[Construction and screening an effective anti-miR-221 RNAi vector in vitro].
OBJECTIVE
To construct and screen an effective anti-miR-221 vector of siRNA.


METHODS
Four hairpin structure of siRNA transcript templates targeting miR-221 and a negative control were synthesized, then ligated with pGCSIL-GFP vector and a pEGFP-miR-221 which express pre-miR-221 was also constructed. All the recombinants were sequenced. The confirmed pGCSIL-GFP recombinants by combining with pEGFP-miR-221 were transfected into 293T cells seperately. The expressed Flag protein was detected by Western blot to evaluate the inhibition effect of targeting sequences. Then a recombinant with the highest anti-miR-221 effect was screened and transfected into U87 glioma cell, and its anti-tumor effect was evaluated by MTT and FCM.


RESULTS
The resulting recombinants were confirmed by sequencing which demonstrated that the recombinant plasmids contained the correct sequences of designed transcript templates. The results of Western blot indicated the expression of Flag of No 1 recombinant plasmid group was inhibited heavily with a 34.3% expression level by compared with control group. The proliferation inhibition and induced apoptosis by this recombinant with an apoptosis ratio of 21.89% in U87 cell were also observed.


CONCLUSIONS
The anti-miR-221 expression siRNA espression recombinants were constructed successfully, and one sequence with the highest inhibition efficiency was screened out, which could inhibit U87 cell proliferation and induce cell apoptosis, and could be used to suppress target gene for further study in tumor biology.
Zhao
Peng
P
Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Fu
Zhen
Z
You
Yong-Ping
YP
Lu
Xiao-Ming
XM
Lu
Ai-Lin
AL
Liu
Ning
N
D001932:Brain Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics*; D005910:Glioma / Q000473:pathology; D006801:Humans; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D035683:MicroRNAs; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C525709:MIR221, human