MicroRNA-29a-3p Regulates SH-SY5Y Cell Proliferation and Neurite Growth through Interaction with PTEN-PI3K/AKT/mTOR Signaling Pathway

Abstract

The effects of microRNA-29a-3p in the proliferation process of nerve cells are unclear. The purpose of this study is to delve into the regulatory role of microRNA-29a-3p, via interaction with phosphatase and tension homolog (PTEN), in the SH-SY5Y cell proliferation process. Different expressions of microRNA-29a-3p in the SH-SY5Y cells were constructed by transfected miRNA-29a-3p mimic and inhibitor. The effects of cell transfection and the mRNA expressions of PTEN, Akt, and mTOR were detected by qPCR. The expressions of PTEN, Akt, and mTOR protein and the phosphorylation levels of Akt and mTOR were examined using Western blotting. Nerve cell proliferation activity and neurite length of each group were measured and examined by the use of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT), and morphological examination. We observed that the levels of PTEN mRNA and protein were distinctly decreased in the microRNA-29a-3p mimic group, but the expressions of the phosphorylated Akt and mTOR mRNA and protein were distinctly upregulated. In the transfected miRNA-29a-3p inhibitor SH-SY5Y cells, the expressions of miRNA-29a-3p were significantly suppressed; however, the expressions of PTEN gene and protein were significantly enhanced. The expressions of phosphorylated Akt and mTOR in the downregulated microRNA-29a-3p group distinctly were suppressed. The SH-SY5Y cell proliferation activity and neurite length in the upregulated microRNA-29a-3p group increased significantly. Our findings revealed that microRNA-29a-3p could enhance the proliferation activity of SH-SY5Y cells and promote neurite growth by inhibiting the expression of PTEN and regulating PI3K/Akt/mTOR signaling pathway.

Figure 1

RT-PCR for the mRNA levels for miRNA-29a-3p, PTEN, Akt, and mTOR. (a) The expression of miRNA-29a-3p increased in the mimic miRNA-29a-3p group and decreased in the inhibitor miRNA-29a-3p group (∗∗∗: p < 0.001). (b) The expression of PTEN was the lowest in the mimic miRNA-29a-3p group and in the inhibitor miRNA-29a-3p group; the expressions of PTEN gene were the highest (∗∗∗: p < 0.001). (c and d) There was no distinct difference in the mRNA expression of Akt and mTOR between the groups (p > 0.05).

Figure 2

Relative expressions of PTEN. The expression of PTEN protein was decreased in the mimic miRNA-29a-3p group, while in the inhibitor miRNA-29a-3p group, the expression of PTEN protein was increased (∗∗∗: p < 0.001). For each different group, the SH-SY5Y cells were separately repeatedly measured 6 times.

Figure 3

Relative expression levels of Akt, p-Akt, and p-Akt/Akt. (a) Western blot assays for the expressions of Akt and p-Akt. (b) The expressions of Akt in different groups. (c and d) The phosphorylation level of Akt in the mimic miRNA-29a-3p group was distinctly increased, while in the inhibitor miRNA-29a-3p group, the phosphorylation level of Akt was significantly decreased, and the trend of the p-Akt/Akt ratio was the same (∗∗∗: p < 0.001).

Figure 4

Relative levels of mTOR, p-mTOR, and p-mTOR/mTOR. (a) Western blot assays for the expressions of mTOR and p-mTOR. (b) There was no significant difference in the expression level of mTOR protein between the groups (p > 0.05). (c and d) The phosphorylation level of mTOR in the mimic miRNA-29a-3p group was the highest, while in the inhibitor miRNA-29a-3p group, the phosphorylation level of mTOR was the lowest. The trend of p-mTOR/mTOR ratio is the same as that of p-mTOR (∗∗∗: p < 0.001).

Figure 5

Quantitative analysis of neurite length and the proliferation activity in different groups. (a) Morphological observation for micrographs of SH-SY5Y cells and neurites. (b and c) The histogram revealed the attenuance 570 values and the neurite length of SH-SY5Y cells in different groups. After transfection with mimic miRNA-29a-3p, the cell proliferation activity was significantly increased, and the neurites became significantly longer. While in the inhibitor miRNA-29a-3p group, the SH-SY5Y cell proliferation activity and neurite length were minimum (∗∗∗: p < 0.001; ∗∗: p < 0.01; ∗: p < 0.05). For each different group, the SH-SY5Y cells was separately repeatedly measured 6 times.