells were not transfected (NT), transfected with miRNA mimics negative control (NC) and 20 nM or 40 nM miR-6126 mimics. (D) Cell number was detected after transfection with microscope 100X (Scale bars: 100 μm). Cell viability was determined using the MTT assay. (E) Protein levels determined by Western blotting in MDA-MB-231 cell lines were transfected with miR-6126 mimics. Values shown are means ± SD. Quantification of the result is shown (n=3) ns: no significant, *p < 0.05, **p < 0.01, ***p < 0.001 versus untreated control cells.

Figure 2

miR-6126 induces cell apoptosis by inhibiting GRP78 expression. (A) Schematic showed reporter constructs of wild-type GRP78 3'UTR (upper panel) and GRP78 3'UTR with mutated miR-6126-binding site (lower panel). (B) Analysis of mRNA GRP78 was detected by qRT-PCR. (C) The activity of miR-6126 promotor was analysis by luciferase gene reporter assay. (D) Protein levels of GRP78 was determined by Western blotting. (E) Immunofluorescent staining of GRP78 in MDA-MB-231 cells by fluorescence microscope 100X (Scale bars: 100μm). (F) Apoptosis rate was examined by Annexin-V FITC/PI staining and flow cytometry.

Figure 3

miR-6126 reduces the PTEN-inhibited Warburg effect by targeting GRP78. (A) The comparison of protein levels determined by Western blotting in MCF-10A and MDA-MB-231 cell lines. β-actin was served as a loading control. (B) MDA-MB-231 cells were not transfected (NC), transfected with empty plasmid (Mock), 2 μg, 4 μg or 6 μg cDNA GRP78. Protein levels were determined by western blotting. β-actin were served as a loading control. (C) MDA-MB-231 cells were transfected siGRP78 with the indicated concentration. Protein levels were determined by western blotting. (D) MDA-MB-231 cells were not transfected (NT), transfected with miRNA mimics negative control (NC) or 20 nM miR-6126 mimics. Protein levels were determined by Western blotting.

Figure 4

miR-6126 suppresses the glycolysis pathway in triple-negative breast cancer (TNBC) cell line. (A) MDA-MB-231 cells were not transfected (NT), transfected with siRNA negative control (NC) or siGRP78. Protein levels were determined by Western blotting. (B) MDA-MB-231 cells were transfected with miRNA mimics negative control (NC) or 20 nM miR-6126 mimics. Protein levels were determined by Western blotting. β-actin and GADPH were served as a loading control. (C) MDA-MB-231 cells were transfected with cDNA negative control (Mock) or 2 μg, 4 μg or 6 μg cDNA of GRP78. Protein levels were determined by Western blotting. (D) MDA-MB-231 cells were transfected with miR-6126 mimics or siGRP78, then incubated with a fluorescent glucose derivative, 2-NBDG, for 30 min before examination by fluorescence microscope 100X (Scale bars: 100μm). Fluorescence intensity of 2-NBDG uptake was analysed by flow cytometry. Phloretin was used as a natural phenol that inhibits glucose uptake. (E) Lactate production was performed by using Lactate Colorimetric/Fluorometric Assay Kit. Values shown are means ± SD. Quantification of the result is shown (n=3) ns: no significant, *p < 0.05, **p < 0.01, ***p < 0.001 versus untreated control cells.

