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. 2015 Sep;36(9):6929-38.
doi: 10.1007/s13277-015-3409-z. Epub 2015 Apr 9.

MicroRNA-199a-3p suppresses glioma cell proliferation by regulating the AKT/mTOR signaling pathway

Liang Shen  1 , Chunming Sun  1 , Yanyan Li  1 , Xuetao Li  1 , Ting Sun  1 , Chuanjin Liu  1 , Youxin Zhou  2 , Ziwei Du  1
Affiliations

MicroRNA-199a-3p suppresses glioma cell proliferation by regulating the AKT/mTOR signaling pathway

Liang Shen et al. Tumour Biol. 2015 Sep.

Abstract

Glioma has been investigated for decades, but the prognosis remains poor because of rapid proliferation, its aggressive potential, and its resistance to chemotherapy or radiotherapy. The mammalian target of rapamycin (mTOR) is highly expressed and regulates cellular proliferation and cell growth. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene transcription and translation via up-regulating or down-regulating the levels of miRNAs. This study was conducted to explore the molecular functions of miR-199a-3p in glioma. We detected the expression of miR-199a-3p in glioma samples by quantitative PCR (qPCR). Then, we transfected the U87 and U251 cell lines with miR-199a-3p. Cellular proliferation, invasion, and apoptosis were assessed to explain the function of miR-199a-3p. PCR confirmed that the expression of miR-199a-3p was lower in glioma samples combined with normal brain tissues. The over-expression of miR-199a-3p might target mTOR and restrained cellular growth and proliferation but not invasive and apoptosis capability. Results indicated that cellular proliferation was inhibited to regulate the AKT/mTOR signaling pathway by elevating levels of miR-199a-3p. MiR-199a-3p in glioma cell lines has effects similar to the tumor suppressor gene on cellular proliferation via the AKT/mTOR signaling pathway.

Keywords: Glioma; Proliferation; mTOR; miR-199a-3p.

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Figures

Fig. 1
Fig. 1
The expression of miR-199a-3p, mTOR, p70S6K, and RhoA in glioma samples and normal brain tissue were assessed by qPCR. Data were reported as 2−△△CT. a miR-199a-3p in human glioma samples compared with normal brain tissues (asterisks, P < 0.01), in low grade (grade II) compared with high grade (grade III, grade IV) (ampersand, P > 0.05). b mTOR in glioma samples compared with normal brain tissues (asterisks, P < 0.01), mTOR expression with the increasing degree of glioma (low-grade glioma vs. high-grade glioma; asterisks, P < 0.01). c, d The expression of p70S6K and RhoA in glioma samples compared with normal brain tissues (asterisks, P < 0.01) and in low grade and high grade (ampersand, P > 0.05)
Fig. 2
Fig. 2
a The expression of miR-199a-3p in U87 and U251 compared with six normal brain tissues (asterisks, P < 0.01). b The expression of miR-199a-3p in cells transfected with miR-199a-3p (asterisks, P < 0.01). c Cellular proliferation was detected in U87 and U251 by cell counting kit (CCK-8) after 6, 24, 48, and 72 h of incubation. Data were reported as means ± SD (asterisks, P < 0.01), compared with cells transfected with miR-199a-3p mimic negative control or untransfected cells. d mTOR in U87 and U251 with miR-199a-3p over-expression compared with miR-199a-3p mimic negative control (asterisks, P < 0.01). e Cell cycle modulation was restrained resulting in G1 phase increase (asterisks, P < 0.01) and S phase decrease (asterisk, P < 0.05)
Fig. 3
Fig. 3
Cellular proliferation was evaluated by cell cycle by flow cytometry analysis and detecting the expression of Ki-67 immunohistochemistry analysis. a Cell cycle modulation was restrained compared with the negative control group. b The expression of Ki-67 was reduced in cells transfected with miR-199a-3p
Fig. 4
Fig. 4
The invasion and apoptosis were assessed and compared with miR-199a-3p mimic negative control. a, b Invasion ability was conducted by transwell invasion system. The number of invasive cells was counted (ampersand, P > 0.05). c, d Apoptosis capability was detected by flow cytometry analysis (ampersand, P > 0.05). e The expression of MMP2 and caspase-3 were conducted (ampersand, P > 0.05)
Fig. 5
Fig. 5
The expression of mTOR, p-mTOR, AKT, p-AKT, p70S6K, p-p70S6K, 4E-BP1, p-4E-BP1, RhoA, and GAPDH in different groups were detected by Western blot analysis
Fig. 6
Fig. 6
a Cellular proliferation was assessed after 72 h of incubation. Cells were treated with NVP-BEZ235 of 100 nmol/L for 24 h and then transfected further with miR-199a-3p (ampersand, P > 0.05), when compared with cells treated with NVP-BEZ235 only. b, c Immunofluorescence analysis showed that the expression of mTOR (asterisk, P < 0.05) and p-AKT (asterisks, P < 0.01) was apparently inhibited in cells by the up-regulation of miR-199a-3p
Fig. 7
Fig. 7
Immunofluorescence analysis showed that the expression of mTOR and p-AKT (asterisk, P < 0.05) was apparently inhibited in cells by the up-regulation of miR-199a-3p in U251
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