miR-34a-5p靶向GMFB抑制细胞增殖对先天性巨结肠的治疗意义

doi:10.3877/cma.j.issn.2095-1221.2018.04.001

目的

探讨miR-34a-5p通过靶基因GMFB调控神经嵴细胞的增殖。

方法

通过荧光定量PCR检测miR-34a-5p在先天性巨结肠症（HSCR）和正常结肠组织中的表达，并通过双荧光素酶报告基因检测miR-34a-5p的靶基因，SH-SY5Y细胞转染miR-34a-5p mimics及对照miR-NC，并转染GMFB表达载体，通过CCK8检测miR-34a-5p和GMFB对神经嵴细胞增殖的影响，并通过Western Blot检测miR-34a-5p对GMFB蛋白表达的影响。采用t检验和单因素方差分析。

结果

荧光定量PCR结果显示，HSCR结肠组中miR-34a-5p的相对表达量为0.43±0.10，低于正常结肠组1.15±0.18，差异具有统计学意义（t = 3.50，P < 0.01）。CCK8结果显示，miR-34a-5p mimics组细胞在培养24?h和48?h后细胞A450值分别为0.53±0.03和0.87±0.04，低于miR-NC对照组0.87±0.03，1.42±0.04。双荧光素酶报告基因实验结果显示，miR-34a-5p mimics与GMFB 3'UTR WT载体共转染组荧光强度为0.44±0.03，低于对照miR-NC与WT载体共转染组1.02±0.06。CCK8结果所示，miR-34a-5p mimics+GMFB组细胞培养24?h和48?h后，A450值分别为0.99±0.02和1.50±0.03，高于miR-34a-5p mimics组0.53±0.03, 0.87±0.04，差异具有统计学意义（t?=?7.07，P < 0.01；t?=?9.14，P < 0.01）。Western Blot检测结果显示，miR-34a-5p mimics组细胞的GMFB蛋白表达量为0.25±0.01，低于miR-NC对照组0.90±0.03，差异具有统计学意义（t?=?35.60，P < 0.01），miR-34a-5p mimics+GMFB组细胞GMFB蛋白表达量为1.03±0.03，高于miR-34a-5p mimics组0.25±0.01，差异具有统计学意义（t?=?42.74，P < 0.01）。

结论

miR-34a-5p能够通过抑制靶基因GMFB的表达，抑制神经嵴细胞SH-SY5Y的增殖。

关键词: 先天性巨结肠, miR-34a-5p, 细胞增殖, 胶质细胞成熟因子

Objective

To investigated the effect of miR-34a-5p on proliferation of SH-SY5Y cells by targeting GMFB in Hirschsprung disease (HSCR).

Methods

The expression of miR-34a-5p in HSCR and normal colon tissues was detected by Q-PCR. The downstream target gene of miR-34a-5p was detected by dual luciferase reporter gene assay. SH-SY5Y cells were transferred with miR-NC, miR-34a-5p mimics and miR-34a-5p mimics+GMFB. The cell viability was detected by CCK8 assay. The expression of GMFB protein was detected by western blot. CCK8 and Western blot results were compared using t test and one-way ANOVA.

Results

The results of Q-PCR showed that the relative expression of miR-34a-5p in the HSCR colon was 0.43±0.10, which was significantly lower than that in the normal colon (1.15±0.18, t = 3.50, P < 0.01). CCK8 results showed that the cell A450 values of miR-34a-5p mimics group was 0.53±0.03 and 0.87±0.04 respectively at 24 h and 48 h, significantly lower than those of the miR-NC control group (0.87±0.03, 1.42±0.04). The dual luciferase reporter assay results showed that the fluorescence intensity of miR-34a-5p mimics co-transfected with GMFB 3'UTR WT vector was 0.44±0.03, which was significantly lower than that of the control miR-NC and WT vector co-transfection group 1.02±0.06. CCK8 results showed that after transfection of miR-34a-5p mimics + GMFB, cells at 24 h and 48 h had significantly higher A450 values 0.99±0.02 and 1.50±0.03, respectively, compared to 0.53±0.03 and 0.87±0.04 in the miR-34a-5p mimics group (t = 7.07, P < 0.01; t = 9.14, P < 0.01). Western blot results showed that the expression of GMFB protein in the miR-34a-5p mimics group was 0.25±0.01, which was significantly lower than that in the miR-NC control group (0.90±0.03, t = 35.60, P < 0.01). The expression of GMFB protein in the miR-34a-5p mimics+GMFB group was significantly higher than that in the miR-34a-5p mimics group (1.03±0.03 versus 0.25±0.01, t = 42.74, P < 0.01).

