下调miR-301a-3p抑制人卵巢颗粒KGN细胞增殖和诱导凋亡的机制研究

doi:10.3877/cma.j.issn.2095-1221.2023.03.002

目的

探讨miR-301a-3p对人卵巢颗粒KGN细胞增殖和凋亡的机制研究。

方法

采用RT-qPCR检测38例多囊卵巢综合征（PCOS）卵巢皮质组织和外周血、人卵巢颗粒KGN细胞内miR-301a-3p表达水平；将KGN细胞分为空白对照、anti-miR-NC、anti-miR-301a-3p，RT-qPCR检测miR-301a-3p表达水平；MTT、流式细胞术检测细胞增殖和凋亡；平板实验检测克隆形成能力；Western blot检测PCNA、Bax、Bcl-2及MAPK信号通路相关蛋白表达。加入MAPK信号通路抑制剂SB203580，并采用以上方法检测其增殖和凋亡的影响。两组间比较采用独立样本t检验，方差不齐性采用t'检验；多组间比较采用单因素方差分析，两两比较组间方差齐时采用LSD-t检验，方差不齐时采用Tamhane's T2检验。

结果

与对照组比较，PCOS组的组织内和外周血内miR-301a-3p表达（2.65 ± 0.44比1.08 ± 0.22，2.54 ± 0.47比1.05 ± 0.22）升高（P均< 0.05）；与人正常卵巢上皮IOSE80a细胞比较，人卵巢颗粒KGN细胞内miR-301a-3p表达（2.38 ± 0.38比0.99 ± 0.10）升高，差异有统计学意义；下调miR-301a-3p可降低细胞活性、克隆细胞数，增加细胞凋亡率、Bax蛋白表达，降低PCNA、Bcl-2、p-p38 MAPK、p-ERK1/2蛋白表达水平，差异有统计学意义（P均< 0.05）。与anti-miR-301a-3p比较，anti-miR-301a-3p+ SB203580克隆细胞数、细胞活性降低，PCNA、Bcl-2蛋白表达水平降低，细胞凋亡率、Bax蛋白表达水平升高，差异有统计学意义（P均< 0.05）。

结论

在人卵巢颗粒细胞内miR-301a-3p高表达，下调miR-301a-3p可抑制人卵巢颗粒KGN细胞增殖和诱导凋亡，其作用机制可能与抑制MAPK信号通路有关。

关键词: miR-301a-3p, 人卵巢颗粒细胞, 增殖, 凋亡

Objective

To investigate the mechanism of miR-301a-3p in proliferation and apoptosis of human ovarian granulocyte KGN cells.

Methods

The expression level of miR-301a-3p was detected by RT-qPCR in tissues and peripheral blood of 38 patients with polycystic ovary syndrome (PCOS) and human ovarian granule KGN cells. KGN cells were divided into control group, anti-miR-NC group and anti-miR-301a-3p group; the expression of miR-301a-3p was detected by RT-qPCR; cell proliferation and apoptosis were detected by MTT and flow cytometry; clone formation ability were detected by plate test; the expressions proliferating cell nuclear antigen (PCNA) , B-cell lymphoma 2 (Bcl2) , BCL2-associated X protein (Bax) , phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) were detected by Western blot. SB203580, an inhibitor of MAPK signaling pathway, was used to investigate the effects on the cell proliferation and apoptosis by the above methods.

Results

Compared with the control group, the expression of miR-301a-3p was increased in PCOS tissues (1.08 ± 0.22 vs 2.65 ± 0.44) and peripheral blood (1.05 ± 0.22 vs 2.54 ± 0.47) , and the difference was statistically significant (both P < 0.05) . Compared with IOSE80a cells of human normal ovarian epithelium, the expression of miR-301a-3p in human ovarian granule KGN cells was increased (0.99 ± 0.10 vs 2.38 ± 0.38) , and the difference was statistically significant (P < 0.05) ; down-regulating miR-301a-3p decreased the cell activity, the number of cloned cells, increased the apoptosis rate and the expression of Bax protein, and decreased the expression levels of PCNA, Bcl-2, p-p38 MAPK and p-ERK1/2 proteins and the difference was statistically significant (all P < 0.05) . Compared with the anti-miR-301a-3p group, the cell number and cell activity of the anti-miR-301a-3p+SB203580 clone were significantly decreased, and the protein expression levels of PCNA and Bcl-2 were significantly decreased, the apoptosis rate and the expression level of Bax protein were significantly increased, and the difference was statistically significant (all P < 0.05) .

