[Construction of periostin shRNA vectors and their effects on the expression of periostin in fibroblasts].
OBJECTIVE
For studying the functions of periostin (POSTN) further, the recombinant plasmid and lentiviral vector for human periostin were constructed. The mRNA and protein expressions of POSTN were tested after the infection with the shRNA plasmid and lentivirus of POSTN in primary keloid fibroblasts (KFb).


METHODS
We designed a specific sequence of small hair RNA targeting POSTN gene which containing both sense and antisense oligo DNA of the targeting sequence. It was cloned into the vectors to construct a plasmid and a lentiviral vector. The plasmid was converted into the competent DH5α E.coli, which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were co-transfected 293T cells to get the lentivirus. KFb was infected with the plasmid and lentivirus. The expressions of POSTN in KFb were detected by RT-PCR and Western blotting. MTT was used to examine the proliferation of KFb and SFb.


RESULTS
The expression of POSTN mRNA in KFb infected with plasmid reduced 43% compared with negative control cells (1.98±0.03 vs 1.12±0.03, F=688.291, P<0.001). The mRNA and protein levels of POSTN in KFb infected with the lentivirus reduced 46% (0.44±0.09 vs 0.81±0.07, F=90.06, P<0.001) and 45% (0.29±0.04 vs 0.53±0.09, F=33.43, P<0.001), respectively, compared with negative control cells. Compared with negative control cells, the decreased proliferation rates were 19%, 14%, 18% in 48 h, 72 h, 96 h, respectively in KFb infected with the lentivirus.


CONCLUSIONS
The expression of POSTN in primary keloid fibroblasts reduced after infecting plasmid or lentiviral shRNA expression vector targeting human POSTN, which will be a basis to the further function study of POSTN.
Liu
Chang
C
Department of Plastic Surgery, Peking University Third Hospital, Beijing 100191, China.
Song
Zhen-hua
ZH
Qin
Ze-lian
ZL
D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007627:Keloid / Q000473:pathology; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D015815:Cell Adhesion Molecules; C512835:POSTN protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins