J Breast Cancer.  2019 Dec;22(4):533-547. 10.4048/jbc.2019.22.e54.

c-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer

• Zhang L
• Fu Y
• Guo H

• KMID: 2470891
• DOI: http://doi.org/10.4048/jbc.2019.22.e54

Abstract

PURPOSE
Recent studies have shown that long non-coding RNA (lncRNA) play an important role in cancer metabolism and development. The lncRNA small nucleolar RNA host gene 7 (SNHG7) was reported to be upregulated in colorectal cancer and contribute to its progression. In the current study, we investigated the role of lncRNA-SNHG7 in breast cancer and explored the underlying mechanism.
METHODS
We monitored the expression of lncRNA-SNHG7 in breast cancer tissues and breast cancer cell lines. We evaluated the effects of lncRNA-SNHG7 on cell proliferation and glycolysis in breast cancer cells by knocking down or overexpressing lncRNA-SNHG7. We searched for the potential microRNA (miRNA) target of lncRNA-SNHG7 and evaluated the effects of the target miRNA on glycolysis. We evaluated the potential regulation of lncRNA-SNHG7 by c-Myc.
RESULTS
LncRNA-SNHG7 was up-regulated in both breast cancer tissues and breast cancer cell lines. Knocking down lncRNA-SNHG7 inhibited breast cancer cell proliferation while overexpressing lncRNA-SNHG7 enhanced cell proliferation. Knocking down lncRNA-SNHG7 resulted in decreased expression of lactate dehydrogenase A (LDHA) and decreased glycolysis. LncRNA-SNHG7 targeted miR-34a-5p to regulate LDHA expression and glycolysis. c-Myc bound to promoter of lncRNA-SNHG7 and positively regulated lncRNA-SNHG7 expression.
CONCLUSION
We demonstrated that c-Myc regulated glycolysis through the lncRNA-SNHG7/miR-34a-5p/LDHA axis in breast cancer cells.

Keyword

Breast neoplasms; Glycolysis; MIRN34 microRNA human; Proto-Oncogene Proteins c-myc; RNA, long noncoding

MeSH Terms

Breast Neoplasms*
Breast*
Cell Line
Cell Proliferation
Colorectal Neoplasms
Glycolysis*
L-Lactate Dehydrogenase
Metabolism
MicroRNAs
Proto-Oncogene Proteins c-myc
RNA, Long Noncoding*
RNA, Small Nucleolar*
L-Lactate Dehydrogenase
MicroRNAs
Proto-Oncogene Proteins c-myc
RNA, Long Noncoding
RNA, Small Nucleolar

Figure

• 

  Figure 1 Elevated expression of lncRNA-SNHG7 in breast cancer. (A) qRT-PCR for the abundance of lncRNA-SNHG7 in 30 pairs of breast cancer samples. (B) The correlation between overall survival and lncRNA-SNHG7 level in breast cancer obtained by TANRIC. (C) qRT-PCR for the abundance of lncRNA-SNHG7 in normal breast cells and various breast cancer cell lines. (D) Western-blot analysis for PARP and β-actin in the nuclear and cytoplasmic fractions, respectively, in MCF-7 cells. Data shown represent 3 independent experiments. (E) Nuclear and cytoplasmic fractions of MCF-7 cells were subjected to qRT-PCR analysis. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; PARP = poly (ADP-ribose) polymerase. *p < 0.05, †p < 0.01, ‡p < 0.001.

• 

  Figure 2 LncRNA-SNHG7 promotes proliferation of breast cancer cells. (A, B) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA-SNHG7 lentivirus. LncRNA-SNHG7 level was assessed by qRT-PCR, and then cells were subjected to cell proliferation analysis. (C) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA-SNHG7 lentivirus and then subjected to cell viability assays. (D, E) MCF-7 cells were transduced with pCDH or pCDH-lncRNA-SNHG7 lentivirus. LncRNA-SNHG7 level was assessed by qRT-PCR, and then the cells were subjected to cell proliferation analysis. (F) MCF-7 cells were transduced with pCDH or pCDH-lncRNA-SNHG7 lentivirus, and then subjected to cell viability assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; qRT-PCR = quantitative real-time polymerase chain reaction; sh = short hairpin. *p < 0.05, †p < 0.01.

