丙泊酚通过上调miR-195抑制胃癌细胞的增殖、迁移和侵袭

doi:10.3760/cma.j.cn371439-20200907-00105

摘要/Abstract

摘要：

目的探讨丙泊酚对人胃癌细胞增殖、迁移和侵袭的影响及其分子机制。方法采用四甲基偶氮唑蓝(MTT)法检测不同浓度丙泊酚对人胃癌MGC-803和HGC-27细胞活力的影响。将MGC-803细胞分为对照组和丙泊酚组,Hoechst 33258染色和电镜检测两组细胞凋亡率,Transwell实验检测两组细胞迁移率和侵袭率。随后将细胞分为对照组、丙泊酚组和丙泊酚+miR-195i组,实时荧光定量PCR(qRT-PCR)检测细胞中miR-195相对表达量,蛋白质印迹法检测细胞中Janus激酶/信号转导与转录激活子(JAK/STAT)信号通路蛋白的表达。结果 0、1、5、10、20 mg/L丙泊酚作用24 h,MGC-803细胞活力分别为(100.00±4.96)%、(94.63±3.15)%、(77.38±6.73)%、(63.82±8.42)%和(35.94±7.01)%,组间差异具有统计学意义(F=5.148,P<0.001),与0 mg/L丙泊酚相比,5、10和20 mg/L丙泊酚作用下细胞活力显著降低(均P<0.05)。同时丙泊酚作用48和72 h也可显著降低MGC-803细胞活力。HGC-27细胞中也检测到类似的结果。Hoechst 33258染色结果显示,对照组阳性细胞百分率为(3.73±1.81)%,丙泊酚组为(25.44±1.05)%,两组间差异具有统计学意义(t=6.415,P<0.001)。电镜结果显示,对照组细胞凋亡率为(4.60±1.36)%,丙泊酚组为(28.15±1.99)%,两组间差异具有统计学意义(t=10.729,P<0.001)。Transwell结果显示,对照组细胞迁移率为(53.94±4.62)%,丙泊酚组为(21.28±3.98)%;对照组细胞侵袭率为(62.38±6.75)%,丙泊酚组为(33.81±4.92)%,两组间差异均具有统计学意义(t=4.628,P<0.001;t=6.418,P<0.001)。qRT-PCR结果显示,对照组、丙泊酚组、丙泊酚+miR-195i组miR-195相对表达量分别为0.58±0.09、1.24±0.22、0.63±0.16,3组间差异具有统计学意义(F=1.547,P=0.001);与对照组相比,丙泊酚组细胞miR-195表达显著增加(P<0.001)。与丙泊酚组相比,丙泊酚+miR-195i组细胞miR-195表达显著降低(P<0.001)。蛋白质印迹检测结果显示,对照组、丙泊酚组、丙泊酚+miR-195i组磷酸化Janus激酶1(p-JAK1)蛋白相对表达量分别为1.18±0.36、0.27±0.08、0.58±0.11,3组磷酸化信号转导与转录激活因子3(p-STAT3)蛋白相对表达量分别为0.83±0.16、0.21±0.07、0.72±0.13,差异均有统计学意义(F=1.655,P<0.001;F=2.520,P<0.001);与对照组相比,丙泊酚组细胞p-JAK1和p-STAT3蛋白相对表达量显著降低(P<0.001;P=0.001);与丙泊酚组相比,丙泊酚+miR-195i组细胞p-JAK1和p-STAT3蛋白相对表达量显著增加(P=0.003;P=0.004)。结论丙泊酚可抑制胃癌细胞MGC-803的细胞增殖、迁移和侵袭,促进其凋亡,其机制可能与丙泊酚促进miR-195表达并抑制JAK/STAT信号通路活性有关。

关键词: 胃肿瘤, 二异丙酚, 细胞增殖, 肿瘤侵润, miR-195

Abstract:

