| Background and Purpose Esophageal cancer(EC)is the fourth leading cause of cancer death in China,and esophageal squamous cell carcinoma(ESCC)is the predominant histologic type(90~95%).The mortality rate of esophageal cancer patients is high and the prognosis is poor.The cancer cells have been invading and metastasizing in the surrounding tissues,and the most patients will lose the opportunity of surgery.Therefore,radiotherapy and chemotherapy are the main treatment methods.At present,platinum represented by cisplatin and fluorouracil are widely applied to chemotherapeutic regimens for esophageal cancer,but they are prone to different degrees of drug resistance,which affects the prognosis of patients.Therefore,it is of great clinical value to explore the drug resistance mechanism of esophageal cancer.DNA polymerase β(DNA pol β)belongs to the X family of DNA polymerases,and it’s mainly involved in base excision repair(BER)in vivo,which is an important pathway of DNA damage repair.DNA pol β is mutated in 30~40% of human tumors.Studies have shown that abnormal expression or mutation of proteins related to DNA damage repair system is closely related to drug resistance of tumors.Our research group previously found 9 kinds of DNA pol β variants in human ESCC,and these mutants had the specificity of esophageal cancer tissue.What are the effects of these mutants on chemotherapy for esophageal cancer? What is the guiding significance in clinical individualized therapy? These problems require in-depth analysis.In this study,DNA pol β variant R183 G was used as the research object to deeply analyze its effect on the sensitivity of esophageal cancer to chemotherapy and explore its mechanism.In the study of DNA pol β variant R183 G,the differentially expressed protein CHD1 L was detected by proteomics.CHD1L(chromodomain helicase/ATPase DNA binding protein 1-like gene),also known as ALC1(amplified in liver cancer 1),is an oncogene,which is highly expressed in many types of tumors and is involved in cell proliferation,migration,invasion,metastasis and tumorigenesis.CHD1 L is a chromatin remodelling enzyme and a DNA damage response protein.During the process of DNA damage repair,CHD1 L binds to DNA repair factors and relies on PARP1 to rapidly recruit to DNA damage sites to initiate a variety of DNA damage repair mechanisms.The chromatin remodeling and DNA repair of CHD1 L are interdependent.Abnormal function of CHD1 L will lead to its inability to be recruited to DNA damage sites or delay of repair kinetics,thus reducing the ability of DNA damage repair.Studies have shown that overexpression of CHD1 L can promote cisplatin resistance in non-small cell lung cancer cells,and CHD1 L knockout can increase the sensitivity of esophageal squamous cell carcinoma and non-small cell lung cancer cells to cisplatin.Therefore,the role of CHD1 L in the effect of DNA polβ variant R183 G on the sensitivity of esophageal cancer to chemotherapy needs further exploration.Based on it,this study is designed to explore the effect of DNA pol β variants on the sensitivity of esophageal cancer to chemotherapy in the molecular and cellular levels.We found DNA pol β variant R183 G decreased the sensitivity of human ESCC cells to chemotherapy drug.Through proteomics,we found that CHD1 L may be involved in the process of chemotherapy resistance caused by DNA pol β variant R183 G.It was further analyzed whether CHD1 L knockdown could reverse the resistance to esophageal cancer chemotherapy induced by DNA pol β variant R183 G,and its mechanism was preliminarily analyzed.This study provides an effective marker for individualized treatment and prognosis assessment of esophageal cancer.Methods 1.Bioinformatics was used to predict the functional changes of DNA pol β variants.2.To investigate the effect of DNA pol β mutation on the sensitivity of human esophageal cancer cells to chemotherapy DNA pol β expression levels in human ESCC cell lines and human immortalized esophageal epithelial cell line SHEE were detected by western blotting.Two human ESCC cell lines(Kyse150 and EC109)expressing low protein levels of DNA pol β were selected to establish stably overexpressing wild-type and mutant DNA pol β cell lines by lentivirus transfection.The effect of overexpressing wild-type and mutant DNA pol β on the growth ability of human ESCC cell lines was detected by cell proliferation assay.The effects of DNA pol β mutation on the sensitivity of human ESCC cell lines to chemotherapy(5-Fluorouracil(5-Fu)and cisplatin)and the ability of cell clone formation were detected by drug toxicity assay and soft agar assay.The effects of DNA pol β mutation on the sensitivity of human ESCC cell lines to DNA damage agent(Methyl Methanesulfonate(MMS))were