• 

  Figure 3 Knocking down lncRNA-SNHG7 inhibits glycolysis in breast cancer cells. (A) MCF-7 cells expressing either control shRNA or lncRNA-SNHG7 shRNA were cultured for 24 hours. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. (B) MCF-7 cells expressing either control shRNA or lncRNA-SNHG7 shRNA were cultured for 24 hours. Levels of lactate in the culture medium were then measured and normalized to cell number. (C, D) ECAR was measured in MCF-7 cells expressing either control shRNA or lncRNA-SNHG7 shRNA by Seahorse XF assays. (E, F) Western blotting and qRT-PCR analysis of glycolysis enzymes in MCF-7 cells expressing either control shRNA or lncRNA-SNHG7 shRNA. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction. *p < 0.01.

• 

  Figure 4 LncRNA-SNHG7 acts as a target of miR-34a-5p to increase LDHA level. (A) Illustration of the base pairing between miR-34a-5p and lncRNA-SNHG7. The base pairing between miR-34a-5p and LDHA 3′UTR is also shown. (B) Lysates from MCF-7 cells were incubated with in vitro-synthesized biotin-labeled sense or antisense DNA probes against lncRNA-SNHG7 for biotin pull-down assay, followed by qRT-PCR analysis to examine miR-34a-5p and lncRNA-SNHG7 level. (C) MCF-7 were transduced with sh-ctrl or sh-lncRNA-SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. Western blot analysis of LDHA and β-actin. (D) MCF-7 were transduced with pCDH or pCDH-lncRNA-SNHG7 lentivirus followed by miR-34a-5p mimics or negative control transfection. Western blot analysis of LDHA and β-actin. (E, F) MCF-7 cells were transduced with sh-ctrl or sh-lncRNA-SNHG7 lentivirus followed by miR-34a-5p inhibitor or negative control transfection. ECAR was measured by Seahorse XF assays. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; ECAR = extracellular acidification rate; qRT-PCR = quantitative real-time polymerase chain reaction; ns = not significant; LDHA = lactate dehydrogenase A; 3′UTR = 3′ untranslated region. *p < 0.01.

• 

  Figure 5 LncRNA-SNHG7 is a direct transcriptional target of c-Myc. (A, B) MCF-7 and MDA-MB-231 cells were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus. Western blot analysis of c-Myc and β-actin. LncRNA-SNHG7 level was assessed by quantitative RT-PCR. (C) Schematic illustration of the consensus c-Myc binding sites in lncRNA-SNHG7 gene promoter. The wild-type and mutant binding sites are shown in the open boxes. The corresponding pGL3-based luciferase reporter constructs generated are shown. (D, E) MCF-7 were transduced with sh-ctrl or sh-c-Myc lentivirus, pCDH or pCDH-c-Myc lentivirus followed by transfection with the indicated pGL3-based luciferase reporter constructs. The reporter activity was measured 24 hours post-transfection, and plotted after normalizing with the Renilla luciferase activity. Western blot analysis of c-Myc and β-actin. (F) MCF-7 cells lysates were analyzed by ChIP assay using anti-c-Myc or IgG rabbit antibody. The ChIP products were amplified by semi-quantitative RT-PCR. (G) Correlation analyses conducted between c-Myc and lncRNA-SNHG7 in breast cancers (n = 50 biologically independent samples). Pearson correlation coefficients (r) and p-values. Data were presented as mean ± standard deviation. lncRNA = long non-coding RNA; sh = short hairpin; RT-PCR = real-time polymerase chain reaction; NS = not significant; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IgG = immunoglobulin G. *p < 0.05, † p < 0.01.

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