Effect Of Double Mutation Of KIT Gene On Cell Proliferation

[Backgound] Gastrointestinal stromal tumors(GISTs)is the most common gastrointestinal tract tumors,accounting for 80% of gastrointestinal tract tumors.Its prognosis by tumor location,tumor diameter and mitosis,only according to tumor diameter ≤2 cm GISTs are called "small GISTs",> 2 cm are called "clinically significant or overt GISTs ",both are originated from ICC(interstitial of Cajal),gastrointestinal pacemaker cells by KIT/PDGFRA(70% ~ 85%)drive gene mutation,but overt and small GISTs of the clinical manifestations,histopathological morphology,biological behavior and prognosis are significantly different,therefore,the genetic differences which need further study.It has been reported in the literature that the median survival time of mice with double mutation of KIT gene is longer and tumor growth is slower than that of mice with single mutation,suggesting that double mutation may block tumor growth,but the mechanism of double mutation of KIT gene is unknown.[Objective] To investigate the differences in KIT gene expression between overt gastric stromal tumor and small gastric stromal tumor,and to explore the mechanism of the inert growth behavior of small gastric stromal tumor,so as to obtain the targeted treatment sites for overt gastric stromal tumor.To investigate the mechanism by which double mutation of KIT gene lead to the stagnation of gastrointestinal stromal tumor and its influence on the expression of mRNA and protein in the PI3 K signaling pathway.[Methods] 1.Establish enrollment criteria,collect the gstric GISTs samples and observe morphological and clinical pathological feature differences by HE staining and immunohistochemical staining.2.The KIT V560 D single mutation,T670 I single mutation and V560D/T670 I double mutation templates were constructed and transfected into tool cell lines to observe the effects of KIT double mutation on cell proliferation and other biological behaviors,as well as the effects on PI3 K mRNA and protein expression.[Results] 1.There was no significant correlation between GISTs and gender,anatomical position and cytological subtype.2.Gastric GISTs are mostly found in patients aged 50 years.3.Gastric small GISTs are often associated with gastrointestinal cancer.4.GISTs are mainly mesenchymal tumors formed after KIT gene mutation,which are mostly seen in the "550-580" hot spot mutation of exon 11.5.There are combined mutations of ATM,APC and other genes in gastric small GISTs with growth potential and gastric overt GISTs with vigorous growth.6.A case of double mutation KIT exon 11W557G/exon 17N822 Y detected by nextgeneration sequencing was grouped into small GISTs with growth potential,suggesting that the double mutation site may have a growth-promoting effect.8.The double mutant group of 293 T cells KIT V560D/T670 I gradually grew slowly after 72 h of culture,which was finally lower than the single mutant group V560 D.9.Protein expression levels of c-kit,PI3 K,AKT,pAKT,pY568+570 and pY721 in the KIT V560D/T670 I double mutant group were lower than those in the V560 D single mutant group.10.The mRNA expression levels of KIT,PI3 K and AKT in the KIT V560D/T670 I double mutant group were all lower than those in the V560 D single mutant group.[Conclusion] 1.Gastric GISTs were mostly found in patients aged 50 years,slightly more in males than females,and the histological subtypes of gastric GIST are mostly spindle cell subtypes.2.High-throughput genetic testing indicates that KIT gene mutation is still the main driving gene for the occurrence and development of GIST.3.Gastric small GISTs with growth potential and gastric overt GISTs with vigorous growth significantly correlated with KIT mutation.4.The lower proliferation level of KIT double-mutant cell lines than that of singlemutant cell lines may be related to the decreased expression levels of genes and proteins in KIT and PI3 K signaling pathways after KIT double-mutation.