Establishment Of Ub-TfR System And Detection Of EGFR Gene Mutation And EML4-ALK Fusion Gene

Establishment of ubiquitin-transferrin receptor expression vectorEndosomal sorting is a key step of endocytosis regulated by ubiquitin. Ubiquitin labeled proteins can be sorted into the lysosomal pathway by the ESCRT. Ubiquitin plays an important signal in endosomal protein sorting and degradation regulation, it can regulate endocytic proteins from endosomes into lysosomes pathway by MVB. Tom1L1 can also bind to ubiquitin. PR-619 is a reversible inhibitor of deubiquitylases (DUBs), which can delay ubiquitin signal. Hrs and TSG101 which are parts of ESCRT complexs also plays an important role in the separation process.Ubiquitin-transferrin receptor (Ubiquitin-Transferrin receptor, Ub-TfR) fusion protein is sorted into multivesicular/lysosomal degradation pathway because of marked by ubiquitin, Ub-TfR will not recycle back to the classical pathway of cell membrane. So Ub-TfR is one of the ideal marker for the study of intracellular protein sorting pathway common mechanism.Therefore, the establishment of Ub-TfR expression vector lays a certain foundation for the study of cell sorting related proteins.Method:1. Construction of expression vector of Ub-TfRThe ubiquitin gene and transferrin receptor protein gene are cloned into the expression vector pCI-neo-HA by enzyme digestion, and the plasmid was extracted after sequencing.2. The expression of Ub-TfR vector.After sequenced correctly, the recombinant expression vector was transfected in Hela cells. After purification of cell supernatant, Ub-TfR was confirmed by Western blot and Immunofluorescence.3. Condition optimization of PR-619 in A431 and HeLa cell linesThe optimal conditions of PR-619 in A431 and HeLa cell lines were detected by western.4. The optimum conditions of Hrs and TSG101 siRNAThe optimal conditions of Hrs and TSG101 siRNA were detected by western.Result:1. Construction, expression and identification of Ub-TfR vector.Connected after enzyme cut, the recombinant expression vector was sequenced exactly and transformed into Hela cell. After purification of cell supernatant, Ub-TfR was confirmed as one band of 95 kDa by Western blot. Which showed that the target protein. Immunofluorescence confirmed that Ub-TfR followsed the endocytosis pathway and entered into the endosomes, Ub-TfR did not recycle back to the membrane after internalization.2. The best stimulating concentration of PR-619 in HeLa cells and A431 cells is 30uM, the best stimulation time is 3 hours; The best interfer time of Hrs and SiRNA TSG10148 hours after transfection.Conclusion:1. PCl-neo-Ub-TfR expression plasmid was successfully constructed, and the expression of Ub-TfR protein was significantly increased after transfection of Hela cells. Immunofluorescence confirmed that ubiquitin transferrin receptor endocytosis is not back to the cell membrane, but to enter the endocytosis pathway and enter the endosome.2. The best stimulating concentration of PR-619 in HeLa cells and A431 cells is 30uM, the best stimulation time is 3 hours;the best interfer time of Hrs and siRNA TSG101 48 hours after transfection. These lay a foundation for the related proteins of the endosomal sorting.Detection of EGFR gene and EML4-ALK fusion gene mutationIn China and throughout the world, Lung cancer became the leading cause death. The main treatments of lung cancer includes surgery,chemotherapy, molecular targed therapy radio and therapy. The main types of lung cancer are small-cell lung carcinoma (SCLC) and nonsmall cell lung cancer (NSCLC). The majority (85%) of lung cancer diagnoses are NSCLC, whereas SCLC consists of 15% of the diagnoses. The resent studies suggest that targeted therapy is a better alternative for treating advanced NSCLC. With the in-depth research in molecular targets of lung cancer, people focus on the molecular targeted drugs which possess high specificity and adverse reaction. EGFR (epidermal growth factor receptor) gene and EML4-ALK(echinoderm microtubule-associated protein-anaplastic lymphoma kinase) fusion gene are the main targeted drug therapy in lung cancer driver gene mutations, so genetic tests is very necessary before targeted drug therapy.EGFR belongs to membrane receptor whose molecular mass is 170kDa, it also belongs to a family of tyrosine kinase receptors. Epidermal growth factor receptor (EGFR) signaling pathway is crucial to the formation and development of NSCLC.EGFR TKIs can selectively inhibit the activation of EGFR tyrosine kinase domain and block its downstream signal transduction. EGFR TKIs has better curative effect in NSCLC.So EGFR mutation is an important predictor in the treatment effect of targeted drug TKIs. EML4-ALK gene which is one of the molecular targets in NSCLC is fused by part gene of EML4 and ALK. Studies confirm that the EML4-ALK fusion gene have carcinogenic activity both in vitro and in vivo. This activity can be effectively blocked by ALK inhibitors. These suggest that EML4-ALK fuse gene mutation plays a key role in the occurrence and development of non-small cell lung cancer.The comprehensive evaluation between EML4-ALK gene mutation, EGFR gene mutation and clinical pathological index can provides a more reliable basis for targeted therapy in NSCLC. Reasonable biomarker screening plan contributes to improve the curative effect and the success rate before the selection of appropriate use of targeted drug therapy. In addition, it not only can avoid some excessive treatment, but also could save medical cost. At the same time, the curative effect is optimized and the toxicity is the lowest.Method:1. Detection of EGFR geneMutations of exons 19 and 21 of EGFR in 252 NSCLCs were detected by PCR amplification and gene sequencing.2. The expression of ubiquitin vector.EML4-ALK fusion gene mutations were detected by Realtime-PCR.Result:1. Correlation between EGFR gene mutation and clinical features casesThe total mutation rate of EGFR gene was 38.8%, including 15.8% of exon 19 and 23.0% of exon 21,respectively,EGFR mutations usually occurred in the female, non-smokers and adenocarcinoma patients (P<0.05). However, there was not any relationship in age (P>0.05).2. Correlation between clinical features cases and EML4-ALK gene mutationThe total mutation rate of EML4-ALK fusion gene was 4.7%, EML4-ALK fusion gene mutations usually occurred in the female and younger age patients (P< 0.05).Mutations were not related to non-smokers and pathological type patients (P >0.05).3. Coexistence phenomenon of EGFR mutation and EML4-ALK mutationNo mutation was detected coexist in EGFR and EML4-ALK gene mutation.Conclusion:1. Due to high mutation frequency of EGFR in Chinese NSCLC patients, it is highly recommended to investigate EGFR gene mutations before treatment with tyrosine kinase inhibitors.2. EML4-ALK fusion gene defines another molecular subset of NSCLC with distinct characteristics, which provides a new option for the clinical treatment of patients with NSCLC.3. The coexistence phenomenon of EGFR mutation and EML4-ALK mutation is rare.