Construction Of GSK-3β-targeting RNAi Adenovirus Vector And The Effect Of Wnt/β-catenin Pathway On Proliferation And Apoptosis Of Human Thyrocytes

Objective: To construct RNAi combinant adenoviral expressive vectors specific to glycogen synthase kinase-3β(GSK-3β)and to observe its gene knockdown effect on the expression of GSK-3β.Secondly, we also wanted to explore the effect of Wnt/β-catenin pathway on proliferation and apoptosis of human thyrocytes using the RNAi adenovirus vector.Methods: The first step was to design and synthesize a pair of complementary single-strand DNA oligos which targeting 1457 site of GSK-3βmRNA. Annealling was used to generate double-strand oligos (ds oligos), and then the ds oligos were cloned into pENTR?/U6, the entry vector, to generate the Entry clone named pENTR. Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT?-DEST, the adenovirus backbone vector, was used to creat the adenovirus plasmid which contains the RNAi cassette. Then, we transfected the adenovirus plasmid digested with PacI into HEK293A cells to product adenovirus, and infected the HEK293A cells with the crude adenovirus to amply the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock .The GSK-3βgene kockdown effect induced by the RNAi adenovirus was detected by western blot analysis.In the last part, we transducted the RNAi adenovirus to inhibit the expression of GSK-3βprotein and to explore the role of Wnt/β-catenin pathway on the regulation of proliferation and apoptosis of primary human thyrocytes, with BrdU cell proliferation assay and Flow CytoMeter.Results: 1) The RNAi adenovirus exppression vector targeting to GSK-3βgene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay. The adenovirus were packaged and amplified in HEK293A cells with high titer. The results of plaque assay showed that the titer of second filial generation adenovirus range from 1.6×10¹⁰pfu to 5.0×10¹⁰pfu. 2) The results of western blot showed the expression of GSK-3βprotein in primary human thyrocytes could be inhibited efficiently by the RNAi adenovirus, and the optimal MOI should be 100; The decrease of GSK-3βexppression in primary human thyrocytes was first observed 48 hours after infection with RNAi adenovirus, and the decrease of GSK-3βexppression was enhanced along with the continuation of infection time, and the inhibition effect would last for more than 6 days after infection; The expression ofβ-catenin protein in primary human thyrocytes could be increased efficiently after RNAi adenovirus infection;3) The results of BrdU assay suggested that inhibiting the GSK-3βprotein with the RNAi adenovirus could stimulate the proliferation of primary human thyrocytes efficiently, but with slight affect on the apoptosis of the cells.Conclusion: RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently. The Wnt/β-catenin pathway plays an important role in the regulation of proliferation of primary human thyrocytes.