补骨脂素抑制膀胱癌T24细胞增殖和迁移的作用及机制研究

doi:10.3877/cma.j.issn.2095-1221.2022.04.001

目的

探讨补骨脂素对人膀胱癌T24细胞存活率、细胞周期、细胞凋亡和迁移的影响及其分子机制。

方法

分别用细胞培养液、3‰二甲基亚砜（DMSO）和不同浓度（10、30、50、100 μg/mL）补骨脂素处理膀胱癌细胞分成对照组、DMSO组和补骨脂素组，CCK-8检测细胞存活率。流式细胞术检测细胞周期和细胞凋亡。划痕实验检测划痕愈合率。RT-qPCR法检测磷脂酰肌醇3激酶（PI3K）和蛋白激酶B （AKT） mRNA表达水平、Western blot法检测PI3K和AKT蛋白的表达及磷酸化情况。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与DMSO组比较，除10 μg/mL补骨脂素作用24 h外，其余浓度补骨脂素作用不同时间的细胞存活率随着补骨脂素浓度增高、作用时间延长而逐渐降低（P < 0.05）。与DMSO组比较，30、100 μg/mL补骨脂素干预24 h后，G1期细胞比例增多，G2/M期比例减少，细胞凋亡率[（9.16±0.97）%、（15.45±1.57）%比（1.02±0.36）%]升高，划痕愈合率[24 h：（45.00±3.44）%、（27.60±2.21）%比（66.10±2.61）%，48 h：（70.00 ± 3.40）%、（45.17±2.44）%比（85.17±3.85）%]降低，PI3K、AKT mRNA表达以及PI3K、AKT蛋白表达水平和磷酸化水平均降低（P均< 0.05）。

结论

补骨脂素降低膀胱癌细胞存活率、阻滞细胞周期、诱导细胞凋亡和抑制细胞迁移，其机制可能与下调PI3K、AKT mRNA、蛋白表达及磷酸化水平有关。

关键词: 补骨脂素, 抗肿瘤, 膀胱癌, 磷脂酰肌醇3-激酶, 丝氨酸/苏氨酸激酶

Objective

To investigate the effects of psoralen on the viability, cell cycle, apoptosis, and migration of human bladder cancer T24 cells and its mechanisms.

Methods

Bladder cancer cells were treated with cell culture medium, 3‰ dimethyl sulfoxide (DMSO) and different concentrations of psoralen and divided into normal control group, DMSO group and psoralen groups (10、30、50、100 μg/mL) . CCK-8 was used to detect cell viabilities. FACS was performed to detect cell cycles and apoptosis. Scratch tests were used to detecte the scratch healing rates. The expression levels of phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) mRNA were detected by RT-qPCR, and the expression and phosphorylation levels of PI3K and AKT protein were detected by Western blot. One-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for comparison between groups.

Results

Compared with DMSO groups, the cell viabilities were decreased gradually with the increase of psoralen concentration and action time (P < 0.05) , except the cells were treated with 10 μg/mL psoralen for 24 h. Compared with DMSO group, the proportion of cells in G1 phase was increased after treatment with 30, 100 μg/mL psoralen for 24 h. The proportion of cells in G2/M phase was decreased, the apoptosis rates were increased [ (9.16±0.97) %、(15.45±1.57) % vs (1.02±0.36) %], the scratch healing rates were decreased [24 h: (45.00±3.44) %, (27.60± 2.21) %vs (66.10±2.61) %, 48 h: (70.00± 3.40) %, (45.17±2.44) %vs (85.17± 3.85) %], PI3K and AKT mRNA expression levels, protein expression levels and phosphorylation levels were all decreased (all P < 0.05) .

Conclusion

Psoralen decreased cell viability, blocked cell cycle, induced apoptosis and inhibited cell migration. Its mechanism may be that psoralen down-regulates the expression levels of PI3K and AKT mRNA and the expression levels and phosphorylation levels of PI3K and AKT protein.

Key words: Psoralen, Antitumor, Bladder cancer, PI3K, AKT

表1 引物序列信息

基因	序列（5'- 3'）	预期扩增产物长度（bp）
PI3K	上游ACCACTACCGGAATGAATCTCT	207
	下游GGGATGTGCGGGTATATTCTTC
AKT	上游CACAGGTCGCTACTATGCC	152
	下游GGTCGTGGGTCTGGAATG
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

表1 引物序列信息

基因	序列（5'- 3'）	预期扩增产物长度（bp）
PI3K	上游ACCACTACCGGAATGAATCTCT	207
	下游GGGATGTGCGGGTATATTCTTC
AKT	上游CACAGGTCGCTACTATGCC	152
	下游GGTCGTGGGTCTGGAATG
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

表2 补骨脂素对膀胱癌T24细胞存活率的影响比较（%，±s）

分组	实验次数	24 h	48 h	72 h	96 h	F值	P值
正常对照	3	99.71±4.70	103.32±2.05	102.65±4.61	102.24±0.99
DMSO	3	100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00
10 µg/mL	3	96.50±2.70	94.53±1.68^a	90.34±3.07^aef	85.50±2.29^aefg	19.062	< 0.001
30 µg/mL	3	93.64±1.67^a	87.29±2.72^abe	83.67±5.76^abe	75.82±2.09^abefg	26.956	< 0.001
50 µg/mL	3	89.72±2.80^abc	83.47±2.12^abce	73.56±2.34^abcef	64.95±4.19^abcefg	79.297	< 0.001
100 µg/mL	3	85.45±2.21^abcd	77.60±2.45^abcde	64.24±3.00^abcdef	55.45±3.06^abcdefg	157.463	< 0.001
F值		21.192	56.974	51.392	124.302
P值		< 0.001	< 0.001	< 0.001	< 0.001

