[Construction of the recombinant adenovirus mediated shRNA to silence PTN in pancreatic carcinoma and the effect of DRGn on neurite in vitro].
OBJECTIVE
To construct the replication-incompetent recombinant adenovirus mediated shRNA to inhibit the neurite growth-promoting factor (Pleiotrophin, PTN) in pancreatic carcinoma and to study the inhibitory effect of shRNA-PTN recombinant adenoviruses on the neurite's growth of dorsal root ganglion neurons (DRGn).


METHODS
Four pairs of complementary single-stranded oligonucleotides (ss oligo) were designed and synthesized and then they were annealed to create a double-stranded oligonucleotide (ds oligo). The ds oligos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into pancreatic carcinoma cells by liposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cell to produce adenovirus. The silence of the recombinant adenovirus against PTN was detected by Western blot. After DRGn was co-cultured with pc-2 cell infected by shRNA-PTN recombinant adenovirus, the morphological changes of DRGn were observed.


RESULTS
The pENTR/U6-shRNA shuttle plasmid was constructed and confirmed by sequencing. The recombinant adenovirus mediated shRNA against PTN was constructed. The best silence effect of the adenovirus against PTN was detected by Western blot on the 7th day after the pancreatic carcinoma cell was transfected. The growth of DRGn neurites was inhibited after the co-culture of DRGn with pc-2 cell which was infected by shRNA-PTN recombinant adenovirus.


CONCLUSIONS
The PTN SiRNA recombinant adenovirus has been constructed and the silence effect against PTN in pancreatic carcinoma cell has been confirmed. The growth of DRGn neurites can be inhibited by shRNA-PTN recombinant adenovirus.
Yao
Jun
J
Department of Hepatobiliary, the First Hospital of Xi'an Jiaotong University, Xi'an 710061, China. yaojun@163.com
Ma
Qing-yong
QY
Wang
Lian-cai
LC
Zhang
Min
M
Shen
Su-gang
SG
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016207:Cytokines / Q000172:deficiency / Q000235:genetics*; D004274:DNA, Recombinant; D005727:Ganglia, Spinal / Q000473:pathology*; D005818:Genetic Engineering; D006801:Humans; D055029:Inverted Repeat Sequences; D016501:Neurites / Q000473:pathology*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction