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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (5): 33-39    DOI: 10.13523/j.cb.20180505
    
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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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     
Received: 24 October 2017      Published: 05 June 2018
ZTFLH:  Q813  
Corresponding Authors: Chun-li LUO     E-mail: luochunli79@126.com
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Yan-ni HAO
Ting LI
Jia-xin FAN
Luo LI
Ling-fang NIU
Li-ping OU
Xiao-hou WU
Chun-li LUO
Cite this article:

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.

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

Lentivirus name Lentivirus sequence(5' - 3')
LV-shPLCε Sense: GGTTCTCTCCTAGAAGCAACC
Antisense:CCAAGAGAGGATCTTCGTTGG
LV-shNC Sense:TTCTCCGAACGTGTCACGT
Antisense:AAGAGGCTTGCACAGTGCA
Table 1 Lentivirus sequence
Plasmid name Plasmid sequence(5' - 3')
pGPU6/GFP/Neo- Sense:CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG
shCDC25A Antisense:GATCCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAC
pGPU6/GFP/Neo-shNC Sense:CACCGCAGGGAATTTCATTCCTCTTTCAAGAGAAGAGGAATGAAATTCCCTGTTTTTTG
Antisense:GATCCAAAAAACAGGGAATTTCATTCCTCTTCTCTTGAAAGAGGAATGAAATTCCCTGC
Table 2 Plasmid vector sequence
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
Table 3 The sequence of primer for PCR
Fig.1 Infection rate of T24 cells infected with lentivirus Microscopic image of T24-NC cells (a)Light microscopy image(×100) (b)Fluorescent photomicrograph(×100) Microscopic image of T24-shPLCε cells (c)Light microscopy image(×100) (d)Fluorescent photomicrograph(×100)
Fig.2 The expression of PLCε inT24 after treated with lentivirus (a)Relative mRNA expression of PLCε detected by q-PCR (b)Expression of PLCε detected by Western blot (c)Relative protein expression of PLCε ** P< 0.01,*** P<0.001 compared with blank control and LV-NC control
Fig.3 The effect of PLCε on glucose consumption and lactate production in bladder cancer T24 cells (a)Glucose consumption in T24 cells treated with LV-shPLCε (b)Lactate production in T24 cells treated with LV-shPLCε ** P< 0.01 compared with blank control and LV-NC control
Fig.4 The effect of PLCε on CDC25A and the molecule related with Warbury effect (a) CDC25A, PKM2, GLUT1, LDHA mRNA expression detected by q-PCR ** P<0.01,compared with blank and LV-NC control (b) CDC25A, PKM2, GLUT1, LDHA protein expression detected by western blot (c)* P<0.05,** P<0.01,*** P<0.001,compared with blank and LV-NC control
Fig.5 The expression of CDCD25A in T24 after treated with plasmid (a)Relative mRNA expression of CDC25A detected by q-PCR *** P<0.001 compared with blank control and shNC control (b)Expression of CDC25A detected by Western blot (c) Relative protein expression of CDC25A ** P< 0.01 compared with blank control and shNC control
Fig.6 Down - regulation of CDC25A inhibits the expression of PKM2, GLUT1, LDHA (a)PKM2, GLUT1, LDHA protein expression detected by Western blot (b) Relative protein expression of PKM2, GLUT1, LDHA * P < 0.05 , ** P< 0.01 compared with blank control and LV-shNC control
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