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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (5): 33-39    DOI: 10.13523/j.cb.20180505
研究报告     
shPLCε通过下调CDC25A抑制T24细胞的瓦伯格效应 *
郝燕妮1,李婷1,范佳鑫1,李罗1,牛凌芳1,欧俐苹1,吴小候2,罗春丽1,**()
1 重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆市重点实验室 重庆 400016
2 重庆医科大学附属第一医院 重庆 400016
Effects of shPLCε on Warburg Effect Through CDC25A in T24 Cells
Yan-ni HAO1,Ting LI1,Jia-xin FAN1,Luo LI1,Ling-fang NIU1,Li-ping OU1,Xiao-hou WU2,Chun-li LUO1,**()
1 Key Laboratory of Clinical Diagnostics Founded by Ministry of Education, College of Laboratory,Chongqing Medical University, Chongqing 400016, China
2 Department of Urinary Surgery, The First Affiliated Hospital of Chongqing, Medical University, Chongqing 400016, China
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摘要：

目的:探讨shPLCε对膀胱癌T24细胞瓦伯格效应的影响及其相关机制。方法:(1)慢病毒感染T24细胞,葡萄糖测定试剂盒和乳酸测试盒分别检测细胞葡萄糖利用和乳酸生成情况;q-PCR、Western blot分别检测PLCε、CDC25A及瓦伯格效应相关分子的表达。(2)转染shCDC25A质粒,q-PCR、Western blot检测CDC25A的表达;Western blot检测瓦伯格效应相关分子的表达情况。结果:(1)慢病毒干扰PLCε后,T24细胞利用葡萄糖和生成乳酸的能力降低,同时下调CDC25A、PKM2、GLUT1、LDHA的表达。(2)干扰CDC25A的表达后可抑制PKM2、GLUT1、LDHA的表达。结论:shPLCε通过下调关键分子CDC25A的表达抑制膀胱癌T24细胞的瓦伯格效应,从而为膀胱癌的治疗提供了新思路。
关键词： PLCε;  CDC25A;  膀胱癌;  瓦伯格效应    
Abstract:

Objective:To study the effect of shPLCε on Warburg effect in bladder cancer and the potential mechanism.Methods:(1) Using lentivirus infecting T24 cells,the glucose consumption and lactate production were detected by the corresponding kit. The expression of PLCε、CDC25A and the molecules related with Warburg effect such as PKM2, GLUT1, LDHA were detected by q-PCR and Western blot. (2)To investigate the related molecular mechanism, using plasimid infecting T24 cells, the expression of CDC25A was detected by q-PCR and Western blot. The expression of molecules related with Warburg effect were detected by Western blot.Result:(1)After reducing the expression of PLCε, glucose consumption and lactate production were significantly decreased in the LV-sh PLCε group,compared with the blank control group and LV-NC group (P<0.01).q-PCR and Western blot showed that the expression of CDC25A, PKM2, GLUT1, LDHA were remarkably lower in LV-shPLCε group than that in blank group and LV-NC group (P<0.05). (2)Western blot showed that the expression of PKM2, GLUT1, LDHA were remarkably lower in shCDC25A group than in blank group and shNC group(P<0.05), after treated with shCDC25A-plasimid.Conclusion:shPLCε can reduce Warburg effect through decreasing the expression of CDC25A in bladder cancer, which can not provide energy for the development and progression of bladder cancer.
Key words: PLCε    CDC25A    Bladder cancer    Warburg effect
收稿日期: 2017-10-24 出版日期: 2018-06-05
ZTFLH:  Q813  
基金资助: * 国家自然科学基金资助项目(81072086)
通讯作者: 罗春丽     E-mail: luochunli79@126.com
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郝燕妮
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牛凌芳
欧俐苹
吴小候
罗春丽

引用本文:

郝燕妮,李婷,范佳鑫,李罗,牛凌芳,欧俐苹,吴小候,罗春丽. shPLCε通过下调CDC25A抑制T24细胞的瓦伯格效应 *[J]. 中国生物工程杂志, 2018, 38(5): 33-39.

Yan-ni HAO,Ting LI,Jia-xin FAN,Luo LI,Ling-fang NIU,Li-ping OU,Xiao-hou WU,Chun-li LUO. Effects of shPLCε on Warburg Effect Through CDC25A in T24 Cells. China Biotechnology, 2018, 38(5): 33-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180505    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I5/33

Lentivirus name Lentivirus sequence(5' - 3')
LV-shPLCε Sense: GGTTCTCTCCTAGAAGCAACC
Antisense:CCAAGAGAGGATCTTCGTTGG
LV-shNC Sense:TTCTCCGAACGTGTCACGT
Antisense:AAGAGGCTTGCACAGTGCA
表1  慢病毒信息
Plasmid name Plasmid sequence(5' - 3')
pGPU6/GFP/Neo- Sense:CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG
shCDC25A Antisense:GATCCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAC
pGPU6/GFP/Neo-shNC Sense:CACCGCAGGGAATTTCATTCCTCTTTCAAGAGAAGAGGAATGAAATTCCCTGTTTTTTG
Antisense:GATCCAAAAAACAGGGAATTTCATTCCTCTTCTCTTGAAAGAGGAATGAAATTCCCTGC
表2  质粒信息
Gene Forward primer(5'- 3') Reverse primer(5' - 3')
PLCε GCAACTACAACGCTGTCATGGAG CCTCATGGTCTCAATATCAGACTGG
CDC25A TTCCTCTTTTTACACCCCAGTCA TCGGTTGTCAAGGTTTGTAGTTC
PKM2 AAATCACGCTGGATAACGC CAGGAAGTCGGCACCTTT
GLUT1 TGCTCATCAACCGCAACGA CCGCATCATCTGCCGACT
LDHA TCAGCCCGATTCCGTTAC CCAATAGCCCAGGATGTG
β-actin GGGACCTGACTGACTACCTC ACGAGACCACCTTCAACTCCAC
表3  引物信息
图1  慢病毒感染膀胱癌细胞T24后感染效率的检测
图2  感染慢病毒后膀胱癌细胞T24细胞内PLCε的表达
图3  下调PLCε对膀胱癌T24细胞利用葡萄糖和生成乳酸的影响
图4  下调PLCε对CDC25A和瓦伯格效应相关部分分子的影响
图5  感染质粒后膀胱癌细胞T24细胞内CDC25A的表达
图6  下调CDC25A抑制PKM2、GLUT1、LDHA的表达
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