The Inhibitory Effect Of RNAi Shp2Gene On The Proliferation Of Chronic Myeloid Leukemia K562Cells

ObjectiveWe constructed pSilencer4.1-shRNA-Shp2recombinant plasmidsthrough RNA interference technology, then transfected them into chronicmyeloid leukemia cell line K562to study the inhibitory effect of downregulation of Shp2gene on chronic myeloid leukemia（CML）. At the sametime, it provides us a further study of the subsequent mechanism of CMLleukemia in vitro and anti-leukemia effect in vivo animal models.Methods13pairs of oligos for short hairpin RNA expression which targetedShp2gene were designed, using online software. Then annealed and insertedthem into pSilencer4.1-CMV neo plasmids to construct recombinantplasmids. K562were transfected with these plasmids. The expression ofShp2mRNA and protein were assayed by real-time PCR and Western blotrespectively. Select the most efficiency group.2Transfect the most efficiency recombinant plasmidspSilencer4.1Shp2/560into K562cells, the mRNA level of bcr/abl weredetected by RT-PCR and real-time PCR; the protein level of bcr/abl were detected by western blot; the cell proliferation were test by methylthiazolyltetrazolium(MTT) and the cell apoptosis were test by flow cytometry(FCM).Results1Enzyme digestion and sequencing both proved that the recombinantplasmids of pSilencer4.1Shp2/560、 pSilencer4.1Shp2/1435、pSilencer4.1Shp2/1783were constructed successfully.2After transfect recombinant plasmids into K562cells, compared withthe negative control group and blank control group of K562cells，theexpression of Shp2gene and protein were reduced.3After transfect the most efficiency recombinant plasmidspSilencer4.1Shp2/560into K562cells, the expression level of bcr/abl fusiongene and fusion protein were significantly decreased; the proliferationability was inhibited significantly (P<0.05); apoptosis was promotedsignificantly (P<0.05), compared with the two control groups.ConclusionThe pSilencer4.1Shp2/560stably transfected group can significantlyreduce the bcr/abl fusion gene and protein expression levels. The biologicaleffect of K562cells may significantly inhibited by down regulation of Shp2gene, which point out that Shp2gene may be play a biological role as atumor suppressor gene.