Effect Of Double Mutation Of Gastrointestinal Stromal Tumor Driver Gene (KIT) On Gene Expression Of Downstream Key Signaling

[Background] Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract(GI),accounting for about 80% of gastrointestinal mesenchymal tumors,and originate from Caja cells of the myenteric plexus.GISTs are classified as very low,low,medium,and high risk based on tumor size,mitotic image count,and tumor site.According to the diameter of the tumor,tumors smaller than 2 cm are defined as small-GIST,and tumors larger than 2 cm in diameter are defined as over-GIST.Gastric large GISTs have a relatively large tumor volume,a high mitotic index,and often have neoplastic necrosis.The biological behavior is more malignant,and recurrence,metastasis,and even death can occur.The current research on small gastric GISTs is mainly based on autopsy or short-term follow-up cases.These make us have insufficient understanding of the clinical and prognosis of small gastric stromal tumors.The biological behavior of small GISTs is controversial.The biological potential of GISTs needs further research.In addition,there are three new double mutations found in small GISTs that are different from large GISTs,and no further studies have been conducted on the effects of these three double mutations on tumor biological behavior.According to a study on KIT gene double protrusions,the median survival time of the KIT gene double mutant mouse model is longer than that of the single mutation mouse model,suggesting that double mutations at specific sites of the KIT gene may affect the function of c-kit and inhibit tumor cells Proliferation hinders tumor growth,but whether double mutations are related to the formation of small GISTs,and its mechanism of blocking growth remains to be further studied.[Objective] To study the clinicopathological characteristics of small gastric stromal tumors,to explore the biological potential of small gastric stromal tumors,in order to guide the treatment strategy of small gastric stromal tumors;to explore the mechanism of the effect of double mutations at specific sites of KIT gene on cell proliferation ability PI3 K / AKT,MEK / ERK,JAK / STAT3 signaling pathway key protein expression and phosphorylation level.[Methods](1)Establish the enrollment criteria,collect gastric stromal tumor cases,observe the morphological differences and clinicopathological characteristics through HE staining,and explore the biological potential of small gastric stromal tumors.(2)Transfect the target gene with specific KIT site mutation into 293 T cell line. Establish KIT V560 D single mutation,T670 I single mutation and V560 D / T670 I double mutation models to observe the effect of KIT gene double mutation on cell proliferation and other biological behaviors,as well as the key downstream pathways PI3 K / AKT,MEK / ERK,JAK / STAT3 Effects of protein expression and phosphorylation levels.[Results]1.161 cases of gastric GISTs,there was no significant difference in the incidence of men and women.GISTs mainly occurred in patients> 50 years of age.There was no significant difference between large and small GISTs in terms of gender and age.2.Histopathologically,the major histological subtypes of gastric GISTs are spindle cell subtypes(92.6%,100/108),followed by epithelioid cell subtypes and mixed cell subtypes.The small gastric GISTs are spindle cell subtypes.Gastric large GISTs interstitial are more prone to mucoid degeneration and necrosis(57.4%,62/108),while small gastric GISTs are not seen.Gastric large GISTs are more likely to have a medium or high risk level(51.9%,56/108),while gastric small GISTs are rarely mitotic,and the NIH risk grade is mainly a very low risk level(96.22%,51/53).3.53 cases of small gastric GISTs were local growth,while 108 cases of large gastric GISTs showed 22 cases of exogenous growth.49 cases of small gastric GISTs were removed by simple endoscopy and laparoscopy,while 78 cases of large gastric GISTs were removed by open surgery.4.Biological behavior is relatively inhert,no recurrence,metastasis and death were found in 53 cases,which is significantly different from GIST.5.KIT V560 D / T670 I double mutation into cells inhibited cell proliferation and weakened the expression and phosphorylation level of key proteins in downstream main pathways PI3 K / AKT,MEK / ERK,JAK / STAT3.1)The cell proliferation capacity of KIT double mutant V560 D / T670 I cell line gradually slowed down after 72 hours of proliferation,and finally it was lower than that of wild type KIT cell line,KIT single mutant V560 D and single mutant T670 I cell line.2)Key signaling molecules c-kit,pY568 + 570,pY703,pY721,pY936,AKT,p-AKT,ERK,P-ERK,STAT3,p-in the main proliferation signaling pathway of KIT double mutant V560 D / T670 I cell line The protein level of STAT3 is lower than that of single mutant KIT V560 D cell line.[Conclusions]1.The biological behavior of small gastric GISTs is relatively inert,and the prognosis is relatively good.2.Double mutations of the KIT gene at specific sites lead to weakened cell proliferation ability,inhibited the signal transduction of the main downstream signaling pathways,and reduced the expression and phosphorylation of key proteins.