Explore The Relationship Between The Calcium-activated Chloride Channels DOG1 And Proliferation Of Gastrointestinal Stromal Tumors(GISTs)

Background:Gastrointestinal stromal tumors(GISTs) are the most common mesenchymal tumors of the digestive tract.Most of these stromal tumors are characterized by mutations in the platalet-derived growth factor receptor a(PDGFRA) or KIT genes,resulting in the constitutive activation of tyrosine signaling pathway.Tyrosine kinase inhibitor(TKI),imatinib mesylate,provides the standard first-line therapy for patients with metastatic、recurrent or unresectable GISTs.But some patients are resistant to IM in clinical practice,including primary resistance and second resistance,which is very sticky to treat.Studies indicate DOG1 is expressed specially in GISTs,which is extremely sensitive,is used as a diagnostic marker to differentiate GIST from other sarcomas,maybe be relate to development and progress of this disease. Objective:To observe GISTs cell proliferation by deregulating DOG1,and initially to explore the relationship between calcium-activated chloride channels DOG1 and proliferation and apoptosis of GISTs,to find the new therapeutic target to treating the primary and second imatinib-resistance GISTs patients. Methods:Silence the DOG1 gene of GIST-T1 cell lines by using RNA interference,then screen the best interfering plasmids by employing the RT-q PCR technology;next,test the GIST-T1 cell lines proliferation before and after silencing the DOG1 gene by applying CCK; meanwhile evaluate whether knocking out the DOG1 gene affects the sensitivity of imatinib by adopting CCK;next,detect the apoptosis and the cell cycle of cells utilizing flow cytometry;examine the KIT protein and other proteins which relate to KIT signal pathway of cells that have been treated with the optimal concentration of imatinib by western blot.ting。Finally,analyze the data statistically by using the Graph Prism program,analyze the GISTs cell proliferation and the proteins expression of KIT signal before and after interfering DOG1 m RNA.Results1.The RT-q PCR of DOG1 m RNA shows DOG1 m RNA in the sh RNA-1 interfering GIST-T1 cell lines is 0.7999 times as much as control;DOG1m RNA in the sh RNA-2 interfering GIST-T1 cell lines is 0.3841 times as much as control; DOG1 m RNA in the sh RNA-3 interfering GIST-T1 cell lines is 0.8526 times as much as control;DOG1m RNA in the sh RNA-4 interfering GIST-T1 cell lines is 0.9669 times as much as control.2、After silencing 0min and 60 min,there is no significant difference in OD value of DOG1 m RNA between the interfered group and the non-interfered group(P>0.5);on the contrary,after silencing 120 min and 240 min,there’s difference(P<0.05).3、The cell viability test shows IC50 of imatinib is between 0.01mg/ml and 0.1mg/ml in GIST-T1 cell lines.4、It shows no difference in OD value between the DOG1 m RNAi GIST-T1 cells that have been treated with 0.01ug/ml、0.1ug/ml、1ug/ml imatinib and the DOG1 m RNAi GIST-T1 cells that haven’t been treated with imatinib(P>0.05); however,here’s remarkable difference in OD value between the DOG1 m RNAi GIST-T1 cells treated with 10ug/ml、100ug/ml imatinib and the DOG1 m RNAi GIST-T1 cells(P<0.001).5、After treated with 0.01ug/ml、0.1ug/ml、1ug/ml imatinib,there’s difference in OD value between the DOG1 m RNAi GIST-T1 cells and the GIST-T1 cells(P<0.01);there’s no difference between the two groups when treated with 10ug/ml、100ug/ml imatinib(P>0.05).6、The flow cytometry shows cell apoptosis of the three groups treated with 100ug/ml imatinib:the early apoptotic cells of control group are increasing from 4.5% to 23.0%;the negative control group are increasing from 4.8% to 19.6%;but SH-2 group are increasing from 4.3% to 12.3%.7、The flow cytometry shows the distribution of cell cycle of three groups:G1 phase is 57.1%,S phase is 42.9% in the control group;G1 phase is 57.5%,S phase is 41.3% in the negative control group;G1 phase is 61.7%,S phase is 37.8% in the SH-2 group.8、Compared with the C group, the relative level of DOG1 is notably down-regulated in the SH-2 group and SH-2(+) group,there is statistical difference(P<0.05),yet,there is no difference in the relative levels of DOG1 between the C(+) group and the C group(P>0.05);it shows no difference in the relative level of p-KIT between the SH-2 group and the C group(P>0.05),but the relative level of p-KIT is obvious decreasing in the C(+) group,it shows significantly difference between C(+) group and the C group(P<0.05).Compared with the C group,the relative level of AKT and p-AKT in C(+) group 、 SH-2 group and SH-2(+) group is no difference(P>0.05);but,the relative level of p-ERK1/2 in the C(+) group 、SH-2 group and SH-2(+) group is obviously less,there is statistical difference(P<0.05). Conclusions:1.The best interfering plasmids of DOG1 m RNA is sh RNA-2,its sequence(5’-3’) is CCTCACGGGCTTTGAAGAG。2.The experiment in vitro shows:silencing the DOG1 m RNA successfully can inhibit proliferation of GIST-T1 cells,it indicates that DOG1 plays an important role in proliferation of GISTs;and the flow cytometry shows down-regulating DOG1 makes the cell cycle stay in the G1/G0 phase,it shows DOG1 may regulate the cell proliferation by regulating the G1/S phase.3.It doesn’t influence the sensitivity of imatinib whether interfering the DOG1 m RNA or not;at the same time,DOG1 doesn’t have double ability to inhibit proliferation of GIST-T1 cells with imatinib.4.The relative level of p-KITY703 and the proteins of KIT signaling pathway is no difference before and after silencing the DOG1 m RNA;however,the relative level of p-ERK1/2 is reduced significantly after interfering the DOG1 m RNA.It shows DOG1 regulates the cell proliferation not by KIT signaling pathway but by ERK1/2 signaling pathway.