Introduction

Breast cancer (BC) registers as the most prevalently occurring malignancy worldwide among women [1]. Despite significant progress in therapy, effective drugs approved for BC remain limited [2]. Therefore, it is crucial to discover new therapeutic targets and biomarkers for BC. Cancer cells exhibit a strong metabolic requirement for energy to sustain their survival and growth [3]. Unlike normal cells, even when the oxygen supply is sufficient, cancer cells predominantly depend on glycolysis for energy, which is known as aerobic glycolysis (Warburg effect) [4, 5]. Aerobic glycolysis, facilitating tumor proliferation with enhanced glucose consumption and lactate concentration, is widely recognized as a hallmark of cancer cells, and targeting this process has been, and continues to be, a focus for therapeutic agent development.

Hexokinase 2 (HK2), which catalyzes the initial rate-limiting and irreversible step of glycolysis reaction, exerts a key role in altered metabolism in various cancers [6,7,8]. HK2 has been shown to be upregulated in a wide range of human cancers, including hepatocellular carcinoma, breast cancer, gallbladder cancer, colorectal cancer, endometrial carcinoma, osteosarcoma, laryngeal carcinoma, etc., and associated with the clinicopathological characteristics and prognostic factors of cancer patients [6,7,8,9,10,11,12,13]. HK2 promotes cancer cell growth, migration, invasion, and metastasis [14,15,16]. Recently, HK2-targeted therapy has displayed beneficial effects in suppressing cancer cell growth in vitro and eradicating tumors in animals [7].

MiRNAs (miRNAs) have been reported to influence various biological behaviors in tumors, such as cellular proliferation, differentiation, apoptosis, cell cycle, and so on [17,18,19,20]. MiRNA dysregulation might play a significant role in cancer pathogenesis and miRNAs are gradually considered to be potential biomarkers for human cancer diagnosis and treatment [21, 22]. In particular, miRNAs have been shown to exhibit a regulatory effect on glucose metabolism in cancer by inhibiting HK2. For instance, miR-202 inhibits pancreatic cancer cell glycolysis and growth by repressing HK2 expression [23]. MiR-3662 suppresses glucose metabolism, growth, and invasion of hepatocellular carcinoma cells (HCC) by targeting HK2 [30]. Resibufogenin regulates the miR-143-3p/HK2 axis to inhibit tumor growth and glycolysis in breast cancer [31]. MiR-3662 and miR-125a act as suppressors for glucose metabolism by HK2 inhibition, and suppress cell proliferation, invasion, or apoptosis in hepatocellular carcinoma cells in vitro [24, 32]. However, the significance of the physiology and pathology of these natural miRNAs molecules is unclear. Our research found that let-7b-5p is a novel inhibitor of HK2, inhibits HK enzyme activity, glucose uptake, lactate level, and ATP concentration, and leads to conversion from aerobic glycolysis to mitochondrial respiration via repressing HK2 in BC cells. HK2 has two isoforms (NM_000189.5 and NM_001371525.1), which share the same 3’-UTR sequence. As let-7b-5p inhibits HK2 expression by targeting its 3’-UTR, it is conceivable that let-7b-5p represses both HK2 isoforms. Let-7b-5p depresses BC proliferation and lung metastasis by suppression of HK2-mediated aerobic glycolysis. Furthermore, let-7b-5p negatively correlates with HK2 in BC tissues. Therefore, these data illustrate the let-7b-5p significance for physiology and pathology in modulating HK2-mediated aerobic glycolysis as well as tumorigenesis and lung metastasis. Upregulation of let-7b-5p could be a promising approach for BC therapy with HK2 overexpression.

Although we show that let-7b-5p regulates BC cell migration and invasion by targeting HK2, we cannot exclude the possibility that it may target other RNAs. It has been reported that let-7b-5p inhibits migration, invasion, and EMT by targeting HMGA2 in head and neck squamous cell carcinoma and HCC cells [25, 26]. We also showed that let-7b-5p could suppress HMGA2 expression in BC cells. Since HMGA2 has been reported to influence cell growth, migration, and invasion in BC cells [33], HMGA2 may be another potential target of let-7b-5p that is involved in these biological processes.

