Resistance Of MiR193 To Esophageal Cancer Cells In Drug-resistant Exosomes And Its Mechanism

IntroductionEsophageal cancer is the eighth most common cancer and one of the most common causes of cancer-related deaths in the world.According to the survey results,Chinese esophageal cancer patients account for more than half of the total number of patients worldwide.Both male and female mortality rates rank first in the world.Treatment options for esophageal cancer include surgery,chemotherapy,and radiation therapy.However,the emergence of drug resistance in esophageal cancer cells is a decisive factor hindering the therapeutic effect.The mechanisms of tumor resistance include:(1)drug efflux,reducing the concentration of drugs in tumor cells.(2)Apoptosis arrest.(3)DNA damage repair response.In tumor cells,the development of drug resistance may be played by one or several mechanisms,and the mechanism of action in different tumor cells is different.Exosomes are nanoscale vesicles with lipid bimolecular membranes that are present in all body fluids.Exosomes secreted by tumor cells are involved in various cellular functions and physiological and pathological events.Exosomes include mRNA,miRNA,proteins,etc.,all of which are biologically active and capable of performing a functional response in target cells.The role of exosome content miRNAs in chemoresistance of esophageal cancer and its related mechanisms are still unclear.In this study,esophageal cancer cisplatin-resistant cell line TE1/DDP was constructed by esophageal cancer cell line TE1.To investigate the effect of exosomes secreted by esophageal cancer cell line TE1/DDP on the sensitivity of cell line TE1 to cisplatin.High-throughput sequencing of miRNAs in the exosomes and transcriptomes of the treated cells.miRNA and mRNA association analysis was performed using bioinformatics software to find miRNAs associated with cisplatin chemoresistance.To study the effects of this miRNA on the biological function(proliferation,apoptosis,clonality,invasion ability,etc.)of esophageal cancer cells,predict related target genes,and analyze the mechanism of its involvement in cisplatin chemotherapy resistance of esophageal cancer.Part ? Cisplatin-resistance effect of cisplatin-resistant exosomes on drug-sensitive cellsPurposeThe cisplatin-resistant cell line of esophageal cancer was constructed to study the effect of exosome of cisplatin-resistant cell line on cisplatin-sensitive cell line.Methods1.The esophageal cancer cisplatin-sensitive cell line TE1 was treated with the lowest tolerated concentration of cisplatin until the cell line was resistant to cisplatin and designated as TEI/DDP cell line.2.Cell viability and apoptosis of TEI/DDP cell line under the stress of minimum cisplatin tolerance were detected by CCK-8 kit and flow cytometry.3.Exosomes were extracted by ultracentrifugation,and the extracted exosomes were identified by transmission electron microscopy and Western blot.4.The effect of exosome from drug-resistant cell lines on the tolerance of cisplatin after interfering with sensitive cells was examined by CCK-8.Results1.The results of CCK-8 kit and flow cytometry showed that the activity of TE1/DDP cell line was significantly higher than that of TE1 cell line under the pressure of IC50 concentration(0.7 ?g/mL)cisplatin(P<0.001),and the apoptosis was significantly decreased.This indicates that the cisplatin-resistant cell line TE1/DDP of esophageal cancer was successfully constructed.2.The morphology of esophageal cancer exosomes can be seen under the transmission electron microscope field of view.The esophageal cancer exosomes are typically circular or elliptical cup-like structures with a size of 50-200 nm.Western blot analysis showed that CD63 protein could be detected in both TE1/DDP cell line and TE1 cell line.3.The CCK-8 results showed that the tolerance of TE1 cells to DDP was significantly increased after the addition of TE1/DDP exosomes(concentration:5.32×109/mL)(P<0.05).SummaryThe cisplatin-resistant cell line TE1/DDP of esophageal cancer was successfully constructed,and its exosomes can increase the resistance of sensitive cells to cisplatin.Part ? Screening of exosome miRNAs and target genes affecting sensitive cell resistancePurposeThe transcriptomes of miRNAs and treated cells in exosomes of esophageal cancer cisplatin-resistant cell line TE1/DDP and cisplatin-sensitive cell line TE1 were subjected to deep sequencing analysis by high-throughput sequencing technology.Look for miRNAs and mRNAs associated with cisplatin resistance in esophageal cancer.To study its relationship with the survival rate of patients with esophageal cancer.Methods1.Exosomes were extracted by ultracentrifugation.Total RNA extraction kit extracts total RNA from exosomes and constructs an RNA sequencing library for