| BackgroundGastric cancer(GC)is one of the most common malignancies worldwide.According to the latest global cancer statistics released by CA Magazine in 2018,there were about 1033,000 new cases of gastric cancer in the world,and about 782,000 deaths due to gastric cancer,which ranked 7th in the incidence of malignant tumors and third in the mortality rate.About 50%of new cases of gastric cancer are concentrated in developing countries,and more than 50%of cases occur in eastern Asia,mainly in China.The incidence of gastric cancer in China is very high.In 2015,there were 679,000 new cases of gastric cancer in China,and about 490,000 death cases because of gastric cancer.Chinese morbidity and mortality of gastric cancer account for 42.6%and 45%of the world respectively.Among them,the incidence rate ranks fifth among 183 countries in the world,and the mortality rate is the sixth,with the number of cases and deaths ranking first in the world.The disease burden of gastric cancer in China is heavy and the prognosis is poor,which seriously threatens the life and health of the people and is the focus of cancer prevention and treatment.Chemotherapy is one of the most important treatments in the treatment of gastric cancer,especially advanced gastric cancer.Although chemotherapy drugs have made great progress,in clinical practice,chemotherapy resistance often occurs and has become one of the important reasons for the treatment failure of gastric cancer.Because most patients with gastric cancer have extensive infiltration,lymph node metastasis,and chemoresistance at the time of diagnosis,these factors have led to poor prognosis and low survival rate of gastric cancer.The limited efficacy and considerable toxicity of gastric cancer chemotherapy have prompted people to seek new systemic treatment strategies.It is now believed that the mechanism of chemoresistance is mainly:1)the "drug pump" effect of the cell membrane;2)the change of the drug target;3)the detoxification effect in the cell;4)the enhancement of DNA repair function;Abnormal apoptosis pathway;6)Changes in cellular microenvironment.Tumor resistance may be the result of multiple factors and multiple mechanisms.Although many studies have been carried out on the structure,function and activation mechanism of drug resistance genes,the molecular genetic mechanism of the pathogenesis and development of gastric cancer and the detailed mechanism of drug resistance in gastric cancer are not fully understood.Gastric cancer gene and molecular variation have become the focus of current research.Little is known about the regulatory factors of chemoresistance in gastric cancer.The chemoresistance of gastric cancer has always been a thorny problem that plagues clinical treatment.If the mechanism of gastric cancer resistance can be fully elucidated,it will promote the development and application of new drugs,improve the survival rate of gastric cancer and improve the quality of life of patients with gastric cancer.The research on the mechanism of drug resistance in gastric cancer is an important topic and has great significance.The transcription factor TAp73,a transcriptionally active isoform of p73,has high structure and function similarities with its homolog p53,therefore,are thought to be a cancer therapy candidate target.However,there is still a controversy about the tumor suppressor role of TAp73,since it has been found in numerous studies that TAp73 expression is elevated in different cancers.In this study,the expression of TAp73 was studied by using multiple gastric cancer cell lines,such as AGS,SNU-1 and SNU-3,and revealed how the overexpression of TAp73 affects the drug resistance and mechanism of chemotherapy.In addition,we explored how TAp73 overexpression affects tumor cell apoptosis in nude mice and their effects on tumors in nude mice.Research purposesIn this study,human gastric cancer cell lines were used as research objects.By constructing human gastric cancer cell lines and TAp73 overexpression and interfering transfected cell lines,the mechanism of TAp73 gene involved in the regulation of gastric cancer chemoresistance was discussed in vitro and in vivo.It is hoped that the predictive indicators of chemotherapy resistance can be screened out,so as to better select the target population of chemotherapy treatment,and lay an experimental foundation for the next step to reverse the clinical research of targeted drug resistance.Methods1.Establishment of drug-resistant cell lines and analysis of drug resistance characteristics:The human gastric cancer cell