The Role Of Autophagy In The Mechanism Of Imatinb-induced Resistant Of Gastrointestinal Stromal Tumors

Background and Objective:Autophagy plays a dual role in tumors, which inhibit tumors on one hand and on the other hand induce drug resistance by aiding tumors to cope with nutritional deficiency and ameliorate chemoradiotherapy reaction. In our preliminary research, we discovered a significant difference in the expression of miR-320aRNA via agilent human microRNA oligonucleoti.de microarray V3 screeming sensitive and resistant GISTss. miR-320a is known to be expressed in certain hepatocellular carcinomas colon cancer and tumors of urinary system and engaged in tumor infiltration, metastasis and drug sensitivity. However, few is known about the role of miR-320a in Imatinib resistance of GISTss, neither is the research of autophagy. So our research aimed at exploring the relationship of miR-320a and autophagy regulation and their role in Imatinib-resistance of GISTss.Method:Target genes and possible pathway genes of miR-320a were predicted through the biological repository data. The protein encoding by those genes has been determined by immunohistochemistry and were expressed significantly between malignant and imatnib-resistant GISTs tissue microarrays. The imatinb-resistant human GISTs cell line (GIST882R) was established and biological function examinations such as cell proliferation、scratch tes、cytotoxicity were conducted to confirm the successful establishment of resistant strain. Quantitative Real-time PCR was used to examine the expression of miR-320a and downstream related genes and immunocytochemistry was applied to qualitatively detect the expression of the proteins encoded by ATG14 and related pAKT,mTOR, Bcl-2 in the sensitive and resistant GISTs cell line.Result:We found that resistant GISTs tissue showed significantly alternations in morphology, the number of mitosis and invasion. We supposed that miR-320a target ATG14 and related AKT, mTOR, BCL-2 gene to regulate autophagy which participate in the drug resistance of Imatinib. Immunohistochemical staining of clinical specimen microarray revealed that the expression of proteins encoded by AKT, mTOR and ATG14 in resistant tissue increased compared with sensitive ones, while BCL-2 protein expression decreased. Moreover, resistant GISTs cell lines displayed morphological changes and enhanced migration capacity. GIST882R had a stonger expression of autophagosome compared with GIST882S. ATK, mTOR ATG14gene expression was detected up-regulated in GIST882R, while BCL-2 were down-regulated by PCR. The same consequence was got in the expression of protein encoding by thoese genes, which was consistant with tissue.Discussion:1. The cells of Imatinb-resistant GISTs had changes in morphology enhancement of invasion and a level of autophagy.2. Autophagy might participate in the induction of GISTs resistance to Imatinib and the down-regulation of miR-320a may enhance autophagy by ATG14 via mTOR-independent pathway.3. In the process of induction, the BCL-2/BECN1/ATG14 and PI3K/AKT/mTOR pathway may paticpate the different stage of autophagy induced by Imatinib.