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| Effects of MiR-145-3p on Proliferation and Differentiation of Enteric Neural Stem Cells |
| CHEN Qin-ming, YANG Liu-cheng, WANG Jian-jun, et al |
| Pediatric Surgery Department, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, China |
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Abstract 【Objective】 To establish the method for culturing and purifying enteric neural stem cells (ENSCs) derived from rat and to investigate the effect of miR-145-3p on proliferation and differentiation of ENSCs. 【Methods】 ENSCs which primarily extracted from 18-days SD rats embryos, were observed by cytological characteristics and identified by Nestin and GFAP immunofluorescence staining. The liposome transfection and lentivirus transfection were used to transfect the ENSCs. Overexpression of miR-145-3p in ENSCs was transfected through liposome and infected with lentivirus with miR-145-3p, which followed by fluorescence microscope and qRT-PCR measurement. Edu cell proliferation assay was used to analyze proliferating cells by recording the proportion of Edu/DAPI in each group. The neurospheres was differentiated by differentiation medium and the GFAP/DAPI ratio of differentiated cells was recorded. 【Results】 Primary culture of ENSCs showed the formation of a large number of neurosphere-like structures (undifferentiated neurospheres). The immunofluorescence showed that Nestin and GFAP were positive in these cells. The qRT-PCR showed that the relative expression level of miR-145-3p in overexpression group was 3.52±0.24 significantly higher than the negative control group 0.01±0.01, the difference was statistically significant (P<0.01). It indicated that lentivirus was successfully transfected into ENSCs. Edu cell infiltration showed that the positive rate of Edu in the miR-145-3p overexpression group was (15.23±1.17) %, while the negative control group was (22.38±1.52) % (P<0.05). After NESCs cells were exposed to the differentiation medium, the proportion of GFAP positive cells in the miR-145-3p overexpression group was (21.47±1.98)%, which was lower than that in the negative cell control group(33.86±2.52)% (P<0.05). 【Conclusions】 Lentivirus transfection is a high efficient transfection method in ENSCs. Overexpression of miR-145-3p inhibits the proliferation and differentiation of ENSCs, which may play an important role in the development of enteric nervous system.
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Received: 16 March 2021
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2022, Vol. 39 
