Normal breast epithelial cells MCF10A and breast cancer cell lines MDA-MMB-436, HS578T, SKBR3, MDA-MB-231, and MCF-7 were purchased from the Shanghai Cell Bank of the Chinese Academy of Science. MCF10A cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium supplemented with 5% horse serum, epidermal growth factor (20 ng/mL), insulin (10 µg/mL), hydrocortisone (0.5 mg/mL), cholera toxin (100 ng/mL), and penicillin (100 U/mL) and streptomycin (100 µg/mL) (Thermo Fisher, Waltham, USA). MDA-MMB-436, HS578T, MDA-MB-231, and MCF-7 were cultured in Roswell Park Memorial Institute 1640 medium containing 10% fetal bovine serum (FBS, Life Technology, Pleasanton, USA), 2mM L-glutamine, 20 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin medium supplemented with 2 mM glutamine and 15% FBS. HS578T cells were cultured in DMEM medium supplemented with 10% FBS, 0.01 mg/mL bovine insulin and 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher). Cells were cultured in a 5% saturated CO₂ atmosphere at 37°C.

A total of 30 paired breast tissues samples (lumA/B, n = 19; basal-like, n = 6; human epidermal growth factor receptor 2 [HER-2], n = 3; normal-like, n = 2) were collected from patients by surgical resection. The diagnosis of breast cancer was confirmed by histopathological evaluation. A total of 50 biologically independent samples (lumA/B, n = 25; basal-like, n = 10; HER-2, n = 9; normal-like, n = 6) were used in correlation analyses conducted between c-Myc and lncRNA-SNHG7 in breast cancers. The use and conduct of all the human materials were approved by the Institutional Review Board (IRB) in the Second Hospital of Hebei Medical University (IRB No. SHHBYU-085-JC1). All participants signed the informed written consent.

We analyzed a total of 837 patients, including 138 stage I, 480 stage II, 180 stage III, 15 stage IV, 15 stage Tis, and 9 stage X patients. These patient samples had been evaluated for histologic characteristics and immunostained for estrogen receptor, HER2/neu, epidermal growth factor receptor, cytokeratin 5/6, p53, and Ki-67. The clinical subtypes of these 837 patients included lumA/B (n = 417+191), basal-like (n = 139), HER-2 (n = 67), normal-like (n = 23).

MCF-7 cells were harvested and washed with phosphate-buffered saline. After centrifugation, the cell pellets were subjected to fractionation using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher) following the manufacture's protocol. The cytoplasmic and nuclear fractions were subjected to RNA extraction or western blot. Poly (ADP-ribose) polymerase (PARP) and β-actin were used as markers for cytoplasm and nucleus in western blot and U1 were used as a marker in real-time polymerase chain reaction (RT-PCR).

Lentivirus expressing control short hairpin RNA (shRNA) or lncRNA-SNHG7 shRNA, and lentivirus expressing lncRNA-SNHG7 (pCDH-lncRNA-SNHG7) or empty vector (pCDH), miR-34a-5p mimics, miR-34a-5p inhibitors, and control miRNAs were purchased from RiboBio (Guangzhou, China). MCF-7 cells or MDA-MB-231 cells were seeded in 24-well or 96-well plates and transduced with lentivirus. The cells were harvested for analysis 48 hours post transduction. In some experiments, MCF-7 cells were transfected with miR-34a-5p mimics or inhibitors using Lipofectamine 2000 (Thermo Fisher) following the manufacturer's protocol and then transduced with lentivirus immediately. Cells were harvested for analysis 48 hours post transfection/transduction.

The CCK-8 assay was used to monitor cell viability. Briefly, MCF-7 cells were seeded in a 96-well plate and transduced with the lentivirus. At 48 hours post transduction, the CCK-8 reagent was added at a final concentration of 100 µL/mL medium (Sigma Aldrich, St Louis, USA) and the cells were incubated for 2 hours. The absorbance was read at 450 nm using BioTek™ PowerWave™ Microplate Spectrophotometer (Thermo Fisher).

MCF-7 cells transduced with lentivirus expressing control shRNA or lncRNA-SNHG7 shRNA were cultured for 24 hours. The lactate levels in the culture medium were determined using the Lactate Assay Kit (Colorimetric; Abcam, Cambridge, USA) according to manufacturer's instructions.

