Construction Of A Lentiviral Vector Of RNAi Targeting Mouse NF-?B/p65 Gene And Study Its Effects In MC3T3-E1 Cells

BackgroundThe transcription factor family, nuclear factor kappa B(NF-?B), is founded in nearly all cells of mammals. The p65/p50 heterodimers are the most important factors in NF-?B families. Activation of NF-?B can modulate expressions of a wide range of cytokines, immune receptors. And its abnormal activation leads to a lot of diseases. NF-?B signaling pathways play an important role in the course of the formation, resorption and remodeling of bone. Several cytokines including RANKL, TNF can activate NF-?B signaling way of pre-osteoclasts and lead to the differentiation of osteoclasts, then bone resorption follow. In the animal model of osteoporosis estrogen deficiency can activate NF-?B pathway, which not only stimulate osteoclastogenesis but also inhibit the normal function of osteoblasts. So we can infer inhibition of NF-?B may be an effective approach to promote bone formation of osteoblast, not just inhibit osteoclast function. And this idea would be another way for osteoporosis treatment. In this study, we designed shRNA sequences aiming at NF-?B/p65 gene of mouse and construction a lentiviral vector of RNAi targeting NF-?B/p65 gene. Then the lentiviral particles were transfected into MC3T3-E1 cells and we assayed the expression changes of several osteogenesis-related genes. ObjectiveTo construct a lentiviral vector of RNA interference targeting mouse NF-?B/p65 gene, and identify the vector through transfecting into MC3T3-E1 cell. And function changes of osteoblasts were assayed after NF-?B/p65 gene expression was obstructed. MethodsThree RNAi target sequences aiming at mouse NF-?B/p65 gene were designed according to the RNAi sequence design principles. In accordance to the three sequences, the sequences of shRNA were synthesized, and the sites for restriction enzymes AgeI and EcoRI were added. And the double-stranded DNA contained short hairpin loop of nineteen bases. The DNA sequences were inserted into the lentiviral vector GV-248 by restriction endonuclease Age I, EcoRI double digestion, and T4 DNA-ligase ligation. The recombinant plasmids were transfected into competent DH5?bacteria. Then the bacteria were cultured in ampicillin medium, and the candidate clones were getted and amplificated. The plasmids were identified to be correct by DNA sequencing. The recombinant and two packaging plasmids(pHelper 1.0, pHelper 2.0) were co-transfected into 293 T cell to produce the lentiviral particles. The lentiviral particles were transfected into MC3T3-E1 cells, and the NF-?B/p65 expression were assayed by Real-time PCR and Western blot analysis. The most efficient vector for inhibition of NF-?B/p65 was obtained. This best vector were transfected into MC3T3-E1 cells, then the proliferation and apoptosis of MC3T3-E1 cells were studied. The osteogenesis-related genes including ALP, OCN, RANKL were assayed by Real-time PCR and Western blot analysis. ResultThe lentiviral RNAi vector GV-248-NF-?B/p65 were constructed successfully. The NF-?B/p65 gene expression in the transfected cells was significantly inhibited at the mRNA and protein levels. Gene RANKL expression levels descended, while gene ALP and OCN expression levels ascended. ConclusionThe lentiviral RNAi vector has been successfully constructed. When NF-?B/p65 gene expressions were inhibited and NF-?B signaling ways were obstructed, the expressions of ALP and OCN ascended, and this led to more bone formation. At the same time, the expressions of RANKL descended, and this led to less bone resorption by osteoclasts.