SHP-2 Gain-of-function Mutation Promotes BEAS-2B Cells Malignant Transformation And Its Molecular Mechanism Exploration

[Backgrounds]Cancer is one of the most important diseases that threaten human health globally.A large number of studies have shown that the occurrence of cancer is affected by both genetic and environmental factors.Gene mutations is one kind of genetic factors.Our laboratory has been working on the study of relationship between gene mutation and cancer development.The SHP-2 gain-of-function mutation is one of the main research subjects in our group.SHP-2（Src homology-2 domain-containing protein tyrosine phosphatases）is a non-receptor protein-tyrosine phosphatases encoded by ptpn11,which is composed of two SH2（Src homology-2）domains and protein-tyrosine phosphatases.Under physiological conditions,binding of the protein-tyrosine phosphatases（PTPs）to the N-SH2 domain inhibits the SHP-2 phosphatase activity.As the SH2 domain binds to other phosphorylated substrates,the binding of the N-terminal domain to the PTP domain is weakened or disappeared.The PTP kinase domain is then exposed,which enables tyrosine phosphatase activity.SHP-2 plays an important role in regulating cell signal transduction and controlling cell activity.Its activation is not only related to the occurrence of malignant diseases in blood system such as Noonan syndrome,juvenile myelomonocytic leukemia（JMML）,but also closely related to the production of many kinds of solid tumors.Previous studies in our laboratory have also found that SHP-2 gain-of-function mutation not only enhances malignancy of breast carcinoma,but also promotes the occurrence of colitis-associated colorectal cancer,and in this process also induces cell EMT（pithelial-mesenchymal transition）.In addition,by searching the database,it was also found that SHP-2 mutations were detected in various solid tumors,and the rate of SHP-2 mutation is 0.87%in lung cancer.According to the World Cancer Report released by the World Health Organization（WHO）,in recent years,lung cancer is the highest incidence and the highest mortality rate of cancer in the world.However,at present,studies on the relationship between SHP-2 mutation and lung cancer were rarely conducted.Based on the above background knowledge,in order to 1)investigate whether SHP-2gain-of function mutation can promote the malignant transformation of normal lung cells,leading to the occurrence of lung cancer;and 2)understand how SHP-2 promotes the malignant transformation of normal lung cells,SHP-2 wild type plasmid and SHP-2^E76K mutation plasmid was transfected into human normal bronchial epithelial cells（BEAS-2B）to identify stable transfected cell lines.Then the changes in malignant biological behavior were examined by in vitro experiments,and further related mechanisms were studied.In addition,the in vivo experiment was carried out by instilling the adenovirus Cre vector into the lungs of the SHP-2^E76Kneo+/+mice in the laboratory to specifically activate the SHP-2^E76K gene in the lungs of mice.To detect the occurrence of lung cancer in mice and the number,size and metastasis of tumors.[Research methodology]1.The SHP-2 wild type and E76K mutant plasmids preserved in our laboratory were sequenced and compared in order to identify the correctness of the sequence.2.The SHP-2 wild type,E76K mutant and empty vector plasmid were transfected into BEAS-2B cell.Then the BEAS-2B cell lines which stably expressed SHP-2 WT,SHP-2^E76K and SHP-2 NC were screened.3.The biological behavior-related experiments were carried out to detect the effect of SHP-2^E76K mutation on BEAS-2B cells by the stable cell lines.（1）Flow cytometry to analyze the periodic changes of BEAS-2B cells before and after transfection.（2）Plate cloning experiment was used to detect changes in colony forming ability of BEAS-2B cells.（3）Soft agar colony forming assay detected the ability of anchor-independent growth of BEAS-2B cells.（4）Transwell assay for detection of the migration and invasion ability of BEAS-2B cells.4.Western blot was used to test the content of ERK/p-ERK protein and EMT associated protein（E-cadherin and Vimentin）.[Research Results]1.Results of SHP-2^E76K and SHP-2 WT plasmid identification showed that the sequence was correct.2.Successfully transfected and screened BEAS-2B cell lines which stably express SHP-2^E76K,SHP-2 WT and SHP-2 NC,and the SHP-2 protein expression of BEAS-2B cells with SHP-2^E76K mutation and SHP-2 wild type（WT）increased.3.The proportion of S phase of BEAS-2B cells with SHP-2^E76K mutation detected by flow cytometry increased.4.The colony forming ability,anchor-independent growth ability,migration and invasion ability of SHP-2^E76K mutant BEAS-2B cells significantly improved.5.SHP-2^E76K mutation promoted the expression of phosphorylated protein in the ERK pathway of BEAS-2B cells,decreased the expression of epithelial marker（E-cadherin） and increased the expression of mesenchymal marker（N-cadherin,Vimentin）.[Conclusion]The SHP-2^E76K mutation promotes the malignant transformation of BEAS-2B cells,activates the ERK signaling pathway and promotes EMT production in BEAS-2B cells.