The ECAR was measured in MCF-7 cells following lentiviral transduction and/or miRNA transfection using the Seahorse XF96e analyzer (Seahorse Bioscience, North Billerica, USA) according to the manufacture's instruction. Briefly, MCF-7 cells were seeded in a 96-well XF cell culture microplate with complete growth medium following lentiviral transduction and/or miRNA transfection. ECAR was measured using an XF96 analyzer in XF base medium containing 4 mM glutamine following sequential addition of 10 µM glucose, 1 µM oligomycin and 50 mM 2-DG. Data were analyzed by a Seahorse XF Glycolysis Stress Test Report Generator.

For antisense oligomer affinity pull down assay, biotin-labeled sense or antisense DNA oligomers corresponding to lncRNA-SNHG7 (RiboBio) were incubated with MCF-7 cell lysate in DNase/RNase free conditions. After incubation for 1 hour, streptavidin-coupled agarose beads (Thermo Fisher) were added to isolate the RNA-RNA complexes. The lncRNA-SNHG7 and miR-34a-5p levels were analyzed by quantitative RT-PCR (qRT-PCR).

Total RNA was extracted from tissues, and the MCF-7 and MDA-MB-231 cells using Trizol reagent (Thermo Fisher) following manufacturer's instructions. The RNA was reverse transcribed into complementary DNA by using PrimeScript RT reagent kit (Takara, Dalian, China). SYBR Green RT-PCR Master Mix was used for qPCR assay which was performed on an ABI 7500 RT-PCR system (Thermo Fisher). U6 was used as the internal control for normalization of the relative miRNA expression. All primers used for RT-PCR were purchased from Qiagen (Germantown, USA).

Total proteins were extracted from MCF-7 and MDA-MB-231 cells using M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher), and protein concentration was measured by using Pierce BCA Protein Assay Kit (Thermo Fisher). A total of 20 µg of protein was loaded onto a sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis gel and later transferred to polyvinylidene fluoride membrane. Following transfer, the membranes were blocked with 5% non-fat milk in 0.1% tween-20 in tris buffered saline (TBST) for 1 hour at room temperature. Then primary antibodies were added and the membranes were incubated at 4°C overnight. Next day, after 3 washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated corresponding secondary antibodies at room temperature for 1 hour. Immunoreactive proteins were detected using Clarity™ Western ECL Blotting Substrates (Bio-Rad, Hercules, USA). The density was quantitated and analyzed using ImageJ. Primary antibodies used in the current study were: anti-HK2 (Thermo Fisher), anti-GPI (Thermo Fisher), anti-PFKL (Thermo Fisher), anti-ALDB (Thermo Fisher), anti-TPI1 (Sigma Aldrich), anti-PGK1 (Thermo Fisher), anti-PGAM1 (Sigma Aldrich), anti-ENO1 (Sigma Aldrich), anti-PKM2 (Sigma Aldrich), anti-lactate dehydrogenase A (LDHA; Thermo Fisher), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Thermo Fisher), and anti-β actin (Sigma Aldrich).

IP-PCR was performed following a modified protocol as described previously [12]. Briefly, MCF-7 cells were lysed in hypotonic buffer (10 mM HEPES [pH 7.6], 1.5 mM MgCl₂, 10 mM KCl, and 0.2 mM phenylmethylsulfonyl fluoride) containing 0.5% NP40 and supplemented with RNase A inhibitor and DNase I. The cell lysates were precleared with protein A/G beads (Sigma Aldrich) and then incubated with protein A/G beads coated with the indicated antibodies for 3 hours at 4°C. Then beads were washed 3 times with wash buffer (10 mM Tris [pH 7.6], 100 mM KCl, 5 mM MgCl₂, and 1 mM dithiothreitol) and eluted using elution buffer (50 mM Tris [pH 8.0], 1% SDS, and 10 mM EDTA) at 65°C for 10 minutes. To isolate protein-associated DNA from the eluted immunocomplexes, the samples were treated with proteinase K at 37°C for 30 min, followed by 2 rounds each of phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform extraction, and then precipitated with ethanol. The purified DNA was subjected to PCR analysis. Primers used for PCR were: lncRNA-SNHG7: Forward: 5′-CTAGGACGTCTGCTCACTGG-3′, Reverse: 5′-CAGGAGGGCTTAGTTACATTGGA-3′. GAPDH: Forward: 5′-TACTAGCGGTTTTACGG GCG-3′, Reverse: 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′.

To determine the effect of c-Myc on lncRNA-SNHG7 promoter, MCF-7 cells and MDA-MB-231 cells were transduced with lentivirus expressing control shRNA or c-Myc shRNA (RiboBio), and co-transfected with the PGL3-WT lncRNA-SNHG7 promoter or mutant constructs and Renilla luciferase reporter plasmid. The firefly and Renilla luciferase activities were measured using a Dual-luciferase reporter assay system (Promega, Madison, USA) 24 hours post transduction/transfection.