Figure 5

miR-6126 and knockdown of GRP78 suppress mitochondrial function by inhibiting mitochondrial fission in triple-negative breast cancer (TNBC) cell line. (A) MDA-MB-231 cells were not transfected (NT), transfected with miRNA mimics negative control (NC), 20 nM miR-6126 mimics, or 100 μM MdiVi-1 for 24 h. Protein levels were determined by Western blotting. (B) Immunofluorescent staining of COX IV in MDA-MB-231 cells with miR-6126 mimics transfection or 100 μM MdiVi-1 treatment (Scale bars: 25 μm). (C) Mitochondria were labelled by using an anti-TOM20 antibody (Scale bars: 25 μm). (D) Immunofluorescent staining of MFN-1 and TOM20 in MDA-MB-231 cells with miR-6126 mimics transfection or 100 μM MdiVi-1 treatment (Scale bars: 25 μm). (E) The mitochondrial respiration profile was analyzed by measuring OCR in MB231 cells transfected with GRP78 siRNA (siG) and the miR-6126 mimic. (F) Quantification of basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in MB231 cells transfected with GRP78 siRNA and the miR-6126 mimic. Values shown are means ± SD. Quantification of the result is shown (n=3) *p < 0.05, **p < 0.01 and ***p < 0.001versus untreated control cells.

Figure 6

GRP78 inhibition enhances doxorubicin (Doxo) cytotoxicity and modulates glutamine-dependent cell viability in triple-negative breast cancer (TNBC) cells. (A) MDA-MB-231 cells were treated with increasing concentrations of Doxo alone or in combination with the GRP78 inhibitor YUM70 (10 µM) for 24 hours. Cell viability was assessed using the MTT assay. (B) Representative images of mitochondrial ROS (mitoROS) detected by mitochondria superoxide detection staining after treatment with Doxo (0.005 µM), YUM70 (10 µM), or the combination for 24 hours. Increased mitoROS was observed in the combination group, indicating enhanced oxidative stress. (C) Western blot analysis showing the effect of YUM70 on AMPK activation. MDA-MB-231 cells were treated with increasing concentrations of YUM70 (0-10 µM) for 24 hours. Phosphorylation of AMPK (p-AMPK) and total AMPK were assessed; α-tubulin served as a loading control. (D) Cell viability of MDA-MB-231 cells treated with YUM70 under high (2.5 mM) or low (0.1 mM) glutamine conditions for 24 hours. YUM70 treatment significantly reduced cell viability in glutamine-replete conditions, suggesting that GRP78 inhibition affects glutamine-dependent metabolic survival. Data are presented as mean ± SD; ***p < 0.001 vs. untreated control.

Figure 7

miR-6126 inhibits tumor growth and metastasis in an orthotopic breast cancer mouse model. (A) The experiment design for evaluating the inhibitory effects of miR-6126 on tumorigenesis and metastasis of TNBC in vivo (B) To monitor primary tumor growth after one-week injection, luciferase substrate luciferin was injected, and bioluminescence emission was measured using in vivo imaging. Mice with luciferase-expressing MDA-MB-231-Luc breast cancer cells implanted in their mammary fat pat. Bioluminescent signals photons/second were quantified using the IVIS imaging system. (C) Tumor volume was recorded weekly after reaching a size of approximately 100 mm³ (D) Representative images of IHC staining of ki-67 in tumor sample using microscope 100X (Scale bars: 100μm). (E) Effect of miR-6126 on inhibition of breast cancer metastasis in vivo. Histological (H&E) staining performed in lung tissue following the different treatments compare with normal mice (Scale bars: 50 μm). (F) Protein level analysis from tumor samples by Western blotting. Values shown are means ± SD. Quantification of the result is shown (n=3) *p < 0.05, **p < 0.01 and ***p < 0.001 versus untreated control cells.

Figure 8

MiR-6126 expression is correlated with GRP78 in human triple-negative breast cancer (TNBC) tumor tissue. (A) Representative images showing immunohistochemical staining of miR-6126 and GRP78 in human TNBC tumor tissues. Scale bars range from 50 to 100 μm. (B) Correlation analysis between miR-6126 and GRP78. Sample size (n) = 50. Statistical calculations were performed on biological replicates using simple linear regression. Expression levels were normalized to the H-score. (C) Proposed mechanism by which miR-6126 enhances PTEN activation through GRP78 inhibition, leading to the reduction of the Warburg effect and mitochondria fission in TNBC.