detected by drug toxicity assay.The effects of DNA pol β mutation on DNA damage repair of human ESCC cell lines were detected by alkaline comet assay.3.Proteomic analysis was conducted to detect the differential proteins in the wild-type and variants human ESCC cell lines with DNA pol β overexpression after Methyl Methanesulfonate treatment.The key molecules involved in the effect of DNA pol β mutation on the sensitivity of human ECSS cells to chemotherapy were preliminarily identified by bioinformatics analysis,and the results was detected by western blotting.4.To investigate the role of CHD1 L in DNA pol β variant R183 G affecting the sensitivity of human ESCC cell lines to chemotherapy Human ESCC cell lines Kyse150 with CHD1 L stably knockdown were established by lentivirus transfection in ESCC cell lines Kyse150 overexpressing wild-type DNA pol β and variant R183 G.The effect of CHD1 L on the growth ability of overexpressing wild-type and mutant DNA pol β human ESCC cell lines was detected by cell proliferation assay.The effect of CHD1 L knockdown on the sensitivity to 5-Fluorouracil,cisplatin and Methyl Methanesulfonate of overexpressing wild-type DNA pol β and variant R183 G human ESCC cell lines was detected by drug toxicity assay,and the effect of CHD1 L knockdown on the clone formation was detected by soft agar assay.The effect of CHD1 L knockdown on cell cycle and DNA damage repair of overexpressing wild-type DNA pol β and variant R183 G human ESCC cell lines was detected by flow cytometry,immunofluorescence assay,western blotting and alkaline comet assay,to explore the mechanism of CHD1 L in DNA pol β variant R183 G mediatied chemotherapy drug resistance.Results 1.Bioinformatics prediction showed in both “open” and “close” states,the G179 R and R183 G variants of DNA pol β play a key role in binding DNA pol β to new d NTP during the BER,which may affect the function of DNA pol β.2.DNA pol β variant R183 G decreased the sensitivity of human ESCC cells lines to chemotherapy drugs(5-Fluorouracil and cisplatin),but increased the sensitivity to DNA damage agent(Methyl Methanesulfonate).The expression levels of DNA pol β in human ESCC cell lines Kyse70,Kyse150,Kyse450 and EC109 were lower than human immortalized esophageal epithelial cell line and other esophageal cancer cell lines,so Kyse150 and EC109 cell lines were selected for subsequent experiments.Human ESCC cell lines overexpressing wild-type and mutant DNA pol β were successfully established,and DNA pol β overexpressing wild-type and mutant DNA pol β had little effect on the growth of human ESCC cell lines.Both DNA pol β variants G179 R and R183 G decreased the sensitivity of human ECSS cells to 5-Fluorouracil,and DNA pol β variant R183 G decreased the sensitivity of human ECSS cells to cisplatin.Compared with the wild-type,DNA pol β variant R183 G could increase the ability of clone formation of human ECSS cells lines.Both DNA pol β variants G179 R and R183 G increased the sensitivity of human ECSS cells to Methyl Methanesulfonate to a certain extent.DNA pol β variants human ESCC cell lines were more damaged and have less DNA repair capacity treated with Methyl Methanesulfonate than the wild-type.3.CHD1 L was a key molecule involved in the effect of DNA pol β variants on the sensitivity of human ESCC cell lines to chemotherapy by proteomic analysis and western bloting.4.The mechanism of CHD1 L in the effect DNA pol β variant R183 G on the sensitivity of human ESCC cell lines to chemotherapy The stable knockdown of CHD1 L in overexpressing wild-type and mutant DNA pol β human ESCC cell lines were successfully established,and CHD1 L knockdown had little effect on the growth of overexpressing wild-type and mutant DNA pol β human ESCC cell lines.Compared with the control group,CHD1 L knockdown increased the sensitivity of DNA pol β overexpressing wild-type and and mutant DNA pol β human ESCC cell lines to 5-Fluorouracil,cisplatin and Methyl Methanesulfonate to different extent,decreased the ability of clone formation of DNA pol β variant R183 G human ESCC cell line,but CHD1 L knockdown had less effect on the cell cycle of DNA pol β variant R183 G human ESCC cell line.CHD1 L knockdown increased the content of γH2AX in DNA pol β variant R183 G human ESCC cell line after cisplatin treatment.DNA pol β variant R183 G human ESCC cell line with CHD1 L knockdown treated with Methyl Methanesulfonate was more damaged compared with the control group.Conclusion DNA pol β variant R183 G could decrease the sensitivity of human ESCC cells to chemotherapy drugs.CHD1 L decreased the sensitivity of DNA pol β variant R183 G human ESCC cells to chemotherapy drugs through its DNA damage repair. |
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