Yonsei Med J.  2019 Feb;60(2):148-157. 10.3349/ymj.2019.60.2.148.

MiR-182-5p Knockdown Targeting PTEN Inhibits Cell Proliferation and Invasion of Breast Cancer Cells

• Zhao YS
• Yang WC
• Xin HW
• Han JX
• Ma SG

• KMID: 2431631
• DOI: http://doi.org/10.3349/ymj.2019.60.2.148

Abstract

PURPOSE
Breast cancer (BC) is one of the most common malignant tumors, affecting a significant number of women worldwide. MicroRNAs (miRNAs) have been reported to play important roles in tumorigenesis. The aim of this study was to determine the roles of miR-182-5p in BC progression.
MATERIALS AND METHODS
The expressions of miR-182-5p and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were measured in BC tissues and cells by quantitative real-time polymerase chain reaction or Western blot. Cell proliferation and invasion were detected by cell counting kit-8 assay and trans-well assay, respectively. The interaction between miR-182-5p and PTEN was probed by bioinformatics analysis, luciferase activity, and RNA immunoprecipitation. A murine xenograft model was established to investigate the role of miR-182-5p in BC progression in vivo.
RESULTS
An abundance of miR-182-5p was noted in BC tissues and cells. High expression of miR-182-5p was associated with poor survival. Abrogation of miR-182-5p inhibited cell proliferation and invasion in BC cells. Interestingly, PTEN was indicated as a target of miR-182-5p, and its restoration reversed miR-182-5p-mediated promotion of proliferation and invasion of BC cells. Moreover, depletion of miR-182-5p suppressed tumor growth via up-regulating PTEN expression in the murine xenograft model.
CONCLUSION
MiR-182-5p exhaustion blocked cell proliferation and invasion by regulating PTEN expression, providing a novel therapeutic avenue for treatment of BC.

Keyword

Breast cancer; miR-182-5p; PTEN; proliferation; invasion

MeSH Terms

Blotting, Western
Breast Neoplasms*
Breast*
Carcinogenesis
Cell Count
Cell Proliferation*
Chromosomes, Human, Pair 10
Computational Biology
Female
Heterografts
Humans
Immunoprecipitation
Luciferases
MicroRNAs
Real-Time Polymerase Chain Reaction
RNA
Luciferases
MicroRNAs
RNA

Figure

• 

  Fig. 1 MiR-182-5p is highly expressed in breast cancer (BC) tissues and cell lines. (A) The abundance of miR-182-5p was examined in BC tissues compared with that in adjacent non-cancer tissues (NC). (B) The survival rate was investigated in BC patients classified according to low miR-182-5p expression and high miR-182-5p expression, p<0.01. (C) The expression of miR-182-5p was detected in BC cell lines (MCF-7, MDA-MB-231, BT20, T47D, and SKBR3), compared with that in normal breast epithelial cell line (MCF-10A). *p<0.05.

• 

  Fig. 2 Depletion of miR-182-5p inhibits cell proliferation and invasion of breast cancer cells. (A) Transfection efficacy was investigated in MCF-7 and MDA-MB-231 cells after transfection of anti-miR-182-5p or anti-NC. (B and C) The effect of anti-miR-182-5p on cell proliferation was investigated in MCF-7 and MDA-MB-231 cells by CCK-8. (D) The invasive ability was evaluated in MCF-7 and MDA-MB-231 cells transfected with anti-miR-182-5p or anti-NC. *p<0.05.

• 

  Fig. 3 PTEN a target of miR-182-5p. (A) Putative binding sites of miR-182-5p and PTEN was predicted by miRTarBase and miRanda. (B–E) The luciferase activity was investigated in MDA-MB-231 and MCF-7 cells co-transfected with WT or MUT luciferase vectors and miR-182-5p, anti-miR-182-5p or their negative control. (F) The abundance of enrichment of PTEN was detected in MDA-MB-231 and MCF-7 cells after Ago1 RNA immunoprecipitation (RIP). (G and H) The expression of PTEN was measured in breast cancer (BC) tissues and cells at the mRNA and protein level, respectively. (I) The effect of miR-182-5p on PTEN protein expression was examined in MDA-MB-231 and MCF-7 cells. *p<0.05.

• 

  Fig. 4 Addition of PTEN reverses miR-182-5p-mediated promotion on cell proliferation and invasion of breast cancer cells. (A) The expression of miR-182-5p was detected in MCF-7 and MDA-MB-231 cells transfected with miR-182-5p or miR-NC. (B) PTEN level was examined in MCF-7 and MDA-MB-231 cells transfected with PTEN or NC. (C) The expression of PTEN was measured in MCF-7 and MDA-MB-231 cells co-transfected with miR-182-5p and NC or PTEN. (D and E) The effect of miR-182-5p and PTEN on cell proliferation was investigated in MDA-MB-231 and MCF-7 cells. (F) The effect of miR-182-5p and PTEN on invasive ability was detected in MDA-MB-231 and MCF-7 cells by trans-well analysis. *p<0.05.

• 

  Fig. 5 MiR-182-5p deficiency suppresses MCF-7 xenograft tumor growth in vivo. (A) Tumor volume was examined every 10 days for five times after cell implantation. (B) Tumor weight was detected at the end point. (C) The expression of miR-182-5p was detected in tumor tissues. (D and E) The abundance of PTEN was evaluated in tumors by qRT-PCR and Western blot, respectively. *p<0.05.

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