The Change Of ErbB Signaling Pathways In Secondary Imatinib Resistant Gastrointestinal Stromal Tumors

ObjectiveImatinib?Imatinib?in the treatment of gastrointestinal stromal tumors?GIST?secondary drug resistance problem has become increasingly prominent,two mutation of Kit/PDGFR can not fully explain the mechanism of drug resistance.Our research objective is to compare the difference of Erb B signaling pathway related genes and protein expression between secondary Imatinib resistant and primary gastrointestinal stromal tumors?GIST?from the transcriptome level observation,which will definite the change of the ErbB signaling pathways and further functional verification was made to illuminate the mechanism of the second dary Imatinib resistance.Methods1?A total of 3 pairs of secondary Imatinib resistant and primary fresh GIST tissue were taken and put them in randomization,cDNA microarray with 41000 genes was used.Genes were considered to be up-or down-regulated when the normalized signal intensityratio Log2 Ratio between secondary Imatinib resistant and primary GIST was >=1 or <=1.The expression of genes related to Erb B signaling pathway was studied.Validation of array results was carried out by RT-PCR and Western blot.2?56 specimens surgically resected from patients with GIST were collected.Then the expression of ErbB4 in GIST and nomal tissue were detected by SABC immunohistochemical method.Anoter 4 recurring GIST and 4 primary GIST specimens surgically resected were collected.After stably passage 5 times since primary culture,GIST cell lines were implanted in oxter of Balb/C mouse.Real-time fluorescent quantitative PCR were used to detect the expression of ErbB4 mRNA after tumorigenesis of GIST cell lines in Balb/C mouse.3?The method of gene silencing was used to exponentially growing GIST867 cells,which were divided into 2 groups : siRNA?siCon?control group,50 nM gene silencing group?si-h-ErbB4?.The a n ti-proliferation effect on GIST867 cells we re determined by M TT assay.Cell cycle and apoptosis were examined by flow cytometer method.RT-P C R and Western technique were used to detect ErbB4,NRG4,PI3 K,AKT3,mTOR,P70S6 K,SHC,SOS,MYC gene expression and protein expression differences.4?The mouse transplanted gastrointestinal stromal tumor model was successfully established.Tumor-bearing mice were divided into several groups : gene silencing group?50nM?,imatinib group?0.1mg/g?,negative control group and saline group.Transplanted gastrointestinal stromal tumor in four groups were respectively intra-tumor injected for 20 days.Tumor inhibitory rate we re evaluated.RT-P C R and Western technique were used to detect ErbB4,NRG4,PI3 K,AKT3,mTOR,P70S6 K,SHC,SOS,MYC gene expression and protein expression differences.5?Affymetrix miRNA chip experiments,Affymetrix lncRNA chip experiments,cyclic RNA sequencing technology to detect the Erb B signaling pathway in the non coding RNA expression differences,which further clarify the regulatory mechanism of ErbB4 gene.Results1?Compared with secondary Imatinib resistant and primary GIST,there were a total of 9 genes up-regulated and 0 down-regulated related to ErbB signaling pathway.Most of the overexpressed genes were those related to cell cycle,cell differentiation,cell growth,protein synthesis,invasion and metastasis of tumor,metabolism,angiogenesis,etc.Difference expression of GIST related gene?c-kit,CD34?were not significant in secondary Imatinib resistant and primary GIST,RT-PCR and Western blot confirmed all the array analysis results.2?The positive rates of ErbB4's expression in GIST and nomal tissue were 64.3% and 0% respectively.There was significant difference of ErbB4's expression between the GIST and nomal tissue?P<0.01?.Overexpression of ErbB4 was correlated with diameter of carcinoma and number of karyokinesis/50HPF?P<0.05?.Expression of ErbB4 mRNA in Imatinib resistant GIST was higher than that in primary GIST?P<0.05?.3?MTT assay shows gene silencing of ErbB4?si-h-ErbB4?could apparently inhibit the GIST867 cell growth.FCM indicate that gene silencing of ErbB4 can Increase the proportion of cells in the G0/G1 phase and reduce the proportion of cells in the S phase,the apoptosis rates of the cells treated by was?15.930±1.678?