high-throughput sequencing.The total RNA was extracted to construct a transcriptome library for transcriptome sequencing.2.MiRNAs with significant differences in expression were selected from the sequencing data for real-time PCR.3.Bioinformatics software was used to analyze miRNAs and mRNAs.MiRNAs associated with cisplatin chemotherapy resistance in esophageal cancer cells are predicted.Results1.A total of 3182 miRNAs were co-targeted to 69,540 genes in the two exosomes.Among them,973 miRNAs were targeted to 67,631 genes in the TE1 group,and 561 miRNAs were targeted to 65,885 genes in the TE1/DDP group.2.Statistical analysis of differential expression showed that there were 493 differential miRNAs in the TE1 group and the TE1/DDP group,of which 189 belonged to up-regulated miRNAs and 304 belonged to down-regulated miRNAs.3.There were 5155 differential genes in the TE1 group and the TE1/DDP group,of which 3446 genes were up-regulated and 1709 genes were down-regulated.4.Correlation analysis of mRNAs and miRNAs by bioinformatics software showed that ESR1 gene and TP53 gene were located at the core of the association analysis network and were related to TFAP2C gene.5.The results of real-time PCR showed that the expression of miRNAs in esophageal cancer was consistent with the expression of the sequencing data.6.Survival curve analysis showed that the overall survival rate of patients with high expression of TFAP2C was higher than that of patients with low expression of TFAP2C.The overall survival rate of esophageal cancer patients with high expression of miR193 was lower than that of patients with low expression of miR193.SummaryAssociation analysis of miRNAs with mRNAs revealed that the TFAP2C gene is associated with chemoresistance in esophageal cancer.miR193 and TFAP2C are associated with survival in patients with esophageal cancer.Part III Mechanism of miR193 involved in cisplatin chemotherapy resistance in esophageal cancerPurposeThe expression of TFAP2C and miR193 in different esophageal cancer cell lines was examined.The expression of resistance-related genes in different esophageal cancer cells was examined.Verify the binding of TFAP2C and miR193.TFAP2C overexpression and miR193 overexpressing esophageal cancer cell lines were constructed.The effects of changes in the expression levels of TFAP2C and miR193 on proliferation,invasion ability,colony forming ability and apoptosis of esophageal cancer cells were studied.Methods1.The expression of TFAP2C and miR193 in different esophageal cancer cells and exosomes was detected by real-time PCR and Western blot.2.Targeted binding of TFAP2C and miR1 93 was detected using a dual-luciferase reporter gene.3.TFAP2C overexpression and miR 193 overexpressing esophageal cancer cell lines were constructed using lentiviral vectors.4.EdU cell proliferation assay,flow cytometry,colony formation assay and Transwell assay were used to investigate the effects of TFAP2C and miR193 expression on the proliferation,cycle,invasion ability,clonality and apoptosis of esophageal cancer cells.5.The expression levels of cell cycle and apoptosis-related proteins were detected by Western blot.6.The TFAP2C and P21 gene promoters were tested for binding by ChIP assay.Results1.The results of real-time PCR showed that the relative expression level of miR193 in the exosomes of TE1 cell line was significantly lower than that in the exosome of TE1/DDP cell line,P<0.001.The relative expression level of miR193 in TE1 cells was significantly lower than that in TE1/DDP cells(P<0.001).The relative expression level of miR193 in TE1 cells(TE1/E)added to the exosomes of TE1/DDP cell line was significantly higher than that in TE1 cells,P<0.01.2.The relative expression level of TFAP2C in TE1/DDP cell line was significantly lower than that in TE1 cell line(P<0.001).The relative expression level of TFAP2C in TE1 cells added to the exosomes of TE1/DDP cell line was significantly lower than that in TE1 cells(P<0.01).3.The dual-luciferase reporter gene assay showed that miR193 binds to the 3'-UTR region of the TFAP2C gene,suggesting that TFAP2C is the target gene of miR193.4.The results of cell cycle assay showed that the proportion of TE1/DDP cell line in the G0/G1 phase of the cell cycle was smaller than that of the TE1 cell line.Exosomes of TE1/DDP cell line can partially block the blockade of cisplatin on esophageal cancer cell cycle.The proportion of miR193 overexpressing cell lines in the G0/G1 phase of the cell cycle was lower than that of the miR193 overexpression control group.The proportion of TFAP2C overexpressing cell lines in the G0/G1 phase of the cell cycle was greater than that in the TFAP2C overexpression control group.5.The relative expression level of TFAP2C,TP53 and P21 gene were lower in TE1/DDP cell lines,and the relative expression