Conclusions

miR-301a-3p is highly expressed in human ovarian granulosa cells, and down-regulation of miR-301a-3p can inhibit the proliferation and induce apoptosis of human ovarian granulosa KGN cells, and its mechanism may be related to inhibition of MAPK signaling pathway.

Key words: miR-301a-3p, Human ovarian granulosa cells, Proliferation, Apoptosis

图1 miR-301a-3p在PCOS组织和外周血内表达（n = 38）注：^aP < 0.05

图2 miR-301a-3p在卵巢颗粒KGN细胞内表达（n = 3）注：^aP < 0.05

图3 下调miR-301a-3p对卵巢颗粒KGN细胞增殖的影响注：a ~ c图为倒置显微镜下观察细胞克隆形成数（×100）；d图为PCNA蛋白表达

表1 下调miR-301a-3p对卵巢颗粒KGN细胞增殖的影响（ ± s）

分组	实验次数	miR-301a-3p	克隆细胞数（个）	细胞活性（%）	PCNA
空白对照	3	1.00 ± 0.09	123.33 ± 15.89	100.03 ± 0.08	0.52 ± 0.06
anti-miR-NC	3	1.02 ± 0.10	127.67 ± 17.21	99.78 ± 0.15	0.54 ± 0.07
anti-miR-301a-3p	3	0.35 ± 0.04^ab	55.67 ± 4.04^ab	47.5 ± 6.89^ab	0.19 ± 0.04^ab
F值		66.381	25.967	173.449	34.426
P值		< 0.001	0.001	< 0.001	0.001

表1 下调miR-301a-3p对卵巢颗粒KGN细胞增殖的影响（ ± s）

分组	实验次数	miR-301a-3p	克隆细胞数（个）	细胞活性（%）	PCNA
空白对照	3	1.00 ± 0.09	123.33 ± 15.89	100.03 ± 0.08	0.52 ± 0.06
anti-miR-NC	3	1.02 ± 0.10	127.67 ± 17.21	99.78 ± 0.15	0.54 ± 0.07
anti-miR-301a-3p	3	0.35 ± 0.04^ab	55.67 ± 4.04^ab	47.5 ± 6.89^ab	0.19 ± 0.04^ab
F值		66.381	25.967	173.449	34.426
P值		< 0.001	0.001	< 0.001	0.001

图4 下调miR-301a-3p对卵巢颗粒KGN细胞凋亡的影响注：a ~ c图分别为流式细胞术实验检测对照、anti-miR-NC和anti-miR-301a-3p细胞的凋亡；d图为Bax、Bcl-2蛋白表达

表2 下调miR-301a-3p对卵巢颗粒KGN细胞凋亡的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	Bax/GAPDH	Bcl-2/GAPDH
空白对照	3	4.95 ± 0.56	0.32 ± 0.05	0.66 ± 0.04
anti-miR-NC	3	5.08 ± 0.82	0.31 ± 0.04	0.67 ± 0.08
anti-miR-301a-3p	3	18.94 ± 2.11^ab	0.78 ± 0.06^ab	0.27 ± 0.05^ab
F值		106.978	84.273	44.600
P值		< 0.001	< 0.001	< 0.001

表2 下调miR-301a-3p对卵巢颗粒KGN细胞凋亡的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	Bax/GAPDH	Bcl-2/GAPDH
空白对照	3	4.95 ± 0.56	0.32 ± 0.05	0.66 ± 0.04
anti-miR-NC	3	5.08 ± 0.82	0.31 ± 0.04	0.67 ± 0.08
anti-miR-301a-3p	3	18.94 ± 2.11^ab	0.78 ± 0.06^ab	0.27 ± 0.05^ab
F值		106.978	84.273	44.600
P值		< 0.001	< 0.001	< 0.001