line resistant to fluorouracil was induced by stepwise increasing dose method.2.Cell viability and apoptotic rate were detected by MTS method and flow cytometry,respectively.3.The expression of TAp73 and its downstream related transcription genes Bax and Noxa and the expression of upstream regulatory gene E2F1 were detected by RT-qPCR and Western blot.4.TAp73 overexpression,E2F1 overexpression,E2F1 siRNA and control empty vector were stably transfected into AGS cells by plasmid transfection to detect the effect of tumor cells.5.Immunoprecipitation was used to detect the relationship between TAp73 and E2F1 in gastric cancer cells.6.Nude mouse tumor formation model:TAp73 overexpression and control vector empty vector transfected cell line,subcutaneous tumor formation in nude mice,nude mice model formation,and PBS,fluorouracil treatment into tumor nude mice;The expression levels of TAp73 protein and mRNA in tumor tissues were detected by Western blot and RT-qPCR respectively.The tumor volume was measured regularly,and the tumor growth curve was drawn.The tumor cells were detected by TUNEL staining.Result1.Chemotherapy inhibits the expression of TAp735-Fu inhibited the expression of TAp73 in gastric cancer cells and activated caspase-3,which significantly induced apoptosis in AGS cells.This suggests that TAp73 may be involved in the inhibition of 5-Fu-induced apoptosis in gastric cancer cells.2.Altering TAp73 expression will affect cell apoptosis and regulation of downstream targetsAfter transfection with TAp73,overexpressed TAp73 inhibited 5-Fu-induced apoptosis.Inhibition of downstream target genes involved in apoptosis,Bax,Noxa expression,these results indicate that TAp73 may affect cell apoptosis by inhibiting the downstream genes of p53,Bax,Noxa.3.E2F1 is an upstream regulator of TAp73,which regulates TAp73 via E2F1E2F1 was inhibited by 5-Fu in GC cells;CHIP binding assay of E2F1 and TAp73 promoter showed that 5-Fu blocked the binding of E2F1 to TAp73 promoter.In addition,E2F1 transfected AGS cells,E2F1 overexpression,TAp73 protein expression and mRNA levels increased,restored the expression of TAp73 protein and mRNA levels after 5-Fu treatment,and inhibited the activation of caspase-3,and inhibited the withering Dead cells.Therefore,these results together indicate that E2F1 is an upstream regulator of TAp73 after chemotherapy drug treatment in GC cell lines.4.The higher the expression of TAp73 and E2F1 the higher the resistance of gastric cancer cells to chemotherapy drugs.The 5-Fu-resistant cells we established were highly resistant to 5-Fu,and showed stronger cell viability and IC50 significantly higher than the parental cells.Treatment with 5-Fu in drug-resistant cell lines did not inhibit the expression of E2F1 and TAp73.Compared with the parental cells,the activation of caspase-3 was decreased.At the same time,the expression of E2F1 was knocked down by transfected siRNA in drug-resistant cells,and the reactivity of drug-resistant cells to 5-Fu was restored,the activation of caspase-3 was increased,and apoptosis was increased.The half-life of E2F1 in AGS-resistant cells is much longer than that of parental cells.These results indicate that TAp73 and E2F1 play an important role in the resistance of gastric cancer cells to chemotherapeutic drugs.E2F1 inhibits the reactivity of GC cells to chemotherapeutic drugs through the expression of TAp73.5.Subcutaneous tumor formation model in nude mice confirmed that enhanced expression of TAp73 promoted fluorouracil resistanceIn the TAp73 transfection group,the difference in tumor volume was not significant in the 5-Fu-treated group compared with the PBS group.In the no-TAp73 transfection group,the tumor volume of the 5-Fu-treated group was significantly lower than that of the PBS group.After 5-Fu treatment,the tumor volume was significantly reduced in the no-TAp73 transfection group compared with the TAp73 transfection group.In addition,transfection of TAp73 significantly reduced the 5-Fu-induced tumor TUNEL signal and reduced caspase-3 activation.It was also confirmed in nude mouse tumor tissues that the expression of TAp73 inhibited the expression levels of Bax and Noxa.Conclusion1.TAp73 is involved in the regulation of resistance to 5-fluorouracil in gastric cancer cells.2.TAp73 affects apoptosis induced by 5-fluorouracil by regulating downstream target genes Bax and Noxa.3.E2F1 is an upstream regulator of TAp73,and the expression of TAp73 is regulated by E2F1,which affects the resistance of gastric cancer cells to 5-fluorouracil. |
PDF Full Text Request