levels of EGFR and Cyclin D1 genes were higher.The TE1/DDP cell line exosomes can change the relative expression levels of these genes in TE1 cell lines.6.The results of EdU proliferation assay showed that the number of cells proliferating in TE1/DDP cells was significantly higher than that in TE1 cells under cisplatin pressure(P<0.01).The number of cells proliferating in TFAP2C overexpressing cell lines was significantly smaller than that in TFAP2C overexpression control group(P<0.01).The number of cells proliferating in miR193 overexpressing cell line was significantly higher than that in miR193 overexpression control group(P<0.01).7.The results of colony formation assay showed that the number of clones of TE1/DDP cell line was significantly increased compared with the number of clones formed by TE1 cell line(P<0.01).Compared with the TFAP2C overexpressing control group,the number of clone formation in the TFAP2C overexpressing group was significantly decreased(P<0.05).Compared with the miR193 overexpressing control group,the number of clones in the miR193 overexpressing group was significantly increased(P<0.01).8.Apoptosis experiments showed that the apoptosis rate of TFAP2C overexpressing cells was significantly higher than that of TFAP2C overexpressing control cells.Compared with miR193 overexpressing control cells,the apoptotic rate of miR193 overexpressing cells was significantly reduced.The apoptosis rate of miR193 overexpression group under cisplatin pressure was significantly higher than that of miR193 overexpression group under normal conditions.9.The results of cell invasion assay showed that the changes of TFAP2C and miR193 expression had no significant effect on the invasive ability of esophageal cancer cells.10.The sequence of the P21 promoter region was amplified by amplification in the DNA obtained from the ChIP experiment,and thus it was found that TFAP2C can bind to P21 to promote cell cycle arrest.Summary1.TFAP2C is the target gene of miR193.2.Overexpression of TFAP2C can significantly inhibit the proliferation,cycle,and colony formation of esophageal cancer cells and promote apoptosis.3.TFAP2C is able to bind to P21 to promote cell cycle arrest miR193 is able to modulate P21 by TFAP2C to relieve cell cycle arrest.Part ? Tumor-bearing assay in nude mice verify miR193 promotes drug resistance in esophageal cancer cellsPurposeThe effect of miR193 on esophageal cancer tumors was examined in nude mice.Methods1.The esophageal cancer cell suspension was injected into the subgingival tissue of both sides of the nude mouse,and the growth of the nodule inoculated with the tumor was observed every day,and the growth of the transplanted tumor at 1 week,2 weeks,3 weeks,and 4 weeks after the inoculation was detected.2.All experimental animals were implanted on both sides.Firstly,according to the difference of left tumor cells,they were divided into three groups.TE1 cells were uniformly inoculated on the right side,and TE1,TE1/DDP and TE1/miRNA193 were inoculated on the left side.After the obvious nodules were touched on both sides,each group of animals was randomly divided into two subgroups for cisplatin intervention,and treated with normal saline as a control.3.After 4 weeks of growth,the nude mice were sacrificed by cervical dislocation,and the tumor entities were separated,photographed,weighed,and the volume was measured.The tumor growth curve was drawn.Results1.The tumor-bearing experiment results in nude mice showed that the tumor volume of the tumor formed by TE1 cell strain under cisplatin pressure was significantly smaller than that under the control condition(P<0.05).2.The tumor volume formed by TE1/DDP cell line under cisplatin pressure was significantly increased compared with the normal control group(P<0.05).3.The tumor volume formed by miR193 overexpressing TE1 cell line under cisplatin pressure was significantly smaller than that of normal control group(P<0.05).4.Under the intervention of cisplatin,the tumor size of TE1/DDP cell line was significantly larger than that of TE1 cell line.The tumor size of miR193 overexpressing TE1 cell line was significantly larger than that of TE1 cell line.SummaryCisplatin can inhibit the growth of esophageal cancer tumors,and overexpression of miR193 can promote the growth of esophageal cancer tumors.Conclusion1.Drug-resistant exosomes can increase the resistance of sensitive cells TE1 to cisplatin.2.TFAP2C is the target gene of miR193.3.The exosomes of TE1/DDP cell line can relieve the cell cycle arrest of cisplatin on esophageal cancer.The mechanism is that TFAP2C,a target gene of miRNA193,is a transcriptional regulator of P21.Cisplatin can inhibit the growth of esophageal cancer tumors.Overexpression of miR193 promotes the growth of esophageal cancer tumors and increases the resistance to cisplatin.