Conclusion

miR-34a-5p inhibited the proliferation of melanoma SH-SY5Y cells by regulating the expression of target gene GMFB.

Key words: Hirschsprung disease, miR-34a-5p, Proliferation, Glia Maturation Factor

图1 miR-34a-5p在HSCR和正常结肠组织中的表达量

表1 miR-34a-5p mimics组和miR-NC组细胞在不同时间细胞活力（A450）的比较结果（ ± s）

分组	样本数	12 h	24 h	48 h
miR-NC组	3	0.47±0.03	0.87±0.03	1.42±0.04
miR-34a-5p mimics组	3	0.46±0.03	0.53±0.03^a	0.87±0.04^a
t值	?	0.45	7.07	9.14
P值	?	0.68	< 0.01	< 0.01

表1 miR-34a-5p mimics组和miR-NC组细胞在不同时间细胞活力（A450）的比较结果（ ± s）

分组	样本数	12 h	24 h	48 h
miR-NC组	3	0.47±0.03	0.87±0.03	1.42±0.04
miR-34a-5p mimics组	3	0.46±0.03	0.53±0.03^a	0.87±0.04^a
t值	?	0.45	7.07	9.14
P值	?	0.68	< 0.01	< 0.01

表2 qRT-PCR引物序列

名称	序列（5’→3’）
miR-34a-5p	F: ACACTCCAGCTGGGTGGCAGTGTCTTAG
?	R: TGGTGTCGTGGAGTCG
U6	F: CGCTTCGGCAGCCACATATACTA
?	R: CGCTTCACGAATTTGCGTGTCA

表2 qRT-PCR引物序列

名称	序列（5’→3’）
miR-34a-5p	F: ACACTCCAGCTGGGTGGCAGTGTCTTAG
?	R: TGGTGTCGTGGAGTCG
U6	F: CGCTTCGGCAGCCACATATACTA
?	R: CGCTTCACGAATTTGCGTGTCA

表3 miR-34a-5p mimics组和miR-NC组细胞的荧光素酶活性（FIR/REN）的比较结果（ ± s）

分组	样本数	GMFB 3'UTR WT	GMFB 3'UTR MUT
miR-NC组	3	1.02±0.06	1.04±0.03
miR-34a-5p mimics组	3	0.44±0.03^a	1.08±0.02
t值	?	8.56	1.10
P值	?	< 0.01	0.33

表3 miR-34a-5p mimics组和miR-NC组细胞的荧光素酶活性（FIR/REN）的比较结果（ ± s）

分组	样本数	GMFB 3'UTR WT	GMFB 3'UTR MUT
miR-NC组	3	1.02±0.06	1.04±0.03
miR-34a-5p mimics组	3	0.44±0.03^a	1.08±0.02
t值	?	8.56	1.10
P值	?	< 0.01	0.33

图2 荧光素酶报告基因检测miR-34a-5p的靶基因

表4 两组细胞在不同时细胞活力(A450)的比较结果（ ± s）

分组	样本数	12h	24h	48h
miR-34a-5p mimics组	3	0.46±0.03	0.53±0.03	0.87±0.04
miR-34a-5p mimics+GMFB组	3	0.47±0.02	0.99±0.02^a	1.50±0.03^a
t值	?	0.28	11.03	12.68
P值	?	0.79	<0.01	<0.01

表4 两组细胞在不同时细胞活力(A450)的比较结果（ ± s）

分组	样本数	12h	24h	48h
miR-34a-5p mimics组	3	0.46±0.03	0.53±0.03	0.87±0.04
miR-34a-5p mimics+GMFB组	3	0.47±0.02	0.99±0.02^a	1.50±0.03^a
t值	?	0.28	11.03	12.68
P值	?	0.79	<0.01	<0.01

图3 miR-34a-5p对人神经嵴细胞GMFB蛋白表达的影响

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Effect of miR-34a-5p on cell proliferation by targeting GMFB in Hirschsprung disease

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Effect of miR-34a-5p on cell proliferation by targeting GMFB in Hirschsprung disease