Recently, let-7b-5p has been identified to have different roles in regulating tumorigenesis and cancer progression. As a tumor suppressor, let-7b-5p inhibits growth and apoptosis by targeting IGF1R in multiple myeloma [34]; let-7b-5p suppresses proliferation and motility by negatively modulating KIAA1377 in squamous cell carcinoma cells [35]. The anti-cancer roles were also confirmed in other cancers, such as human glioma and gastric cancer [36, 37]. As a tumor-promoting factor, let-7b-5p is overexpressed in ovarian cancer, and its silence dampens ovarian cancer cell proliferation [38]. Suppression of let-7b-5p is conducive to an anti-tumorigenic macrophage phenotype in prostate cancer by SOCS1/STAT pathway [39]. The findings show that let-7b-5p plays a tissue-specific role in different types of cancer. Previous research have presented that let-7b-5p was downregulated in BC [28] and overexpression of let-7b-5p was associated with better OS and disease-free survival (DFS) in all breast cancer cases [40] by TCGA dataset analysis. However, the influence of let-7b-5p on the Warburg effect and its mechanism in regulating breast cancer is still unclear. We showed that let-7b-5p suppresses not only aerobic glycolysis but also the growth and metastasis of breast tumors by inhibiting HK2-mediated glycolysis. Therefore, our research presents a molecular explanation which links the anti-cancer effect of let-7b-5p in inhibiting breast tumor progression with its ability to dampen glycolysis. In addition, let-7b-5p associates glycolysis with breast tumor proliferation and lung metastasis in vivo.

Estrogen receptor (ER) and breast-cancer susceptibility gene (BRCA) are widely recognized as important markers for BC. ER is not only a powerful predictive and prognostic marker but also a valuable target for the treatment of hormone-dependent breast cancer. BRCA, which includes BRCA1 and BRCA2, is a critical tumor suppressor gene for BC. Mutations in BRCA can cause chromosomal instability, promote cell proliferation, and hinder normal cell differentiation, leading to the development of BC. Recent discoveries have indicated that there are some correlations between such BC markers, let-7b and HK2. Let-7b has been shown to inhibit the expression of ER-α, which is inversely correlated with let-7b in BC tissues [41, 42]. Estradiol (E2) treatment has been found to promote HK2 expression in paclitaxel-resistant BC cells [43]. Dysregulation of let-7b has also been observed in BRCA2 germ-line mutation carriers between invasive breast cancer and asymptomatic normal breast tissue [44]. Furthermore, BRCA1 has been found to repress HK2 expression, reducing glycolysis and attenuating BC cell migration [45].

Overall, our study demonstrates that let-7b-5p dampens BC cell growth and metastasis in vitro and in vivo by suppressing glycolysis via inhibiting the expression of HK2. Let-7b-5p negatively correlates with HK2 in patients with breast cancer. These results verify the significance of the let-7b-5p/HK2 axis in aerobic glycolysis as well as breast tumorigenesis and progression. Therefore, let-7b-5p could be valuable for treating HK2-overexpressing breast cancer patients.

Materials and methods

Cell culture

MDA-MB-231, ZR75-1, and HEK293T cell lines were obtained from American Type Culture Collection (ATCC). MDA-MB-231 cell line labeled with firefly luciferase was a gift from Professor Yongfeng Shang. All cells were cultured in DMEM (Gibco) appended to 10% FBS (Everygreen) and 100 μg/ml penicillin and streptomycin (Biomed) at 37˚C with 5% CO2.

RNA oligonucleotides, plasmids, lentivirus, regents

Let-7b-5p mimic/inhibitor was purchased from GenePharma. Wild-type and mutated sequences of the HK2 3′-UTR were inserted into a pcDNA3-luciferase expression vector, generating HK2 3′-UTR WT and HK2 3′-UTR MUT, respectively. HK2 expression vector was constructed by inserting PCR-amplified fragments into pcDNA3 (Invitrogen). HK2 shRNA stable cell line was established by lentiviral transduction using pSIH-H1-Puro (System Biosciences) carrying HK2 shRNA. The target sequence of HK2 shRNA was ATAAGCTACAAATCAAAGA. Stable cells that were infected with lentiviruses were screened using puromycin. Reagents for miRNAs and plasmids transfection were, respectively, Lipofectamine RNAiMAX and Lipofectamine 3000 (Invitrogen). Anti-HK2 antibody was obtained from Cell Signaling Technology and an anti-β-actin antibody was obtained from Santa Cruz Biotechnology.

