| Objective:1. Establish Gefitinib-resistant EGFR mutant lung adenocarcinoma H3255 and HCC827 cell lines and explore the epidermal growth factor receptor?EGFR?alternative activation signaling pathway2.Establish B7-H3 knockout H3255 and HCC827 cell lines,and explore the cross-linking regulation of B7-H3 signal on EGFR signal.Methods:1. The drug-resistant H3255 and HCC827 lung cancer lines were induced step by step by Gefitinib concentration gradient increasing method.The cell survival rate was calculated according to the OD value.According to the VR,the 50%inhibitory concentration?IC50?was calculated by linear regression between the logarithm of drug concentration and VR,and the drug resistance index?RI?was calculated.2. The OD values of H3255 group,H3255/GR group,HCC827 group and HCC827/GR group were detected by CCK8 method at 0h,24h,48h and 72h,respectively.The proliferation of Gefitinib resistant cell lines H3255/GR,HCC827/GR,H3255 and HCC827 were compared.3. The expression of EGFR downstream signal molecules AKT,ERK1/2,Stat3,p-AKT,p-ERK1/2 and p-Stat3 in H3255,HCC827,H3255/GR and HCC827/GR cells were detected by Western blot method,and the differences between drug-resistant and non-drug-resistant strains were analyzed and compared.4. The B7-H3 on H3255,HCC827 and A549 lung cancer cells were knocked out by CRISPR/Cas9 technology,and the knockout verification was conducted by flow cytometry and Western Blot analysis.5.The proliferation difference between the B7-H3 knockout lung cancer cell lines and the control cell lines was detected by CCK8 technology,and the change of its sensitivity to Gefitinib drug was detected.The apoptosis ratio between the B7-H3 knockout lung cancer cell lines and the control cell lines was detected by flow cytometry?7-aad/annexin-v-apc double staining?after 8h of CPT induction.6.Western blot was used to detect the expression of EGFR downstream signaling molecules AKT,erk1/2,Stat3,p-akt,p-erk1/2 and p-stat3 in H3255/KO,HCC827/KO and control cells,and to analyze and compare the signal differences between the two cell lines.Results:1.In this study,by gradually increasing the dose of Gefitinib,HCC827/GR cells and H3255/GR cells were successfully obtained from EGFR-sensitive mutant HCC827?Del E746-A750?cells and H3255?L858R?cells.The CCK8 results show that the IC50 of H3255 is 0.047±0.018?mol·L-1,the IC50 of H3255/GR is 0.45±0.17?mol·L-1?RI=9.566±3.679?,and the IC50 of HCC827 is 0.013±0.003?mol·L-1 The IC50 of HCC827/GR was 0.18±0.03?mol·L-1?RI=13.242±2.848?,and the drug-resistant strain was successfully established.2.The proliferation of H3255/GR and HCC827/GR cells was significantly lower than that of non-drug-resistant strains at 24 h,48 h and 72 h.3.No significant changes were observed in pmurakt,p-ERK1/2 and p-Stat3 of H3255/GR and HCC827/GR compared with non-resistant strains.In H3255 cells,Gefitinib had obvious inhibitory effect on p-AKT and p-STAT3,moderate inhibitory effect on t-STAT3and t-ERK1/2,and only slight inhibitory effect on p-ERK1/2.There was no significant change in the expression of p-ERK1/2 and p-Stat3 in H3255/GR cells,but the expression of p-AKT was significantly down-regulated.In Gefitinib-treated HCC827 cells,the expression of pmur ERK1 and p-STAT3 decreased significantly,and the expression of t-STAT3 and t-ERK1/2 also decreased to some extent,in which p-AKT and p-STAT3almost lost expression.In HCC827/GR cells,after Gefitinib treatment,the expression of p-AKT and p-STAT3 was almost not inhibited,p-ERK1/2 was moderately inhibited,and Gefitinib almost lost its inhibitory effect on its signal.4. B7-H3 gene was successfully