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. 2018 Nov 27;37(1):289.
doi: 10.1186/s13046-018-0945-6.

Long noncoding RNA LINC00511 contributes to breast cancer tumourigenesis and stemness by inducing the miR-185-3p/E2F1/Nanog axis

Guanming Lu  1 , Yueyong Li  2   3 , Yanfei Ma  1 , Jinlan Lu  4 , Yongcheng Chen  1 , Qiulan Jiang  3 , Qiang Qin  1 , Lifeng Zhao  3 , Qianfang Huang  1 , Zhizhai Luo  1 , Shiqing Huang  5   6   7 , Zhongheng Wei  8
Affiliations

Long noncoding RNA LINC00511 contributes to breast cancer tumourigenesis and stemness by inducing the miR-185-3p/E2F1/Nanog axis

Guanming Lu et al. J Exp Clin Cancer Res. .

Abstract

Background: Emerging evidence have illustrated the vital role of long noncoding RNAs (lncRNAs) long intergenic non-protein coding RNA 00511 (LINC00511) on the human cancer progression and tumorigenesis. However, the role of LINC00511 in breast cancer tumourigenesis is still unknown. This research puts emphasis on the function of LINC00511 on the breast cancer tumourigenesis and stemness, and investigates the in-depth mechanism.

Methods: The lncRNA and RNA expression were measured using RT-PCR. Protein levels were measured using western blotting analysis. CCK-8, colony formation assays and transwell assay were performed to evaluate the cell proliferation ability and invasion. Sphere-formation assay was also performed for the stemness. Bioinformatic analysis, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were carried to confirm the molecular binding.

Results: LINC00511 was measured to be highly expressed in the breast cancer specimens and the high-expression was correlated with the poor prognosis. Functionally, the gain and loss-of-functional experiments revealed that LINC00511 promoted the proliferation, sphere-formation ability, stem factors (Oct4, Nanog, SOX2) expression and tumor growth in breast cancer cells. Mechanically, LINC00511 functioned as competing endogenous RNA (ceRNA) for miR-185-3p to positively recover E2F1 protein. Furthermore, transcription factor E2F1 bind with the promoter region of Nanog gene to promote it transcription.

Conclusion: In conclusion, our data concludes that LINC00511/miR-185-3p/E2F1/Nanog axis facilitates the breast cancer stemness and tumorigenesis, providing a vital insight for them.

Keywords: Breast cancer stem cells; E2F1; LINC00511; Nanog; miR-185-3p.

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Conflict of interest statement

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki principles. It was approved by the Medical Research Ethics Committee of Youjiang Medical College Affiliated Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
LncRNA LINC00511 is ectopically over-expressed in breast cancer tissue compared to normal tissue. a LINC00511 expression in the breast cancer tissue compared with normal adjacent tissue. b LINC00511 expression in the breast cancer tissue with TNM stage. c High and low expression groups according to the mean value. d Survival rate of patients with high or low LINC00511 expression. *** indicates p value less than 0.001. * indicates p value less than 0.05
Fig. 2
Fig. 2
LncRNA LINC00511 promotes cell proliferation and invasion in vitro and vivo. a, b Colony formation assay in MDA-MB-231 and MCF-7 cells transfected with sh-LINC00511 or sh-NC. c, d CCK-8 assay presented the proliferation of MDA-MB-231 and MCF-7 cells transfected with sh-LINC00511 or sh-NC. e, f Number of invaded MDA-MB-2 31 cells and MCF-7 cells. g, h Tumor growth (volume and weight) in vivo. ** indicates p value less than 0.01. * indicates p value less than 0.05
Fig. 3
Fig. 3
LncRNA LINC00511 contributes to the maintenance of breast cancer CSC characteristic. a LINC00511 expression in breast cancer cell lines MDA-MB-468, MDA-MB-231, MDA-MB-453, and MCF-7 and normal cell lines (MCF-10A). b, c Breast cancer cell MDA-MB-231, and MCF-7 LINC00511 expression. d Stem factor (Oct4, Nanog, SOX2) expression in MDA-MB-231 cells transfected with shRNA for LINC00511. e The stem factors (Oct4, Nanog, SOX2) expression in MCF-7 cells transfected with enhanced LINC00511 plasmids. f, g Mammosphere diameter and quantity when transfected with shRNA for LINC00511. h, i Mammosphere diameter and quantity when transfected with enhanced LINC00511 plasmids. ** indicates p value less than 0.01. * indicates p value less than 0.05
Fig. 4
Fig. 4
LINC00511 targeted the miR-185-3p/E2F1 in the breast cancer cells as competing endogenous RNA. a The subcellular position of LINC00511 on the cytoplasm or nucleus. GAPDH and U1 acted as the cytoplasm and nuclear control. b The binding sites of miR-185-3p and LINC00511. c miR-185-3p expression in the breast cancer cells MDA-MB-231, and MCF-7 measured by RT-PCR. d miR-185-3p expression in the transfection of LINC00511 silencing and enhanced LINC00511 plasmid. e miR-185-3p and E2F1 3’-UTR binding sites. f E2F1 mRNA expression in the breast cancer cells MDA-MB-231, and MCF-7 measured by RT-PCR. g E2F1 mRNA expression in the miR-185-3p inhibitor (miR-185-3p inhib) transfection and enhanced LINC00511 plasmid transfection. ** indicates p value less than 0.01. * indicates p value less than 0.05
Fig. 5
Fig. 5
E2F1 enhanced Nanog expression. a The Nanog gene promoter region. b Chromatin immunoprecipitation (ChIP) assay was performed to identify which region functioned as the effective binding site of Nanog promoter region. c The luciferase reporter plasmid having wild type or mutant sequences of Nanog promoter region (− 586 ~ − 576) were established. d The luciferase activity of wild type sequences and mutant of Nanog promoter region. e, f Nanog protein expression when transfected with enhanced E2F1 plasmid or E2F1 silencing
Fig. 6
Fig. 6
The mechanism by which LINC0051/miR-185-3p/E2F1 axis promoted breast cancer stemness and tumorigenesis
See this image and copyright information in PMC

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