Objective To investigate the effect of propofol on the proliferation, migration and invasion of human gastric cancer cells and its molecular mechanism. Methods The cell viabilities of human gastric MGC-803 and HGC-27 cells under different concentration of propofol were detected by methyl thiazolyl tetrazolium (MTT) method. MGC-803 cells were divided into control group and propofol group. Hoechst 33258 staining and electron microscopy were used to detect the apoptosis rates of the two groups of cells. Transwell experiment was used to detect the migration and invasion rates of the two groups of cells. The cells were then divided into control group, propofol group and propofol + miR-195i group. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-195 in the cells. Western blotting was used to detect the expressions of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway proteins. Results At 24 h, the cell viabilities of MGC-803 cells under the action of 0, 1, 5, 10 and 20 mg/L propofol respectively were (100.00±4.96)%, (94.63±3.15)%, (77.38±6.73)%, (63.82±8.42)% and (35.94±7.01)%, with a statistically significant difference (F=5.148, P<0.001). The cell viabilities of MGC-803 cells under the action of 5, 10 and 20 mg/L propofol were decreased significantly compared to that under the action of 0 mg/L propofol (all P<0.05). At the same time, the effects of propofol for 48 and 72 h could also significantly reduce the viabilities of MGC-803 cells. Similar results were also detected in HGC-27 cells. The results of Hoechst 33258 staining showed that the percentage of positive cells in the control group was (3.73±1.81)%, and that in the propofol group was (25.44±1.05)%, with a statistically significant difference (t=6.415, P<0.001). The results of electron microscopy showed that the apoptosis rate in the control group was (4.60±1.36)%, and that in the propofol group was (28.15±1.99)%, with a statistically significant difference (t=10.729, P<0.001). Transwell results showed that the cell migration rate in the control group was (53.94±4.62)%, and that in the propofol group was (21.28±3.98)%; the cell invasion rate in the control group was (62.38±6.75)%, and that in the propofol group was (33.81±4.92)%, and there were statistically significant differences (t=4.628, P<0.001; t=6.418, P<0.001). qRT-PCR results showed that the relative expressions of miR-195 in the control group, propofol group and propofol + miR-195i group were 0.58±0.09, 1.24±0.22 and 0.63±0.16, with a statistically significant difference (F=1.547, P=0.001). miR-195 expression was increased significantly in the propofol group compared to the control group (P<0.001). Compared with the propofol group, miR-195 expression in the propofol + miR-195i group was decreased significantly (P<0.001). Western blotting results showed that the relative expressions of phosphorylase Janus kinase 1 (p-JAK1) protein in the control group, propofol group and propofol + miR-195i group were 1.18±0.36, 0.27±0.08 and 0.58±0.11; the relative expressions of phosphorylase signal transducer and activator of transcription 3 (p-STAT3) protein in the three groups were 0.83±0.16, 0.21±0.07 and 0.72±0.13, and there were statistically significant differences (F=1.655, P<0.001; F=2.520, P<0.001). The expressions of p-JAK1 and p-STAT3 protein in the propofol group were decreased significantly compared to the control group (P<0.001; P=0.001). The expressions of p-JAK1 and p-STAT3 protein in the propofol + miR-195i group were increased significantly compared to the propofol group (P=0.003; P=0.004). Conclusion Propofol can inhibit the cell proliferation, migration and invasion of gastric cancer MGC-803 cells, and promote its apoptosis. Its mechanism may be related to the promotion of miR-195 expression and inhibition of JAK/STAT signal pathway activity.

Key words: Stomach neoplasms, Propofol, Cell proliferation, Neoplasm invasiveness, miR-195

毛智军, 刘栋, 张玉明, 肖琪, 马鹏, 蒋延安. 丙泊酚通过上调miR-195抑制胃癌细胞的增殖、迁移和侵袭[J]. 国际肿瘤学杂志, 2020, 47(12): 705-711.

Mao Zhijun, Liu Dong, Zhang Yuming, Xiao Qi, Ma Peng, Jiang Yan'an. Propofol inhibits the proliferation, migration and invasion of gastric cancer cells by up-regulating miR-195[J]. Journal of International Oncology, 2020, 47(12): 705-711.

