| Prostate cancer(PCa)has become the second leading cause of death and malignancy in men worldwide.Prostate cancer is also one of the most common malignant tumors in developed countries.The incidence of prostate cancer in China’s mainland in recent years Rose sharply.Prostate cancer diseases have higher morbidity and mortality.Once the lymph node or(and)other tissues and organs metastasize to the prostate cancer disease,the patient’s prognosis is poor,and the patient’s overall survival period is very short.In today’s world,there are many mature treatments for prostate cancer,such as waiting for observation and active monitoring,surgical resection,radiotherapy,endocrine therapy,and chemotherapy.Androgen deprivation therapy(Androgen deprivation therapy,ADT)method is one of the most effective methods for the treatment of prostate cancer,but many patients will develop drug resistance during treatment,and eventually disease progression due to biochemical recurrence will lead to castration resistance The occurrence of Castration-resistant prostate cancer(CRPC)disease.At present,there are two main clinical treatment methods for castrationresistant prostate cancer: one is chemotherapy and the other is new endocrine therapy.The main drugs of the new endocrine therapy are enzalutamide and abiraterone.Although this treatment can effectively prolong the survival time of patients,it is not uncommon for primary resistance to treatment: Abiraterone and enzalutamide account for approximately one-third and four of all patients,respectively.One in ten,and drugs that are initially sensitive to CRPC treatment all develop secondary resistance within 24 months.Next,this article will describe the process,functional and molecular biological level verification tests for establishing castration-resistant prostate cancer enzalutamide-resistant cell lines,and the whole exome sequencing results for parental and drug-resistant cells.ObjectiveIn this project,the C4-2 cell line was used to establish a model of castrationresistant prostate cancer enzalutamide-resistant cell line(hereinafter referred to as drug-resistant cells),and the whole exome sequencing was performed to analyze various mutations in the cells.After screening out known related mutations,mutations related to drug resistance are obtained.By measuring IC50 and detecting cell survival and growth(MTT),it proved that drug-resistant cells were successfully established.In vitro functional experiments were used to observe the proliferation,invasion and migration ability of drug-resistant cells,and molecular biology experiments to understand the expression of drug-resistant related genes and proteins.Through whole exome sequencing,we obtained the results of three different mutations in drug-resistant cells,screened out mutations that may be related to drug resistance,and provided new methods and methods for clinical treatment of patients with castration-resistant prostate cancer resistance Ideas.MethodIn this study,the castration-resistant prostate cancer(C4-2)cell line cryopreserved at-80 ° C was recovered from the sex hormone laboratory of the Second Hospital of Tianjin Medical University.After multiple fluid changes and passages,stable growth of prostatic prostate cancer cells was obtained.Cells in good condition and similar numbers were selected for grouping treatment.The experimental group was given a continuous culture of 10 microliters per liter,and the control group was given DMSO with the corresponding concentration for cultivation and culture.During the period,the cells were changed and passaged to observe their growth.When the growth rate of the cells in the experimental group is similar to that in the control group,some cells in the two groups are selected for MTT experiments to quantify the survival and growth of the cells.Amplify and freeze the cell lines of the experimental and control groups that can be stably inherited.The cells of the remaining experimental group were cultured at 20 microliters per liter,and the control group was given DMSO culture and culture at the corresponding concentration,and the above operation was repeated until the drug rose to 40 microliters per liter.The successful establishment of drug-resistant cell lines was confirmed by detecting the IC50 of cells in the experimental group and the control group.In vitro experiments through cloning formation experiment,Transwell experiment and scratch experiment,respectively observe the difference in the proliferative ability,the invasion ability and the migration ability of parental cells and drug-resistant cells.RT-q PCR was used to detect the difference in the expression of related resistance genes(AR,AR-V7,Malat1 and CYTOR)in parental and resistant cells.Western Blot experiments were used to verify the difference in expression of resistance-related proteins(AR,AR-V7)in parental and resistant cells.Whole exome sequencing,using probe hybrid capture technology to capture and enrich the DNA sequence of the exon region of the genome,and then perform high-throughput sequencing genome analysis.In this way,related mutation results of parental cells and drug-resistant cells are obtained.ResultsThe method of increasing the concentration gradient successfully induced the castration-resistant prostate cancer enzalutamide-resistant cell line.Under light microscopy,compared with the parental cells,the drug-resistant cell line had increased particulate matter in the cytoplasm and the surrounding cells The antennae increased significantly,and the cells showed island-like growth characteristics.It can be seen from the MTT experiment that drug-resistant cell lines can stably proliferate in common medium and enzalutamide medium with a concentration of 40 micromoles per liter,and the proliferation rate is similar,while the parental cells can be stable in common medium Proliferation,but proliferation was inhibited in enzalutamide medium at a concentration of 10 micromoles per liter.The IC50 measurement results show that the parental and drug-resistant cells have significant differences in the drug sensitivity to enzalutamide.The IC50 of the parental and drug-resistant cells to enzalutamide is 62.71 micromoles per liter and 17.10 micromoles,respectively.Each liter,the resistance of drug-resistant cells is about 3.667 times that of parent cells.It was confirmed that the castration-resistant prostate cancer enzalutamide-resistant cell line had the characteristics of low resistance to enzalutamide.Cell clone formation experiments confirmed that the proliferation capacity of drug-resistant cells was significantly higher than that of parent cells.Cell Transwell-Invsion experiments confirmed that the invasion ability of drug-resistant cells was enhanced,which was significantly higher than that of parent cells.Cell scratch healing experiments confirmed that the migration ability of drug-resistant cells was significantly higher than that of parent cells.The q PCR results showed that the expression levels of resistancerelated genes AR-FL,AR-V7,Malat1,and CYTOR were significantly increased.WB results showed that with the increase of drug stimulation concentration,AR and ARV7 protein expression in drug-resistant cells gradually increased.ConclusionsThis experiment successfully established an enzalutamide-resistant cell line of castration-resistant prostate cancer,and successfully verified the biological function and molecular biological changes of drug-resistant cells.This drug-resistant cell line can simulate the tumor characteristics of CRPC patients who are clinically resistant to enzalutamide.The whole exome sequencing results found mutations that may be related to drug resistance,and this part still needs subsequent verification. |
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