Silencing Of MKK6 With Lentivirus-mediated RNAi

Mitogen-activated protein kinase (MAPK), an important signaling pathway in cells, could transduce multiple extracellular signals into the nuclei. It plays an important role in cell growth, development, differentiation and apoptosis. In mammalian cells, there are four major MAPK subfamilies, i.e., extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 and ERK5. The p38 signaling pathway regulates cell growth, differentiation and apoptosis, playing an important role in the occurrence and development of various diseases including inflammation and cancer. MKK6, an upstream kinase of p38, regulates cell responses through phosphorylating p38 under the stimulation of pro-inflammatory cytokines, growth factors and stress stimuli.RNA interference (RNAi) is an important gene silencing technology developed in recent years, and it suppresses the expression of target genes through post-transcriptional gene silencing (PTGs). During RNAi, the key functional molecule is the 21-23 bp small interfering RNA (siRNA), which decreases the gene expression level by binding and degrading the target mRNA. RNAi is of great importance to the research of gene functions and cellular signaling pathways. Currently, three major RNAi technologies, i.e., chemically synthesized siRNAs, shRNA-expressing plasmids and shRNA-expressing virus vectors are used to knockdown genes. In host cells, plasmid and virus vectors can continuously generate shRNAs, which can be further cut into siRNAs by Dicer to exert the RNAi effect. Compared with siRNAs, plasmid and virus vectors could induce gene silencing stably. So, they are more advantageous in long-term RNAi research.The lentivirus vector is developed on the basis of human immunodeficiency virus type 1(HIV-1), and it has a strong ability in infecting both dividing cells and non-dividing cells (e.g., mature neurons). After infecting host cells, its genome can be integrated into the host genome, resulting in the stable expression of exogenous genes. Compared with other virus vectors like adenovirus and retrovirus vectors, the lentivirus vector has low immunogenicity and high integration rate, making it a better vector for exogenous genes.To further investigate the signaling pathways regulated by MKK6, lentivirus-mediated RNAi was applied to knockdown MKK6 in Hela cells. The shRNA sequence targeting MKK6 mRNA was designed with RNAi designing software and protocols provided by Invitrogen. Then, the shRNA sequence was synthesized and cloned into the pENTRTM/H1/TO entry vector. The RNAi cassette in the entry clone was transferred into the pLenti4/BLOCK-iTTM dest vector by LR recombination reaction to construct the expression clone. After that, the expression plasmids were co-transfected into 293FT producer cells with ViraPowerTM Packaging Mix to produce lentivirus. Meanwhile, lentivirus expressing LacZ shRNA was also produced and used as the negative control in the follow-up experiments. Lentivirus was collected forty-eight and seventy-two hours after transfection, respectively. Then, the lentivirus was tittered and condensed for further research.Hela cells were infected with MKK6 lentivirus and LacZ lentivirus, respectively. Then, Zeocin was used to select cells integrated with lentivirus genome. Western blot analysis was applied to detect MKK6 protein levels in different cell clones. In this way, cell clones with decreased levels of MKK6 were identified.Through this study, we reached the following conclusions:1. The annealing method was improved. Temperature gradient annealing was found to be effective in the synthesis of double-stranded oligos.2. Entry clones and expression clones of MKK6 and LacZ were successfully constructed and corresponding lentivirus was also successfully packaged and condensed.3. Lentivirus was collected twice after package, which increased the production of lentivirus.4. Western blot analysis showed that cells infected with MKK6 lentivirus had decreased levels of MKK6 while MKK6 levels in cells infected with LacZ lentivirus remained as high as that in untreated cells.5. Cell lines with decreased MKK6 levels were established with Zeocin, which formed the basis for the further study of MKK6-regulated signaling pathways.