图/表 7

表1

图1

图2

图3

图4

表2

图5

参考文献 20

[1]	Marghalani AM, Bin Salman TO, Faqeeh FJ, et al. Gastric carcinoma: insights into risk factors, methods of diagnosis, possible lines of management, and the role of primary care[J]. J Family Med Prim Care, 2020,9(6):2659-2663. DOI: 10.4103/jfmpc.jfmpc_527_20. doi: 10.4103/jfmpc.jfmpc_527_20 pmid: 32984103
[2]	Chidambaran V, Costandi A, D'Mello A. Propofol: a review of its role in pediatric anesthesia and sedation[J]. CNS Drugs, 2015,29(7):543-563. DOI: 10.1007/s40263-015-0259-6. doi: 10.1007/s40263-015-0259-6 pmid: 26290263
[3]	Oren-Ziv A, Hoppenstein D, Shles A, et al. Sedation methods for intra-articular corticosteroid injections in juvenile idiopathic arthritis: a review[J]. Pediatr Rheumatol Online J, 2015,13:28. DOI: 10.1186/s12969-015-0021-0. doi: 10.1186/s12969-015-0021-0 pmid: 26141717
[4]	Wang J, Cheng CS, Lu Y, et al. Novel findings of anti-cancer pro-perty of propofol[J]. Anticancer Agents Med Chem, 2018,18(2):156-165. DOI: 10.2174/1871520617666170912120327. doi: 10.2174/1871520617666170912120327 pmid: 28901262
[5]	李正民, 张玉明, 张振, 等. HIF-1介导的线粒体能量代谢参与丙泊酚调控肾透明细胞癌细胞增殖及凋亡[J]. 国际肿瘤学杂志, 2019,46(12):711-717. DOI: 10.3760/cma.j.issn.1673-422X.2019.12.002.
[6]	Yang C, Gao J, Yan N, et al. Propofol inhibits the growth and survival of gastric cancer cells in vitro through the upregulation of ING3[J]. Oncol Rep, 2017,37(1):587-593. DOI: 10.3892/or.2016.5218. doi: 10.3892/or.2016.5218 pmid: 27840947
[7]	Peng Z, Zhang Y. Propofol inhibits proliferation and accelerates apoptosis of human gastric cancer cells by regulation of microRNA-451 and MMP-2 expression[J]. Genet Mol Res, 2016,15(2). DOI: 10.4238/gmr.15027078. doi: 10.4238/gmr.15028258 pmid: 27421007
[8]	Hammond SM. An overview of microRNAs[J]. Adv Drug Deliv Rev, 2015,87:3-14. DOI: 10.1016/j.addr.2015.05.001. doi: 10.1016/j.addr.2015.05.001 pmid: 25979468
[9]	Zhang J, Wu GQ, Zhang Y, et al. Propofol induces apoptosis of he-patocellular carcinoma cells by upregulation of microRNA-199a expression[J]. Cell Biol Int, 2013,37(3):227-232. DOI: 10.1002/cbin.10034. doi: 10.1002/cbin.10034
[10]	Deng H, Guo Y, Song H, et al. MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer[J]. Gene, 2013,518(2):351-359. DOI: 10.1016/j.gene.2012.12.103. doi: 10.1016/j.gene.2012.12.103
[11]	Singh Bajwa SJ, Bajwa SK, Kaur J. Comparison of two drug combinations in total intravenous anesthesia: propofol-ketamine and propofol-fentanyl[J]. Saudi J Anaesth, 2010,4(2):72-79. DOI: 10.4103/1658-354X.65132. doi: 10.4103/1658-354X.65132 pmid: 20927266
[12]	Lundstrom S, Twycross R, Mihalyo M, et al. Propofol[J]. J Pain Symptom Manage, 2010,40(3):466-470. DOI: 10.1016/j.jpainsymman.2010.07.001. doi: 10.1016/j.jpainsymman.2010.07.001 pmid: 20816571
[13]	Bergmann TK, Barraclough KA, Lee KJ, et al. Clinical pharmacokinetics and pharmacodynamics of prednisolone and prednisone in solid organ transplantation[J]. Clin Pharmacokinet, 2012,51(11):711-741. DOI: 10.1007/s40262-012-0007-8. doi: 10.1007/s40262-012-0007-8
[14]	Patankar M. Sildenafil citrate[J]. J Indian Med Assoc, 2000,98(12):800-803, 815. pmid: 11394480
[15]	Dulskas A, Al Bandar M, Choi YY, et al. A case of gastric cancer metastasis to the breast in a female with BRCA2 germline mutation and literature review[J]. Acta Chir Belg, 2019,119(1):59-63. DOI: 10.1080/00015458.2017.1411554. doi: 10.1080/00015458.2017.1411554 pmid: 29202657
[16]	Zhang L, Chen X, Liu B, et al. MicroRNA-124-3p directly targets PDCD6 to inhibit metastasis in breast cancer[J]. Oncol Lett, 2018,15(1):984-990. DOI: 10.3892/ol.2017.7358. doi: 10.3892/ol.2017.7358 pmid: 29387242
[17]	Fabian MR, Sundermeier TR, Sonenberg N. Understanding how miRNAs post-transcriptionally regulate gene expression[J]. Prog Mol Subcell Biol, 2010,50:1-20. DOI: 10.1007/978-3-642-03103-8_1. doi: 10.1007/978-3-642-03103-8_1 pmid: 19841878
[18]	Pencik J, Pham HT, Schmoellerl J, et al. JAK-STAT signaling in cancer: from cytokines to non-coding genome[J]. Cytokine, 2016,87:26-36. DOI: 10.1016/j.cyto.2016.06.017. doi: 10.1016/j.cyto.2016.06.017 pmid: 27349799
[19]	Khanna P, Chua PJ, Wong BSE, et al. GRAM domain-containing protein 1B (GRAMD1B), a novel component of the JAK/STAT signaling pathway, functions in gastric carcinogenesis[J]. Oncotarget, 2017,8(70):115370-115383. DOI: 10.18632/oncotarget.23265. doi: 10.18632/oncotarget.23265 pmid: 29383166
[20]	Ouyang J, Pan X, Lin H, et al. GKN2 increases apoptosis, reduces the proliferation and invasion ability of gastric cancer cells through down-regulating the JAK/STAT signaling pathway[J]. Am J Transl Res, 2017,9(2):803-811. pmid: 28337309

丙泊酚浓度(mg/L)	MGC-803细胞			HGC-27细胞
丙泊酚浓度(mg/L)	24 h	48 h	72 h	24 h	48 h	72 h
0	100.00±4.96	100.00±5.31	100.00±4.12	100.00±5.38	100.00±6.17	100.00±4.18
1	94.63±3.15	88.32±4.61	82.76±7.14^a	95.61±8.46	91.75±7.54	86.48±7.08
5	77.38±6.73^a	68.26±4.18^a	54.41±4.19^a	82.42±9.18^a	75.48±3.87^a	62.52±3.48^a
10	63.82±8.42^a	52.72±5.85^a	33.57±7.34^a	71.92±4.75^a	61.19±4.57^a	46.83±9.40^a
20	35.94±7.01^a	26.84±4.95^a	20.46±8.01^a	52.75±6.38^a	34.72±4.35^a	21.89±8.04^a
F值	5.148	4.624	5.688	6.952	6.415	7.415
P值	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001