Quantitative real-time PCR (RT-qPCR)

Total RNA, including mRNA and miRNA, was extracted with TRIzol reagent (Invitrogen). miRcute Plus miRNA First-Strand cDNA Kit (Tiangen) was used to transcribe miRNA into cDNA. RT-qPCR analysis was determined with 2 × Taq Pro Universal SYBR qPCR Master Mix (Vazyme) using the BioRad CFX96. The relative fold expression of the targets was normalized to U6 or β-actin (endogenous control) and calculated by the 2−∆∆Ct method. Primer sequences used are listed in Table S2.

Luciferase reporter assay

Cells seeded in a 24-well plate were co-transfected with negative control (NC) or let-7b-5p mimic, in combination with luciferase reporters HK2 3′-UTR WT/ Mut and pRL-TK (internal control) using Lipofectamine 3000. Luciferase activities analysis were performed 48 h later following the manufacturer’s instruction (Promega).

Cell proliferation, migration, and invasion assays

Cell proliferation was performed using a CCK-8 kit and colony formation assay. Cell migration was examined by scratch test. Cell invasion was assessed by transwell assay with Matrigel Invasion Chambers. These assays were conducted according to the methods described previously [46].

Glycolytic phenotype assay

Hexokinase Colorimetric Assay Kit, Glucose Uptake Colorimetric Assay kit, ATP Colorimetric Assay kit and Lactate Assay Kit II were purchased from Biovision and used to detect HK activity, glucose uptake, ATP, and lactate production, respectively. These assays were detected following the manufacturer’s protocols as described previously [47].

ECAR and OCR assays

ECAR were examined by Seahorse XF Glycolysis Stress Test Kit and OCR were examined by Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies). Samples were detected via Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience). The assays were performed referring to manufacturer-provided protocols as described previously [48].

Tumorigenesis and metastasis in nude mice

Animal experiments were approved by the Institutional Animal Care Committee of the Bei**g Institute of Biotechnology. For tumorigenesis analysis, ten million MDA-MB-231 cells stably carrying control or HK2 shRNA treated with 1 μmol antagomiR-let-7b-5p (anti-let-7b-5p) or antagomiR-NC (scramble) for 3 days were subcutaneously inoculated into female BALB/c nude mice (6 to 8 weeks old) which were randomly selected seven into each group without blinding. Tumor size was detected by vernier caliper every 5 days and tumor volume was calculated as the formula: (length × width2)/2. After 45 days, the mice were sacrificed and dissected tumors were imaged, and then frozen in liquid nitrogen for further study.

For the metastasis experiment, one million of these treated MDA-MB-231 cells were injected into female BALB/c nude mouse (n = 5/group) by lateral tail vein [47]. Thirty days later, these mice images were captured by the IVIS200 imaging system (Xenogen Corporation) and metastatic foci of lung tissues was analyzed by H&E staining.

Clinical samples, miRNA FISH, and IHC

Samples of 144 human breast cancer and 114 normal tissues were obtained from the PLA General Hospital, with the informed consent of patients and approval of the Institutional Review Committees of the Chinese PLA General Hospital. The expression level of let-7b-5p was determined following miRNA FISH instructions (Exonbio). Let-7b-5p probe (FITC labeled) sequence was AACCACACAACCTACTACCTCA. The scramble probe (negative control) sequence was GTGTAACACGTCTATACGCCCA. The level of HK2 expression was determined by IHC and cyanine 3 system (K1051, APExBIO). Anti-HK2 antibody (Cell Signaling Technology) was used as the primary antibody. IHC of specimens was analyzed as previously described [49]. The fluorescence intensity was examined using a microscope (BX53F; Olympus, Tokyo, Japan). The let-7b-5p or HK2 score was calculated by multiplying staining intensity (1, low; 2, medium; 3, strong) by stained cells percentage (0–100%).

Statistical analysis

Statistical analyses were processed with GraphPad Prism 7 software. Comparisons among multiple groups were analyzed by One-way ANOVA. Means between the two groups were compared by Student’s t-test. Correlation analysis between HK2 and let-7b-5p expression was represented using Spearman rank correlation. P < 0.05 was considered statistically significant. All experiments in vitro were performed in triplicates.