shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 <sup>*</sup>

doi:10.13523/j.cb.20191101

中国生物工程杂志

2019, Vol. 39

Issue (11): 1-12 DOI: 10.13523/j.cb.20191101

研究报告

shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 ^*

段李梅¹,杨锦潇¹,刘佳渝²,郑永波²,吴小候²,罗春丽^1,^**(

)

1 重庆医科大学检验医学院重庆医科大学临床检验诊断学教育部重点实验室重庆 400016
2 重庆医科大学附属第一医院重庆 400016

shPLCε Inhibits Serine/Glycine Metabolism and Proliferation of Prostate Cancer via YAP Signaling Pathway

DUAN Li-mei¹,YANG Jin-xiao¹,LIU Jia-yu²,ZHENG Yong-bo²,WU Xiao-hou²,LUO Chun-li^1,^**(

)

1 Key Laboratory of Clinical Diagnostics Founded by Ministry of Education, College of Laboratory,Chongqing Medical University, Chongqing 400016, China
2 The First Affiliated Hospital of Chongqing, Medical University, Chongqing 400016, China

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摘要：

目的该研究旨在探讨磷脂酰肌醇特异性磷脂酶C epsilon(phospholipase C epsilon, PLCε)对前列腺癌细胞丝氨酸/甘氨酸代谢及细胞增殖的影响。方法慢病毒及质粒转染LNCAP、PC3细胞,q-PCR、Western blot分别检测LNCAP、PC3细胞中 PLCε、Yes相关蛋白(yes associated protein,YAP)、丝氨酸/甘氨酸生成酶[包括磷酸丝氨酸转氨酶1(phosphoserine aminotransferase1,PSAT1)、磷酸丝氨酸磷酸酶(phosphoserine phosphatase,PSPH)、丝氨酸羟甲基转移酶2(serine hydroxymethyltransferase2,SHMT2)及增殖相关基因细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)]的表达情况;克隆形成实验及MTT实验检测细胞的克隆形成率及增殖活性。结果 (1)感染LV-shPLCε可显著下调前列腺癌细胞LNCAP、PC3中的PLCε、YAP、PSAT1、PSPH、SHMT2及增殖相关基因的mRNA及蛋白质水平,同时抑制细胞的克隆形成能力和增殖活性;(2)在shPLCε组细胞中加入过表达YAP质粒后,能明显逆转YAP、PSAT1、PSPH、SHMT2及增殖相关基因的下调,但加入干扰YAP质粒后结果相反。结论 shPLCε可通过下调YAP的表达抑制前列腺癌细胞的丝氨酸/甘氨酸生成,从而抑制细胞的增殖。

关键词： 前列腺癌; 磷脂酰肌醇特异性磷脂酶C; epsilon; Yes相关蛋白; 丝氨酸/甘氨酸代谢; 增殖

Abstract:

Objective: To investigate the effects of phospholipase C epsilon on serine/glycine metabolism and cell proliferation in prostate cancer cells.Methods: Lentivirus and plasmid were transfected into LNCAP and PC3 cells. The expression of YAP, serine/glycine producing enzyme (PSAT1,PSPH,SHMT2) and proliferation-related genes (Cyclin D1,PCNA) were detected by q-PCR and Western blot. The cloning formation experiment and MTT assays were used to detect the clone formation rate and cell proliferation activity.Results: (1) Infection with LV-shPLCε significantly down-regulated the mRNA and protein levels of PLCε,YAP,serine/glycine producing enzymes (PSAT1,PSPH,SHMT2) and proliferation genes (Cyclin D1,PCNA) in prostate cancer cells LNCAP and PC3. At the same time, it inhibits the clone formation ability and proliferative activity of LNCAP and PC3 cells. (2) After adding over-expressing YAP plasmid to shPLCε group, YAP,serine/glycine producing enzymes and proliferation genes were significantly reversed. but the results of the interference with the down YAP plasmid were reversed.Conclusion: shPLCε inhibits the serine/glycine metabolism and proliferation in prostate cancer cells by down-regulating the expression of YAP.

Key words: Prostate cancer PLCε YAP Serine/Glycine metabolism Proliferation

收稿日期: 2019-04-15 出版日期: 2019-12-17

ZTFLH:

Q814

基金资助: * 国家自然科学基金(81072086)

通讯作者: 罗春丽 E-mail: luochunli79@126.com

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引用本文:

段李梅,杨锦潇,刘佳渝,郑永波,吴小候,罗春丽. shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 ^*[J]. 中国生物工程杂志, 2019, 39(11): 1-12.

DUAN Li-mei,YANG Jin-xiao,LIU Jia-yu,ZHENG Yong-bo,WU Xiao-hou,LUO Chun-li. shPLCε Inhibits Serine/Glycine Metabolism and Proliferation of Prostate Cancer via YAP Signaling Pathway. China Biotechnology, 2019, 39(11): 1-12.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191101 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/1

表2 q-PCR引物信息

表3 质粒信息

图1 转染shPLCε慢病毒后LNCAP和PC3细胞中PLCε的mRNA和蛋白质水平

图2 LNCAP和PC3细胞的克隆形成实验 (a)Clone formation experiments of LNCAP (b)Clone numbers of LNCAP (c)Clone formation experiments of PC3 (d)Clone numbers of PC3 * P<0.05, ** P<0.01,*** P<0.001

图3 LNCAP和PC3细胞的MTT实验(72h)

图4 转染shPLCε后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平

图5 转染shPLCε后LNCAP和PC3细胞中YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平

Fig.6 mRNA and protein levels of YAP in LNCAP and PC3 cells infected with over-expression or knockdown of YAP plasmid (a)mRNA level of YAP in LNCAP infected with over-expression or sh-YAP plasmid by q-PCR (b)mRNA level of YAP in PC3 infected with over-expression or sh-YAP plasmid by q-PCR (c)Western blot of YAP in LNCAP (d)Relative protein level of LNCPA (e)Western blot of YAP in PC3 (f)Relative protein level of PC3 * P<0.05, ** P<0.01, *** P<0.001

图7 转染过表达或敲低YAP质粒后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平

图8 转染过表达或敲低YAP质粒后LNCAP和PC3中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平

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