表2 补骨脂素对膀胱癌T24细胞存活率的影响比较（%，±s）

分组	实验次数	24 h	48 h	72 h	96 h	F值	P值
正常对照	3	99.71±4.70	103.32±2.05	102.65±4.61	102.24±0.99
DMSO	3	100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00
10 µg/mL	3	96.50±2.70	94.53±1.68^a	90.34±3.07^aef	85.50±2.29^aefg	19.062	< 0.001
30 µg/mL	3	93.64±1.67^a	87.29±2.72^abe	83.67±5.76^abe	75.82±2.09^abefg	26.956	< 0.001
50 µg/mL	3	89.72±2.80^abc	83.47±2.12^abce	73.56±2.34^abcef	64.95±4.19^abcefg	79.297	< 0.001
100 µg/mL	3	85.45±2.21^abcd	77.60±2.45^abcde	64.24±3.00^abcdef	55.45±3.06^abcdefg	157.463	< 0.001
F值		21.192	56.974	51.392	124.302
P值		< 0.001	< 0.001	< 0.001	< 0.001

表3 补骨脂素作用24 h时对膀胱癌T24细胞周期的影响比较（%，±s）

分组	实验次数	G1	S	G2/M
DMSO	3	57.91±0.84	25.73±0.17	16.37±1.00
30 µg/mL	3	62.34±1.13^a	32.77±1.35^a	4.89±1.38^a
100 µg/mL	3	72.08±2.75^ab	25.56±0.83^b	2.36±1.97^a
F值		49.678	59.844	73.895
P值		< 0.001	< 0.001	< 0.001

表3 补骨脂素作用24 h时对膀胱癌T24细胞周期的影响比较（%，±s）

分组	实验次数	G1	S	G2/M
DMSO	3	57.91±0.84	25.73±0.17	16.37±1.00
30 µg/mL	3	62.34±1.13^a	32.77±1.35^a	4.89±1.38^a
100 µg/mL	3	72.08±2.75^ab	25.56±0.83^b	2.36±1.97^a
F值		49.678	59.844	73.895
P值		< 0.001	< 0.001	< 0.001

图1 补骨脂素作用24 h时对T24细胞周期的影响

表4 补骨脂素对膀胱癌T24细胞凋亡和迁移率的影响比较（%，±s，n = 3）

分组	凋亡率（24 h）	划痕愈合率（24 h）	划痕愈合率（48 h）
DMSO	1.02±0.36	66.10±2.61	85.17±3.85
30 µg/mL	9.16±0.97^a	45.00±3.44^a	70.00±3.40^a
100 µg/mL	15.45±1.57^ab	27.60±2.21^ab	45.17±2.44^ab
F值	133.451	142.294	113.707
P值	< 0.001	< 0.001	< 0.001

表4 补骨脂素对膀胱癌T24细胞凋亡和迁移率的影响比较（%，±s，n = 3）

分组	凋亡率（24 h）	划痕愈合率（24 h）	划痕愈合率（48 h）
DMSO	1.02±0.36	66.10±2.61	85.17±3.85
30 µg/mL	9.16±0.97^a	45.00±3.44^a	70.00±3.40^a
100 µg/mL	15.45±1.57^ab	27.60±2.21^ab	45.17±2.44^ab
F值	133.451	142.294	113.707
P值	< 0.001	< 0.001	< 0.001

图2 补骨脂素对膀胱癌细胞凋亡的影响

图3 补骨脂素对膀胱癌细胞划痕愈合率的影响

表5 补骨脂素对膀胱癌细胞PI3K、AKT mRNA表达的影响比较（±s）

分组	实验次数	PI3K	AKT
DMSO	3	1.00±0.00	1.00±0.00
30 µg/mL	3	0.56±0.11^a	0.45±0.03^a
100 µg/mL	3	0.31±0.04^ab	0.26±0.06^a
F值		86.107	288.978
P值		< 0.001	< 0.001

表5 补骨脂素对膀胱癌细胞PI3K、AKT mRNA表达的影响比较（±s）

分组	实验次数	PI3K	AKT
DMSO	3	1.00±0.00	1.00±0.00
30 µg/mL	3	0.56±0.11^a	0.45±0.03^a
100 µg/mL	3	0.31±0.04^ab	0.26±0.06^a
F值		86.107	288.978
P值		< 0.001	< 0.001

表6 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响比较（±s，n = 3）

分组	PI3K	AKT	p-PI3K	p-AKT
DMSO	1.83±0.17	1.75±0.12	3.59±0.42	2.82±0.25
30 µg/mL	1.35±0.07^a	1.34±0.09^a	1.98±0.07^a	1.60±0.20^a
100 µg/mL	1.00±0.10^ab	1.00±0.16^ab	1.00±0.27^ab	1.00±0.32^ab
F值	35.359	26.746	61.081	37.389
P值	< 0.001	0.001	< 0.001	< 0.001

表6 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响比较（±s，n = 3）

分组	PI3K	AKT	p-PI3K	p-AKT
DMSO	1.83±0.17	1.75±0.12	3.59±0.42	2.82±0.25
30 µg/mL	1.35±0.07^a	1.34±0.09^a	1.98±0.07^a	1.60±0.20^a
100 µg/mL	1.00±0.10^ab	1.00±0.16^ab	1.00±0.27^ab	1.00±0.32^ab
F值	35.359	26.746	61.081	37.389
P值	< 0.001	0.001	< 0.001	< 0.001

图4 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响

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Inhibitory effect and mechanisms of psoralen on proliferation and migration of bladder cancer T24 cells

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Inhibitory effect and mechanisms of psoralen on proliferation and migration of bladder cancer T24 cells