%,significantly higher than that in the control group and the imatinib group.The expression of ErbB4,PI3 K,AKT3,mTOR,P70S6 K,SHC,SOS,MYC gene and protein expression were significantly down regulated after ErbB4 silencing in the GIST867 cells?P<0.01?.There was no significant difference in NRG4?P>0.05?.4?Tumor inhibitory rate in gene silencing group?si-h-ErbB4?was 60.663%,significantly higher than that in the imatinib group?12.545%?and negative control group?3.638%?.The expression of Erb B4,PI3 K,AKT3,m TOR,P70S6 K,SHC,SOS,MYC gene and protein expression were significantly down regulated in the tumor-bearing mice after ErbB4 silencing?P<0.01?.There was no significant difference in NRG4?P>0.05?.5?the expression of non coding RNA in the ErbB signaling pathway was detected by microarray experiments?Affymetrix miRNA,Affymetrix lncRNA?and cyclic RNA high-throughput sequencing,We found that:1.There are 39 different miRNA related to ErbB signaling pathway between imatinib resistant GIST group and primary GIST group,there are 10 different mi RNA related to ErbB4 signaling pathway regulation.They are,respectively,hsa-miR-193a-5p,hsa-miR-103a-3p,hsa-miR-570-3p,hsa-miR-769-5p,hsa-miR-1226-3p,hsa-miR-124-3p,hsa-miR-107,hsa-miR-150-5p,hsa-miR-16-5p,hsa-miR-103a-3p.We consider that hsa-miR-103a-3p may be the most important regulator of miRNA,which paired with target gene ErbB4,degradated ErbB4 mRNA,inhibited its translation,mediated down-regulation of transcription and regulation of ErbB signaling pathway.Its low expression is closely related to the over expression of ErbB4.2.There are 4 different lncRNA related to ErbB signaling pathway between imatinib resistant GIST group and primary GIST group,the differentially regulated lncRNA associated with ErbB4 signaling pathway were lnc-GABPA-21,lnc-PRR20A-6,lnc-ZNF333-4,which may be related to abnormal expression of PI3 K and P70S6 K and abnormal activation of ErbB4 downstream mTOR signaling pathway.3.There are 15 different circRNA related to ErbB signaling pathway between imatinib resistant GIST group and primary GIST group,They are,respectively,circRNA₀0817;circRNA₀8911;circRNA₀8912;circRNA₀8914;circRNA₀9169;circRNA₀9413;circRNA₁0278;circRNA₁0279;circRNA₁4875;circRNA₁7652;circRNA₂5090;circRNA₂5870;circRNA₂8117;circR NA₂8118;circRNA₂8764.The different circRNA related to ErbB4 signaling pathway is circRNA₀8911;circRNA₀8912;circRNA₀8914.They act as a "sponge" of miRNAs,which competitively bind to mi RNAs,leads to over expression of ErbB4,AKT3,P70S6 K and leads to abnormal activation of ErbB4 signaling pathway.ConclusionFrom the above experiment,we speculate that in imatinib resistant GIST,circRNA₀8911;circRNA₀8912;circRNA₀8914,acted as a "sponge" of miRNAs,overexpressed and competitively binded hsa-miR-103a-3p and reduced its function of inhibiting ErbB4 expression,which leaded to over expression of Erb B4 and abnormal activation of downstream mTOR and RAS signaling pathways with over expression of NRG4,PI3 K,AKT3,mTOR,p70s6 k,SHC,SOS,MYC as well.Then,the synthesis of protein and the invasion and metastasis of tumor were induced.Finally it lead to imatinib secondary resistance.Overexpression of lnc-ZNF333-4 may induce overexpression of PI3 K and P70S6 K and induce abnormal activation of ErbB4 downstream mTOR signaling pathway.These non coding RNA may be a new target for the treatment of imatinib resistant GIST.In addition,the expression of ErbB4 was widely found in GIST,and expression of ErbB4 mRNA in Imatinib resistant GIST was higher than that in primary GIST.ErbB4 gene silencing may down-regulation of ErbB4,PI3 K,AKT3,mTOR,P70S6 K,SHC,SOS,MYC gene expression,which induced the downstream mTOR and RAS signal pathways closed and induced cell apoptosis and as well inhibit GIST867 cell proliferation and tumorigenicity in mice,therefore the overexpression of Erb B4 may be associated with tumor development,malignant gastrointestinal and recurrence.Expression of Erb B4 and the related non coding RNA intervention may be an effective way for the treatment of malignant imatinib resistant GIST.Therefore,the secondary resistance to imatinib in GIST may be mediated by a pathway other than the kit/ PDGFR mediated signaling pathway--ErbB4 signaling pathway.Overexpression of ErbB4 induces activation of their common downstream signaling pathways,leading to the occurrence of IM resistance.