A Novel Tumor Gene Therapeutic Strategy Based On Targeting Ubiquitylation-induced Degradation Of Insulin-like Growth Fcator Receptor And Insulin Receptor

Insulin-like growth factor-I receptor (IGF-1R), is a member of receptor tyrosine kinase (RTK) family, IGF-1R is overexpressed in many types of cancer cell. IGF-1R is made up withαsubunit andβsubunit, which are located in the extracellular and intracellular respectively, and the two parts are connected with disulfide linkage. Theαsubunit can combine with the ligand,βsubunit contains kinases catalytic domain and many phosphoric sites. Ligand binding or receptor overexpress activate the receptor, which lead to the activation ofβsubunit, and then recruit the downstream adaptor called IRS-1, which contains PTB domain, and activate MAPK and PI-3K signaling pathway next, which boost cell proliferation, metastasis and invasion, meanwhile inhibite apoptosis, accelerate tumor malignant transformation. Insulin receptor (InsR), which acts as an accomplice in this process, has a same adaptor in cytoplasm with IGF-1R, they induce same signaling pathway, lead to similar effects. Therefore, both IGF-1R and InsR are key targets in tumor therapy.Ubiquitylation-induced degradation is a main negative regulating method of protein in cytoplasm. ubiquitylation is a process that finished by ubiquitin-activating enzyme (E1), ubiquitin-conjunction enzyme (E2) and ubiquitin-ligase (E3), ubiquitylate the substrate and make it degraded in proteasome. E3 plays a key role in this process, it can identify and combine with the substrate specifically, and transfer ubiquitin to it. In this research, we use U-box domain of CHIP and RING domain of Cbl, both of them have the activation of E3, catalyze and degrade IGF-1R and InsR, to achieve the aim of cancer therapy.In the first part of this research, we used recombinant PCR to fuse PTB gene of IRS-1 and U-box gene of CHIP or RING gene of Cbl, then cloned these fusion genes into eukaryotic expression vector pFLAG-CMV4, structured recombinant ubiquitin ligases PTB-U-box and PTB-RING. In the second part, we investigated the functions of recombinant ubiquitin ligases to downregulate the two target protein. And in the third part, we examined the effects of recombinant ubiquitin ligases on proliferation and metastasis in HeLa cells.Part 1: designed primers, used PCR amplify PTB gene, U-box gene and RING gene respectively, there are several continuous reserve complement base pairs between the reverse primer of PTB and the forword primer of U-box or RING. Fuse PTB and U-box or RING with recombinant PCR, named the production PTB-U-box and PTB-RING. Treated the production with restriction enzyme EcoR I,EcoR V, then cloned them into eukaryotic vector pFLAG-CMV4. After identifing and sequencing, named the recombinant plasmid pFLAG-CMV4-PTB-U-box, pFLAG-CMV4-PTB -RING respectively, and pFLAG-CMV4-PTB, whose functional domain was deleted.Part 2, transfected several types of cancer cell with high-expression of IGF-1R and InsR with recombinant ubiquitin ligases, including SK-Br-3, HepG2, HHCC and HeLa, pFLAG-CMV4-PTB-U-box and pFLAG -CMV4-PTB-RING were experimental groups, pFLAG-CMV4-PTB and pFLAG-CMV4 were control groups, examined their expressions and the downregulation of target protein with western blot. Meanwhile, mRNA abundance of the two target proteins were detected with real-time PCR, ensure that their downregulation occur in post-translational level.Part 3, after that, we invetigated the effects of recombinant ubiquitin ligases on proliferation and metastasis in HeLa cells, whose effect was the best in part 2. The experimental groups and control groups were same with part 2. MTT and Flat cloning formation assey examined the effects of cell proliferation after transfected with recombinant ubiquitin ligases, and transwell assey reflected the metastasis change in HeLa. The results showed that the two recombinant ubiquitin ligases could inhibite proliferation and metastasis in HeLa cells obviously.In conclusion, because of the abnormal activation and overexpression of RTK in many types of cancer, targeted degradation may be an effective method. Both IGF-1R and InsR are important members in RTK family, participate in cancer malignant transformation. Their downregulation can inhibit the development of cancer, therefore, both of them are ideal targets in cancer therapy. Ubiquitylation is a common modification in post-translational level, classic ubiquitylation of protein induces ubiquitin-proteasome pathway, lead to the degradation of ubiquitylated substrates. Use the binding domain of the adaptor which can bind to IGF-1R and InsR specifically to achieve the specificity and selectivity of recombinant ubiquitin ligases, while the catalytic domain achieve the functionality, thereby reach our goal that targeted degradation of IGF-1R and InsR, inhibit cell proliferation and metastasis. It is a novel and effective cancer therapy strategy.