Data are presented as mean ± standard deviation and analyzed using GraphPad Prism 8 software (GraphPad Software, San Diego, USA). Student's t-test, 1- or 2-way analysis of variance with Bonferroni post hoc test was used for analysis. The difference was considered as significant when p < 0.05.

To investigate the potential role of lncRNA-SNHG7 in breast cancer, we monitored the level of lncRNA-SNHG7 in breast cancer tissues. As shown in Figure 1A, we detected significantly increased level of lncRNA-SNHG7 in tumor tissues when compared to normal breast tissue. In addition [13], we analyzed a total 837 patients cases by TANRIC and found that increased level of lncRNA-SNHG7 was correlated with decreased patient survival (Figure 1B). Next, we monitored the level of lncRNA-SNHG7 in several breast cancer cell lines including MDA-MMB-436, HS578T, MDA-MB-231, MCF-7, and HS578T. As shown in Figure 1C, compared to normal breast epithelial cells, all the breast cancer cell lines had significantly higher level of lncRNA-SNHG7, which is consistent with the results from patient tissues. We monitored the expression pattern of lncRNA-SNHG7 by cell fractionation. The quality of the fractions was confirmed by western blotting. As expected we detected the nuclear marker, PARP, mainly in the nuclear fractions and β-actin was detected only in the cytoplasmic fraction (Figure 1D). RT-PCR analysis of fractionated nuclear and cytoplasmic RNA revealed that lncRNA-SNHG7 localized mainly in the cell cytosol while the small nuclear RNA U1 localized only in nucleus and β-actin mRNA mainly localized in cell cytosol (Figure 1E). Taken together, our data demonstrated that lncRNA-SNHG7 was up-regulated in breast cancer and mainly localized in cell cytosol.

To evaluate the effect of lncRNA-SNHG7 on cell proliferation of MCF-7 cells, we transduced MCF-7 cells with lentivirus expressing shRNA against lncRNA-SNHG7 or lentivirus expressing control shRNA. Transduction of lentivirus expressing shRNA against lncRNA-SNHG7 significantly decreased the endogenous level of lncRNA-SNHG7 (Figure 2A). Correspondingly, the total cell number in MCF-7 cells transduced with lentiviruses expressing lncRNA-SNHG7 shRNA1 or shRNA2 was lower than the total cell number in MCF-7 cells transduced with lentivirus expressing control shRNA, and the difference became significant on day 5 (Figure 2B). In addition, MCF-7 cell transduced with lentiviruses expressing lncRNA-SNHG7 shRNA1 or shRNA2 had significantly decreased cell viability compared to MCF-7 cells transduced with lentivirus expressing control shRNA (Figure 2C). Transduction of MCF-7 cells with lentivirus expressing lncRNA-SNHG7 resulted in significantly higher lncRNA-SNHG7 level (Figure 2D) compared to the non-transduced cells or cells transduced with the control lentivirus. The overexpression of lncRNA-SNHG7 resulted in higher cell number and the difference was significant on day 5 post lentiviral transduction (Figure 2E), which was due to the significantly increased cell viability of MCF-7 cells overexpressing lncRNA-SNHG7 (Figure 2F). Similar results were obtained using MDA-MB-231 cells (Supplementary Figure 1). Taken together, our data demonstrated that lncRNA-SNHG7 promoted breast cancer cell proliferation.

Next, we investigated the functional role of lncRNA-SNHG7 in glycolysis in MCF-7 cells. We observed acidification of the culture medium of MCF-7 cells. In contrast, knocking down lncRNA-SNHG7 by transducing lentivirus expressing lncRNA-SNHG7 shRNA1 or shRNA2 in MCF-7 cells prevented the acidification of culture medium (Figure 3A). We also detected significantly decreased lactate production in MCF-7 cells transduced with lentivirus expressing lncRNA-SNHG7 shRNA1 or shRNA2, when compared to MCF-7 cells transduced with lentivirus expressing control shRNA (Figure 3B). Knocking down lncRNA-SNHG7 also significantly decreased cell glycolysis assessed by the ECAR (Figure 3C and D). As knocking down lncRNA-SNHG7 affected glycolysis, we next investigated which of the glycolysis enzymes were affected by the knockdown. Using western blotting and RT-PCR, we identified that knocking down lncRNA-SNHG7 did not affect the protein and mRNA levels of most glycolysis enzymes including HK2, GPI, PFKL, ALDB, TPI1, PGK1, PGAM1, ENO1, and PKM2 (Figure 3E and F). In contrast, we detected significantly decreased protein and mRNA levels of LDHA in MCF-7 cells transduced with lentivirus expressing lncRNA-SNHG7 shRNA1 or shRNA2 (Figure 3E and F), suggesting that lncRNA-SNHG7 regulated the expression of LDHA. Similar results were obtained using MDA-MB-231 cells (Supplementary Figure 2). Collectively, we demonstrated that lncRNA-SNHG7 regulated LDHA expression and glycolysis.

