| Background:Esophageal cancer is a common malignant tumor in the digestive system,and my country is also an area with a high incidence of esophageal cancer.Esophageal cancer mainly has two subtypes:ESCC and EAC.The United States and European countries are mainly EAC,and more than 90%of esophageal cancer patients in my country are ESCC.Currently,surgery,radiotherapy and chemotherapy are the three conventional treatments for ESCC.Among them,surgery is the primary treatment for patients with localized lesions in the early stage.However,due to the occult onset of esophageal cancer,the early clinical symptoms are not obvious,many patients lose the best chance of surgery when they are found in the middle and late stage.Radiotherapy,as one of the main means of local treatment for esophageal cancer,plays an irreplaceable role in cervical esophageal cancer and non-surgical esophageal cancer,but has limited effect on patients with long lesion length and recurrence after radiotherapy.Chemotherapy and targeted drug therapy are the main means to prolong the survival time of patients with advanced or postoperative esophageal cancer,but their effect is limited by the drug resistance of tumor.Systematic analysis of the results of several randomized controlled trials shows that the response rate of advanced esophageal cancer to standard chemotherapy is only about 30%,and the targeted drug sorafenib can only prolong the survival of patients for 3 months.Therefore,to overcome the drug resistance of tumor cells to chemotherapy drugs is of great significance to improve the clinical efficacy of esophageal cancer.There are two forms of drug resistance in tumor cells,including single drug resistance and multi drug resistance,most of which are multi drug resistance.Drug resistance is mainly related to the changes of intracellular drug concentration and distribution,the activation of intracellular antioxidant system,DNA damage repair system and extracellular matrix.Verapamil(VER)is an L-type calcium channel inhibitor,which is mainly used in the treatment of cardiovascular and cerebrovascular diseases.However,more and more studies have shown that it is a drug that can reverse multidrug resistance of tumor cells.Ver has been found to reduce the drug resistance of tumor by changing the intracellular drug concentration,with the effective concentration of 6.0-10.0 μM.However,intravenous administration not only can not effectively reverse the drug resistance concentration,but also may lead to cardiovascular side effects(the safe concentration in peripheral venous blood is 1.0-2.0 μM).The concentration in local tissue of intra-arterial infusion of drugs can reach 3-10 times of that in peripheral blood.Studies have shown that VER can reverse the MDR of tumors and can improve the clinical efficacy of chemotherapy including hepatocellular carcinoma,colorectal cancer,gastric cancer,lung cancer and malignant ascites.Initial studies have shown that VER is anti-tumor MDR by regulating the expression of P-gp protein.VER can inhibit or down-regulate the expression of P-gp,prevent drug efflux and improve the killing effect of tumor chemotherapy drugs.Recent studies have shown that the mechanism of VER reversing tumor multidrug resistance is likely to have nothing to do with P-gp.Our previous high-throughput screening experiments showed that the expression of SLIT3,KCNMA1,KLK1,LPAR1,CHRDL1 and NID1 gene were related to the reversal of drug resistance by verapamil.In particular,KCNMA1 gene expression was significantly increased in ver reversing cisplatin resistance of ESCC cells.This topic is mainly to study the relationship between KCNMA1 gene expression and VER reversing tumor multidrug resistance in vitro and vivo.Part 1:The role of KCNMA1 expression in VER reversing the drug resistance of ESCC cellsObjective:In this study,by studying the expression difference of KCNMA1 gene in ESCC tissues and cells,to study the possible relationship between the expression difference of KCNMA1 gene and VER reversing ESCC tumor resistance,and to provide a theoretical basis for targeted therapy of ESCC patients with tumor resistance.Based on these experiments,we tested the expression of KCNMA1 gene in ESCC;overexpression or silencing of KCNMA1 gene to study the sensitivity of tumor cells to cisplatin;and the role of KCNMA1 gene expression in tumor cell proliferation and apoptosis.Through these experiments,we have a deeper understanding of the mechanism of VER reversing tumor MDR,and provide clinical evidence for the treatment of ESCC patients.Method:1.CCK-8 and colony formation experiment detects the effect of VER combined with cisplatin on the viability of KYSE150,KYSE180 and KYSE450 three ESCC cells;2.High-throughput screening and sequencing,comparing the difference in gene expression between the DPP group and the VER+DPP group in KYSE150 and KYSE180,and verifying this difference with qRT-PCR;3.WB detects the