knocked out in H3255,HCC827 and A549 lung adenocarcinoma cells using CRISPR/Cas9 technology,respectively.B7-H3 gene was confirmed to be knocked out by flow cytometry and Western Blot analysis.5. The deletion of B7-H3 caused the proliferation of HCC827 and H3255 cells with EGFR mutation to decrease significantly.After 24h,48h and 72h of culture,the proliferation of H3255,HCC827 and A549 cells was significantly reduced,and the apoptosis induced by CPT was significantly increased.6. When treated with a gradient concentration of Gefitinib,B7-H3 KO resulted in a significant reduction in cell survival of H3255 strain and HCC827 strain and control strain.In addition,Gefitinib has a dose-dependent inhibitory effect on the viability of HCC827/KO cells.In contrast,H3255/KO cells showed a stable inhibitory effect at a Gefitinib concentration of 120?M.In summary,this indicates that B7-H3 knockout increases the susceptibility of lung adenocarcinoma cells to EGFR-TKI.7. We tested the total levels and phosphorylation levels of ERK1/2,AKT and STAT3 in H3255/KO and HCC827/KO cells treated with Gefitinib.B7-H3 KO H3255 cells significantly reduced the expression of t-ERK1/2,p-AKT,p-STAT3,and the expression of t-STAT3 and p-ERK1/2 also decreased to a certain extent.B7-H3 KO combined with gefitinib caused p-AKT and p-STAT3 were undetectable,while t-STAT3,t-ERK1/2,and p-ERK1/2 were unchanged compared to H3255 KO cells.In HCC827 cells,whether B7-H3 KO or gefitinib treatment regulates t-STAT3,t-ERK1/2,p-AKT,p-ERK1/2 and p-STAT3 are almost the same,but not exactly the same.However,B7-H3 KO combined with gefitinib further reduced the expression levels of p-AKT,p-STAT3,p-ERK1/2 and total STAT3,ERK1/2 in HCC827 cells.Conclusion:1.In this study,we successfully established the Gefitinib resistant strains H3255/GR and HCC827/GR strains by gradually increasing the concentration of Gefitinib.PI3K/AKT,JAK2/STAT3 and Raf/MEK/ERK1/2 signal pathways have different degrees of involvement in the EGFR downstream signal pathway between H3255 and HCC827 cells.The JAK2/STAT3 cascade in H3255/GR and HCC827/GR cells,the PI3K/AKT pathway in HCC827/GR cells can be fully activated,while the PI3K/AKT pathway in H3255/GR cells and Raf/MEK/ERK1 in HCC827/GR cells The cascade/2 can be at least partially activated by EGFR signaling.In H3255 cells,the RAS/RAF/MEK/ERK1/2 pathway does not play a significant functional advantage in the EGFR downstream signaling pathway.Difference in bypass activation between EGFR mutant subtypes Del E746-A750 and L858R.2.B7-H3 induced signaling pathways include RAF/MEK/ERK1/2,PI3K/AKT/m TOR and JAK2/STAT3,which overlap with the EGFR signaling pathway in lung adenocarcinoma.In this study,we found that CRISPR/cas9-mediated B7-H3 knockout?KO?significantly reduced the proliferation and proliferation of H3255?L858R?,HCC827?Del E746-A750?and A549?EGFR wild-type?lung adenocarcinoma cells Apoptosis.B7-H3 was more sensitive to Gefitinib than H3255 and both.B7-H3 KO significantly reduced the total amount and phosphorylation level of ERK1/2,AKT and STAT3 in H3255 cells,while its effect was comparable to that of gefitinib in HCC827cells.Gefitinib in combination with specific inhibitors against ERK1/2,AKT or STAT3further showed that EGFR signaling was insufficient in H3255 cells and functionally overlapped with cross-linking signals in HCC827 cells.In summary,our study revealed that B7-H3-induced and EGFR signals differ in signal transduction and cross-linking in lung adenocarcinoma cells with two mutant alleles,Del E746-A750 and L858R. |
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