图5 Western blot检测MAPK信号通路相关蛋白表达

表3 下调miR-301a-3p对MAPK信号通路相关蛋白表达的影响（ ± s）

分组	实验次数	p-p38 MAPK/p38MAPK	p38 MAPK/GAPDH	p-ERK/GAPDH	ERK/GAPDH
空白对照	3	1.12 ± 0.11	0.98 ± 0.10	0.83 ± 0.08	0.75 ± 0.06
anti-miR-NC	3	1.09 ± 0.10	1.02 ± 0.09	0.80 ± 0.10	0.72 ± 0.09
anti-miR-301a-3p	3	0.75 ± 0.07^ab	0.97 ± 0.08	0.38 ± 0.04^ab	0.73 ± 0.08
F值		14.078	0.257	31.650	0.116
P值		< 0.001	0.781	0.001	0.892

表3 下调miR-301a-3p对MAPK信号通路相关蛋白表达的影响（ ± s）

分组	实验次数	p-p38 MAPK/p38MAPK	p38 MAPK/GAPDH	p-ERK/GAPDH	ERK/GAPDH
空白对照	3	1.12 ± 0.11	0.98 ± 0.10	0.83 ± 0.08	0.75 ± 0.06
anti-miR-NC	3	1.09 ± 0.10	1.02 ± 0.09	0.80 ± 0.10	0.72 ± 0.09
anti-miR-301a-3p	3	0.75 ± 0.07^ab	0.97 ± 0.08	0.38 ± 0.04^ab	0.73 ± 0.08
F值		14.078	0.257	31.650	0.116
P值		< 0.001	0.781	0.001	0.892

图6 下调miR-301a-3p可能通过MAPK信号通路对卵巢颗粒KGN细胞增殖和凋亡的影响注：a ~ c图为倒置显微镜下观察细胞克隆形成数（×100）；d ~ f图为凋亡图

图7 Western blot检测PCNA、Bax、Bcl-2蛋白表达

表4 下调miR-301a-3p可能通过MAPK信号通路对卵巢颗粒KGN细胞增殖和凋亡的影响（ ± s，n = 3）

分组	克隆细胞数（个）	细胞活性（%）	PCNA/GAPDH	细胞凋亡率（%）	Bax/GAPDH	Bcl-2/GAPDH
空白对照	134.87 ± 10.24	99.69 ± 0.74	0.54 ± 0.05	5.42 ± 0.68	0.34 ± 0.05	0.65 ± 0.06
anti-miR-301a-3p	53.15 ± 6.88^a	46.57 ± 5.78^a	0.21 ± 0.02^a	18.71 ± 2.46^a	0.77 ± 0.07^a	0.29 ± 0.04^a
anti-miR-301a-3p+SB203580	34.85 ± 2.46^ab	29.42 ± 4.52^ab	0.11 ± 0.04^ab	26.85 ± 3.69^ab	1.24 ± 0.16^ab	0.12 ± 0.04^ab
F值	161.306	222.125	101.267	52.319	55.264	96.926
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表4 下调miR-301a-3p可能通过MAPK信号通路对卵巢颗粒KGN细胞增殖和凋亡的影响（ ± s，n = 3）

分组	克隆细胞数（个）	细胞活性（%）	PCNA/GAPDH	细胞凋亡率（%）	Bax/GAPDH	Bcl-2/GAPDH
空白对照	134.87 ± 10.24	99.69 ± 0.74	0.54 ± 0.05	5.42 ± 0.68	0.34 ± 0.05	0.65 ± 0.06
anti-miR-301a-3p	53.15 ± 6.88^a	46.57 ± 5.78^a	0.21 ± 0.02^a	18.71 ± 2.46^a	0.77 ± 0.07^a	0.29 ± 0.04^a
anti-miR-301a-3p+SB203580	34.85 ± 2.46^ab	29.42 ± 4.52^ab	0.11 ± 0.04^ab	26.85 ± 3.69^ab	1.24 ± 0.16^ab	0.12 ± 0.04^ab
F值	161.306	222.125	101.267	52.319	55.264	96.926
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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Mechanism of miR-301a-3p in proliferation and apoptosis of human ovarian granulosa KGN cells

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Mechanism of miR-301a-3p in proliferation and apoptosis of human ovarian granulosa KGN cells