Korean J Physiol Pharmacol.  2016 Mar;20(2):139-145. 10.4196/kjpp.2016.20.2.139.

Myosin VI contributes to malignant proliferation of human glioma cells

• Xu R
• Fang XH
• Zhong P

• KMID: 2388669
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.139

Abstract

Previously characterized as a backward motor, myosin VI (MYO6), which belongs to myosin family, moves toward the minus end of the actin track, a direction opposite to all other known myosin members. Recent researches have illuminated the role of MYO6 in human cancers, particularly in prostate cancer. However, the role of MYO6 in glioma has not yet been determined. In this study, to explore the role of MYO6 in human glioma, lentivirus-delivered short hairpin RNA (shRNA) targeting MYO6 was designed to stably down-regulate its endogenous expression in glioblastoma cells U251. Knockdown of MYO6 signifi cantly inhibited viability and proliferation of U251 cells in vitro. Moreover, the cell cycle of U251 cells was arrested at G0/G1 phase with the absence of MYO6, which could contribute to the suppression of cell proliferation. In conclusion, we firstly identified the crucial involvement of MYO6 in human glioma. The inhibition of MYO6 by shRNA might be a potential therapeutic method in human glioma.

Keyword

Cell cycle; Glioma; Myosin VI; Proliferation; ShRNA

MeSH Terms

Actins
Cell Cycle
Cell Proliferation
Glioblastoma
Glioma*
Humans*
Myosins*
Prostatic Neoplasms
RNA, Small Interfering
Actins
Myosins
RNA, Small Interfering

Figure

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  Fig. 1 Lentivirus-delivered shRNA targeting MYO6 depleted its endogenous expression in U251 cells.(A) Evaluation of the lentivirus transduction rate, which was more than 80% as calculated by cellular enumeration using fluorescence and light microscopy. (B) Quantitative analysis of MYO6 knockdown efficiency in U251 cells assessed by qRTPCR. β-actin gene was used as an internal control. (C) Representative immunoblot showing MYO6 knockdown efficiency determined in U251 cells. GAPDH protein was used as an internal control. Data are mean±SD (n=3; t-test). *p<0.001; scale bar, 100 µm.

• 

  Fig. 2 Knockdown of MYO6 inhibits viability and proliferation of glioblastoma U251 cells.(A) MTT showing growth curves determined in U251 cells. The number of viable cells was much fewer in the shMYO6 group than in the shCon group. (B) Representative colony formation showing clonogenic survival determined in U251 cells. (C) The number of colonies was much fewer in the shMYO6 group than in the shCon group. Data are mean±SD (n=3; t-test). *p<0.001; scale bar, 250 µm.

• 

  Fig. 3 Knockdown of MYO6 arrests cell cycle progression in U251 cells.(A) Comparison of the cell population in G0/G1, S and G2/M phase between shCon and shMYO6 groups assessed by flow cytometry. (B) The percentage of cells in G0/G1 phase was significantly higher in the shMYO6 group than in the shCon group, while the percentages of cells in S phase was simultaneously reduced. Data are mean±SD (n=3; t-test). ***p<0.001.

• 

  Fig. 4 Effect of MYO6 knockdown on cell apoptosis.(A) Cytogram of Annexin V-APC binding vs. 7-AAD uptake in U251 cells following lentivirus infection in shCon and shMYO6 groups. (B) Quantification of apoptotic cells in U251 cells by FACS. Annexin V+/7-AAD-: early apoptotic cells; Annexin V+/7-AAD+: late apoptotic cells; Annexin V-/7-AAD-: viable cells; Annexin V-/7-AAD+: cells in necrosis.

• 

  Fig. 5 Knockdown of MYO6 regulate the expression of cell cycle markers.The expression alterations of cell cycle markers in glioma cells was confirmed by using western blotting, including CDK4, Cyclin D1 and PARP.

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