Gain-of-function E76K-Mutation Of Ptpn11 And MiR-100 Inactivation Synergistically Facilitate Malignant Transformation Of Mouse Embryonic Fibroblasts

[Backgrounds]Cancer is a serious threat to human health,remains a high morbidity and mortality worldwidely.Furthermore,the rate of malignancy,recurrence and metastasis is very high.Therefore,there is a great significance to study the mechanism of tumorigenesis and development.A large number of studies have shown that tumorigenesis are a chronic process affected by multiple genes and multiple factors.Regulation of gene mutations on tumorigenesis is not only an emerging hot topic in the fields,but also a potential candidate for treating tumor.Previous researches of our laboratory have shown the effects of gene mutations on tumorigenesis and development.The role of SHP-2 protein-activated mutation encoded by the proto-oncogene Ptpn11 in tumorigenesis attract our interest.Here,in this project,we propose to use mouse embryonic fibroblasts as a molecular mechanism model for in vitro studies of tumorigenesis.Finally,we hope to find new potential tumor markers and raise the pobssibility of improving prognosis.SHP-2 is a ubiquitous non-receptor protein tyrosine phosphatase（PTP）,which contains two tandem Src homology domains（N-CH2 terminus and C-CH2 terminus）that act as a phosphotyrosine-binding domain and mediate the interaction with substrate,playing a critical role in a variety of signal transduction pathways.Dyfunction of SHP-2 induce cancer,Noonan syndrome,leukemia,lung cancer and so on.Previous researches of our laboratory reported that SHP-2 mutations promotes the malignant transformation of breast cancer cells and occurrence of colitis-related colorectal cancer.Micro RNAs（miRNA）are belong to endogenous non-coding small RNAs（about 22 nucleotides）that bind to the 3’-UTR region target genes,which subsequently degrading target mRNAs,inhibiting their translation,and mediating post-transcriptional of genes.Our previous study found that in MEFs with SHP-2 D61G functional mutant induced by arsenic,a variety of miRNA expression profiles and the expression of miR-100significantly decreased assayed using gene chip,which attracts our interest for further study.It has been documented that miR-100 that abnormally expressed in many cancers is involved in a variety of cellular processes,including cell cycle,differentiation,migration and invasion,and apoptosis.Based on previous reports,we wondered whether the functional mutation of SHP-2and the inactivation of miR-100 could induce tumors?If so,which kind of cancer?our preliminary data found that the cell morphology changed following passage,and the primary MEFs aged.Howvere,the MEFs containg Ptpn11^E76Kmutation and miR-100inactivation will spontaneously immortalize without aging.Thus,we want to determine the effect of Ptpn11^E76Kfunctional mutations and miR-100 inactivation in the malignant biological behavior of MEFs and their tumorigenesis.[Methods]1. Primary mouse embryonic fibroblasts（MEFs）were prepared from pregnant mice of different genotypes at 13.5d,and required MEFs were obtained and purified after genotype identification of the corresponding embryo tissue.2. We infected Cre recombinase adenovirus（Ad-Cre-GFP）with four groups of MEFs,Under the action of Cre recombinant enzyme,the neo cassette in front of E76K can be knocked out to express E76K.In addition,the recognition of loxp site can also be used to remove miR-100 by Cre recombinant enzyme to achieve the functional inactivation of miR-100.Therefore,Ptpn11^E76Kactivation mutation and miR-100 inactivated MEF cell lines were obtained,and the expression of Cre,SHP-2,miR-100 in each group of cells was detected by q PCR to verify the transfection efficiency.3.Perform aging-relatedβ-galactosidase staining and related cell behavior experiments to discover the effects of Ptpn11 E76K and miR-100 knockout on the malignant biological behavior of MEFs.a.Proliferation experiments:MTT,Ki67 immunofluorescence and flow cytometry,cell cycle analysisb.Cell colony forming ability:plate cloningc.Unanchored cell growth:soft agar colony formationd.Migration and invasion capabilities:Transwell,Wound Healing4.In vivo experiments,Nude mouse tumor formation model to detect the proliferation of these four MEFs in nude mice and pass the xenografts subcutaneously in C57/BL6mice.[Results]1.The morphology of the primary MEFs is spindle-shaped,the cell body is widen and branched,the nucleus is oval-shaped,and the cell viability is good.2.The four groups of MEF cells successfully infected and obtained fot experiments are And the SHP-2 mRNA level in the two groups of cells recovered to the same level as that in WT（+/+）MEFs.The expression level of miR-100 mRNA in3. P10 WT cells became senescent,the cell body became flat,the nucleus became larger,growthwasretarded,β-Galstainingwasblue.However,accelerates over aging,and the cell morphology changes significantly.4. Ptpn11^E76K activation mutation and miR-100 inactivated MEF cells had significantly improved proliferation,colony forming ability,migration and invasion ability.5. Ptpn11^E76Kactivation mutation and miR-100 inactivation promoted the increase of telomerase activity in MEF cells,and the cells spontaneously immortalized.6. Ptpn11^E76K mutation and miR-100 inactivation promoted tumor formation under the skin of nude mice and can be passaged continuously without significant difference in organ appearance.[Conclusion]Ptpn11^E76K activational mutation and miR-100 inactivation promote malignant transformation of MEF cells.