Construction of a CTGF and RFP-coexpressed renal tubular epithelial cell and its application on evaluation of CTGF-specific siRNAs on epithelial-mesenchymal transition.
OBJECTIVE
To construct a connective tissue growth factor (CTGF) and red fluorescent protein (RFP)-coexpressed renal tubular epithelial cell that can be used to quantitatively evaluate the CTGF-induced epithelial-mesenchymal transition (EMT).


METHODS
The CTGF and RFP coding sequences were linked with a "2A" peptide, cloned into an expressing vector, and the HK-C2AR cell clone, which could express CTGF and RFP, were selected from the HK-2 cells after the recombinant plasmid transfection. Then, the CTGF-induced EMT in this cell line was determined, to further determine the association of RFP fluorescence intensity with the CTGF expression or CTGF-induced EMT. CTGF-specific ribonucleic acid (RNA) interference was also used to reconfirm the association.


RESULTS
The constructed HK-C2AR cells could stably express RFP and CTGF proportionally, and the CTGF expressed in the cell line could induce EMT of cells, whereas the RFP expressed in the cell could exhibit bright red fluorescence after excitation. After the silence in CTGF, the RFP expression was also decreased, and the CTGF-induced EMT was also inhibited.


CONCLUSIONS
The CTGF and RFP-coexpressing renal tubular epithelial cell, HK-C2AR, could be used to quantitatively evaluate CTGF-specific small interfering RNAs on EMT by quantitatively detecting the RFP expression. Also, this cell line could be used to quantitatively determine the mechanism of CTGF-induced EMT in renal tubular epithelial cells.
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Meichu
M
Cheng
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Fuyou
F
Liu
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Youming
Y
Peng
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Junxiang
J
Chen
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Guochun
G
Chen
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China.
Li
L
Xiao
Department of Nephrology, Second Xiangya Hospital of Central South University, Changsha, Hunan, China; Institute of Nephrology, Central South University, Changsha, Hunan, China. Electronic address: liuhxy2013@163.com.
Hong
H
Liu
D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D000070584:Tripartite Motif Proteins
D055513:Connective Tissue Growth Factor; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C064419:TRIM27 protein, human; D000070584:Tripartite Motif Proteins