Whole Exome Sequencing Analysis And The Establishment Of Imatinib Resistance Cells Of GIST

Objective To establish Imatinib-resistant cell line from primary human gastrointestinal stromal tumor?GIST-FR?and identify Imatinib resistance related gene mutation in GIST-FR cell line and the clinical samples of GIST.Methods Primary cells were extracted from fresh GIST specimen by dissecting to small tissue piece and were immortalized?GIST-F?.Immunohistochemical staining and western bloting were used to indentify the biological characters of GIST-F.Acquired Imatinib resistance was induced in GIST cells?GIST-FR?using the intermittent concentration gradient method.The cell morphology changes of parents cells GIST-F and resistant cells GIST-FR were observed under the optical microscopy.Cell Counting Kit-8?CCK-8?assay was used to calculate the IC₅₀0 and draw the cell proliferation curve.The cell cycle of GIST-FR cells was detected by flow cytometer.GIST and Imatinib resistance related gene mutations were detected in 50GIST clinical samples,GIST-F and GIST-FR cells by whole exome sequencing?WES?.Result?1?The GIST-F cell line was established successfully,and passaged successfully for more than 40 generations.GIST-F showed short fusiform shape,and expressed CD117 protein.?2?Imatinib-resistant GIST cell line GIST-FR was established successfully.The IC₅₀ values of GIST-F and GIST-FR were 15.91?g/mL and 55.95?g/mL,respectively.The resistance index of GIST-FR cells to Imatinib was 3.52?P<0.01?.The cell proliferation ability of GIST-FR was significaanlty lower than GIST-F.The cell cycle distributions of GIST-F and GIST-FR were24.59±5.63%and 44.76±2.11%in G0/Gl phase,69.60±4.53%and 52.64±0.60%in S phase,5.82±1.10%and 2.60±1.50%in G2/M phase,respectively?P<0.05?.?3?The results of WES analysis showed that mutations in KIT were present in 48 of the 50 GIST.New mutations of exon 11 were observed in 22 cases.New insertion mutations of exon 9 were found in 9 cases.Exon 16 and exon 17 had one mutation in S65 and S30 case,respectively.Only a deleterious mutation of PDGFRA was detected in one case.We analyzed 368 cancer-related genes in these 50 GIST cases,GIST-F and GIST-FR cells.Mutations were detected in 37 loci of 34genes,including IGF1R.WES analysis also revealed 59 loci of 47 new genes mutations in two cases of Imatinib-resistant GIST samples.There were 35 new mutations in 31 genes in Imatinib-resistant GIST-FR cells.Imatinib-resistant related genes were subjected to GO function enrichment analysis.The functions of these target genes were enriched in“positive regulation of osteoblast proliferation?GO:0033690?”and“negative regulation of transcription,DNA-templated?GO:0045892?”,et al.Conclusion?1?The GIST-F cell line was successfully established in vitro and showed specific biological characters of GIST.?2?The sensitivity of GIST-FR to Imatinib targeted therapy was decreased significantly.?3?Twenty-five novel mutations were revealed in KIT gene,and 37 mutations in 34 cancer-related genes were also observed for the first time.Ninety-one new genomic alterations were found in Imatinib-resistant GIST samples.These data contribute to our understanding of the detailed molecular mechanisms of pathogenesi and drug resistance of GIST,which may provide molecular basises for the clinical diagnosis and therapy for GIST.