Next, we investigated the mechanism of lncRNA-SNHG7 regulated LDHA expression. LncRNAs have been shown to function as miRNA sponges. LncRNA-SNHG7 was identified to binding to miR-34a (Figure 4A), which has been described previously [14]. To confirm the interaction between lncRNA-SNHG7 and miR-34a, we performed the pull-down assay. We found that a biotin-labeled antisense DNA probe of lncRNA-SNHG7 enriched the endogenous lncRNA-SNHG7 while the sense DNA probe did not (Figure 4B). In addition, lncRNA-SNHG7 antisense DNA probe was able to pull down miR-34a-5p (Figure 4B). These results indicated that lncRNA-SNHG7 interacted with miR-34a-5p. To test whether the regulation of LDHA by lncRNA-SNHG7 depended on miRNA. LncRNA-SNHG7 shRNA lentivirus transduced-MCF-7 cells were transfected with miR-34a-5p inhibitors and the LDHA protein level was monitored. As shown in Figure 4C, the expression of lncRNA-SNHG7 shRNA resulted in decreased LDHA expression. In contrast, overexpression of miR-34a-5p inhibitor in MCF-7 cells expressing lncRNA-SNHG7 shRNA rescued the expression of LDHA. Overexpression of lncRNA-SNHG7 resulted in up-regulation of LDHA expression while this upregulation was inhibited by overexpression of miR-34a-5p (Figure 4D). Therefore, these results indicated that lncRNA-SNHG7 regulated LDHA expression through miR-34a-5p. Knocking down lncRNA-SNHG7 resulted in decreased cell glycolysis as measured by ECAR. In contrast, overexpressing miR-34a-5p inhibitor rescued the decreased cell glycolysis mediated by lncRNA-SNHG7 knockdown (Figure 4E and F). Taken together, our data demonstrated that lncRNA-SNHG7 targeted miR-34a-5p and regulated LDHA expression and glycolysis.

Next, we investigated whether c-Myc regulated lncRNA-SNHG7 expression as it has been previously reported that the promoter of SNHG7 was strongly bound by endogenous c-Myc [15]. Transduction of lentivirus expressing c-myc shRNA significantly decreased endogenous lncRNA-SNHG7 level and c-Myc expression in both MCF-7 cells and MDA-MB-231 cells (Figure 5A). In contrast, transduction of lentivirus expressing c-myc significantly increased lncRNA-SNHG7 level and c-Myc expression in MCF-7 cells and MDA-MB-231 cells (Figure 5B). These results indicated that c-Myc positively regulated lncRNA-SNHG7 expression. We further evaluated whether the promoter of lncRNA-SNHG7 conferred c-Myc dependent transcriptional activity. DNA fragments of lncRNA-SNHG7 promoter containing a predicted wild-type or mutant c-Myc binding region were inserted into the promoter region of a luciferase reporter plasmid pGL3 (Figure 5C). As was expected, significantly increased luciferase activity was only obtained in MCF-7 cells that were transfected with luciferase reporter plasmid containing the wild type lncRNA-SNHG7 promoter but not the mutant promoter. Knocking down c-Myc by lentiviral transduction significantly decreased the luciferase activity (Figure 5D). Overexpression of c-Myc by lentivirus transduction significantly increased the luciferase activity in MCF-7 cell transfected with reporter plasmid containing wild type promoter (Figure 5E). These results indicated that c-Myc could regulate transcription through lncRNA-SNHG7 promoter. The binding of c-Myc to lncRNA-SNHG7 promoter was further verified by ChIP assay. As shown in Figure 5F, anti-c-Myc antibody successfully pulled down the chromatin fragments containing the lncRNA-SNHG7 promoter. Correspondingly, we detected a positive correlation between lncRNA-SNHG7 expression and c-Myc expression. Taken together, our data demonstrated that c-Myc positively regulated lncRNA-SNHG7 expression.