expression of KCNMA1 protein in the VER group,DPP group,and DPP+DPP group;4.Immunohistochemical method to detect the protein expression of KCNMA1 in tissue samples of ESCC patients treated with VER combined with cisplatin;5.Detect the cell viability of ESCC cells against cisplatin by silencing or over-expression of KCNMA1 gene expression,and then using CCK-8 method;6.Through anti-proliferation experiments and apoptosis experiments,study the relationship between the expression of KCNMA1 gene and ESCC cell proliferation and apoptosis.Result:1.CCK-8 and colony formation experiment results show that VER increases the sensitivity of ESCC cells to cisplatin,especially to KYSE150 cells,while VER has little effect on KYSE180 cells.Therefore,in this experiment,KYSE150 cells were selected as VER reversal sensitive cells,and KYSE180 cells were selected as anti-reversal model cells for further research;2.The results of high-throughput transcription sequencing showed that the gene expression of SLIT3,KCNMA1,NID1,KLK1,LPAR1 and CHRDL1 all showed differences,and this difference was verified by qRT-PCR.Compared with the KYSE150 DDP group,the KCNMA1 expression in the KYSE150 DDP+VER group was significantly up-regulated,and the expression level of KCNMA1 in KYSE150 cells was higher than that in KYSE180 cells;3.WB results showed that between the KYSE180 DDP group and the KYSE180 DDP+VER group,the protein expression of KCNMA1 was not significantly different,while in the KYSE150 DDP+VER group,KCNMA1 was significantly up-regulated;4.The results of immunohistochemistry showed that the expression of KCNMA1 was significantly increased in patients with positive VER reversal protocol,but there was no significant difference in the expression of KCNMA1 in negative patients;5.Overexpression of KCNMA1 gene can significantly increase the sensitivity of KYSE150 cells to cisplatin,while silencing the expression of KCNMA1 gene reduces the sensitivity of KYSE150 cells to cisplatin;6.The results of proliferation and apoptosis experiments show that the expression of KCNMA1 can enhance the anti-proliferation effect of VER on ESCC cells,and can also enhance the pro-apoptotic effect of VER on ESCC cells.Conclusion:VER can promote the expression of KCNMA1 and enhance the sensitivity of KYSE150 cells to cisplatin.The mechanism may be that the expression of KCNMA1 can enhance the anti-proliferation and pro-apoptosis effects of cisplatin on KYSE150 cells.Part 2: The expression of KCNMA1 promotes the inhibitory effect of cisplatin on transplanted tumor in nude miceObjective:This study aimed to analyze the correlation between the expression of KCNMA1 in nude mice with esophageal squamous cell transplantation tumors and the reversal of tumor sensitivity to cisplatin by VER? and to explore the role of KCNMA1 in the treatment of ESCC.Method:1.Construct overexpression and silence KCNMA1 KYSE150 cell model,2.Inject ESCC cells subcutaneously into nude mice.The esophageal squamous cell carcinoma model was constructed and divided into five groups including Control group,DPP group,DPP+VER group,siRNA-KCNMAl group and Over-KCNMAl+DPP group.When the tumor volume grows to 50mm2,intraperitoneal injection of 2.0mg/kg cisplatin and l.Omg/kg VER,compare the tumor size and tumor inhibition rate of each group of nude mice;3.Comparison of the expression of KCNMA1 protein in the tissues of nude mice in each group by immunohistochemical experiment;4.WB and qRT-PCR experiments to detect the expression of KCNMA1 in the three groups of nude mice;Result:1.KCNMA1 overexpression and expression silenced KYSE150 cell beads were prepared by plasmid transfection and lentivirus transfection.The results of qRT-PCR and WB showed that the expression of KCNMA1 protein in Over-KCNMAl group was significantly higher than that in vector group,while siRNA-KCNMAl The expression of KCNMA1 protein in the group was significantly lower than that in the sh-NC group,indicating that the overexpression and silent expression of KCNMA1 cell models were successfully constructed;2.In this experiment,all nude mice developed tumors,and the tumor volume of nude mice in each group increased with time during the treatment period.The tumor volume of the Over-KCNMAl+DPP group and the DPP+VER group was obvious after14 days.It is smaller than that in the DPP group alone,and after silencing the expression of KCNMA1,the tumor tissue volume increases significantly;3.The results of WB and qRT-PCR in tumor tissues of nude mice showed that the expression of KCNMA1 protein in Over-KCNMAl group and DPP+VER group was significantly higher than that in Control group and DPP group.Conclusion:Overexpression of KCNMA1 can increase the sensitivity of esophageal squamous cell carcinoma tissue to cisplatin and inhibit the growth of tumor tissue volume;while silencing the expression of KCNMA1 reduces the inhibitory effect of cisplatin on tumors in nude mice. |
PDF Full Text Request