Investigation On The Anti-tumor Efficacy And Mechanisms Of Small Molecule Kinase Inhibitors In Non-small Cell Lung Cancer

Objective: 1)To investigate the relationship between EGFR exon 19 and 21 gene mutations and clinicopathological features in patients with advanced non-small cell lung cancer(NSCLC),explore the relationship between EGFR gene mutation types and EGFR-TKIs in NSCLC patients,and guide individualized treatment of patients with non-small cell lung cancer;2)The high-throughput sequencing method was used to sequence the mutations of 13 driving genes related to targeted therapy in patients with non-small cell lung cancer,and to analyze the relationship between different types of mutations and clinicopathological features and prognosis.Provide theoretical basis for clinical precision treatment;3)To investigate the anti-tumor effect of the second-generation proteasome inhibitor Oprozomib on lung cancer and its preliminary molecular mechanism by studying the role of small protease inhibitor Oprozomib in the development of lung cancer cells.Methods: 1)Collect 140 patients with non-small cell lung cancer confirmed by histopathology of the Affiliated Tumor Hospital of Xinjiang Medical University,and analyze the clinical data and EGFR mutation types.The chi-square test and the Fisher test were used to analyze the correlation between the patient's clinical data and the type of EGFR mutation.In addition,Kaplan-Meier survival analysis was used to compare the median progression-free survival(mPFS)and median overall survival(mOS)in NSCLC patients with EGFR19 exon mutations or exon EGFR21 exon mutations.;2)Samples of 69 patients with NSCLC from the Cancer Hospital affiliated of Xinjiang Medical University were collected.Targeted high-throughput sequencing analysis of EGFR,KRAS,ALK,ROS1,RET,PIK3 CA,MET,ERBB2,BRAF,FGFR1,DDR2,NRAS,and MAP2K1 driver gene mutations in patients,Combining bioinformatics methods to identify mutations and types of tumor-driven genes associated with tumor-targeted therapy.We analyzed the relationship between the type ofdriver gene mutation and clinicopathological features,the correlation between patient prognosis and different types of driver gene mutations;3)MTT assay was used to detect the inhibitory effect of different concentration gradient OPZ on the proliferation of EGFR wild-type lung cancer cells with different genotypes(A549,HOP62,NCI-H226,NCI-H1299 and NCI-H1373).At the same time,MTT assay was used to detect the combination of OPZ and cisplatin drugs for lung cancer cells.Whether OPZ improve the sensitivity of tumor cells to cisplatin,whether the OPZ can inhibit anchorage-independent growth of lung cancer cells in vitro by plate cloning assay and soft agar colony formation assay;Five different genotypes of lung cancer cells were treated with different concentrations of OPZ,and the expression of signaling pathway-related proteins(p53,p21,PUMA,BAX,NOXA,Caspase-3)was detected by Western Blot.Results: 1)EGFR gene mutation accounted for 49.29%of the total number of people,including 21 exons L858 R mutation(37.68%)and 19 exon deletion(24.64%);There were 16 cases(23.19%)of EGFR-compliant mutations.Mainly EGFR-sensitive mutations combined with EGFR 20 exon T790M mutations,suggesting that these patients may develop resistance to EGFR-TKI;Exon 19 deletion mutations are more likely to be female,family history,stage III-IV non-small cell lung cancer patients,Exon 21 mutations are more likely to be male,adenocarcinoma,and non-small cell lung cancer patients over 60 years of age;The median PFS of patients with exon 19 deletion mutations in non-small cell lung cancer was significantly longer than that of patients with exon 21 mutations.The difference was statistically significant(P < 0.05).This indicates that EGFR-TKI drugs have a good therapeutic effect on non-small cell lung cancer patients with 19 exon deletion mutations;2)High-throughput sequencing of 13 drug-targeted therapeutic-driven genes,in which the EGFR mutation has the highest mutation rate among all driving gene mutations,It has a higher mutation rate in women,adenocarcinoma,and advanced clinical patients(P<0.05);The KRAS gene mutation rate was 11.59%,which was more likely to occur in patients with advanced adenocarcinoma of grade III-IV who were older than 60 years,male,family history of tumor,and clinical stage.ALK gene mutation,PIK3 CA gene mutation,BRAF gene mutation and ROS1 gene mutation were not associated with clinicopathological features of NSCLC patients(P>0.05);Double-driver gene mutations and three-driver gene mutations in patients with non-small cell lung cancer,In patients with EGFR-mutant non-small cell lung cancer,the EGFR gene was accompanied by other gene mutations such as TP53 and ERBB2,and the PFS of patients was significantly lower than that of patients with EGFR single gene mutation,but no biostatistical significance(P>0.05);3)By reducing cleaved Caspase 3 and PARP protein expression,OPZ stabilizes p53 protein and its downstream transcriptional targets,thereby significantly reducing cell proliferation and promoting apoptosis.In addition,OPZ can improve the sensitivity of lung cancer cells to the traditional chemotherapy drug cisplatin,thereby improving the effect of chemotherapy.Conclusion:1)In patients with non-small cell lung cancer,EGFR19 exon deletion mutation is more likely to be female,family history,tumor stage in stage III-IV non-small cell lung cancer patients,and exon 21 mutation is more likely to be male,adenocarcinoma patients Non-small cell lung cancer patients over the age of 60 years.The median PFS of 19 exon-mutant non-small cell lung cancer patients was significantly longer than the median PFS of 21 exon mutation patients;2)Mutations in the EGFR gene along with other driver gene mutations may be the cause of EGFR-TKI resistance in patients and may be one of the predictors of poor prognosis.Patients with EGFR-sensitive gene mutations may need to select an effective individualized treatment model based on the type of accompanying gene mutation;3)The protease inhibitor OPZ has an inhibitory effect on the proliferation of different genotypes of lung cancer cells,and thus can be used as a targeted therapeutic drug alone or in combination with other chemotherapy or targeted drugs,and is a potential therapeutic drug for lung cancer patients.