Exnerimcntal Study Of The Function Of TNIK In The NF-κB Signaling Pathway And Cancer Cell Growth Inhibition

The NF-кB signaling pathway is involved in modulation of many important cellularfunctions such as cell survival, development and immune response. TNIK is of criticalimportance for both canonical NF-κB signaling pathway and JNK signaling pathway.Consequently, TNIK knockdown using shRNA or siRNA has enormous influence on many kindsof cell signalings and cell survival.In this work, we used the pShuttle-CMV plasmid which was inserted Amino-terminal Flagtagged TNIK and mTNIK (K56A) cDNA to study whether TNIK was necessary for NF-κBactivation by TNFα. We investigated the antitumor effect of Ad-shTNIK in vivo and in vitrousing adenovirus harboring short hairpin RNA (shRNA) targeting TNIK mRNA.MethodsIn our study, firstly, we analysed the expression of TNIK in HeLa useing western blottingand co-IP. By combining RNA interference, western blot and reporter activity assay, we studiedwhether TNIK was necessary for NF-κB activation by TNFα. Secondly, We investigated theantitumor effect of Ad-shTNIK in vitro using MTT assay, crystal violet staining assay Hochest33342staining assay, flow cytometry detection of apoptosis. After experiments in vitro, thenaked mice harboring Bel-7404xenograft tumor were taked as model to research the antitumoreffect of Ad-shTNIK in vivo.ResultsBy combining western blotting and coimmunoprecipitation assays, we found that TNIKunderwent ubiquitin-and proteasome-mediated degradation in response to TNFα. Furtherinvestigation through RNA interference, western blot and reporter activity assay disclosed thatTNIK was necessary for NF-κB activation by TNFα. Experiments in vitro showed thatAd-shTNIK had obvious antitumor effect in cancers but no in normal cells, otherwise, unitingTNFα, the cell viability of cancer cells was lower than only using Ad-shTNIK. Ad-shTNIKinfection prevents the growth of xenograft tumor. HE staining and TUNEL staining indicatedthat Ad-shTNIK caused evident apoptosis in response to TNFα. Moreover,immunohistochemistry assay indicates that TNIK was necessary for NF-κB activation by TNFα. ConclusionThe results demonstrated that TNIK was necessary for NF-κB activation by TNFα andAd-shTNIK had obvious antitumor effect. TNIK can be a new target of gene-virus therapy forcancer.