| Part Ⅰ The clinical correlation observation between TFAP2E methylation and the response to fluorouracil in gastric cancerObjective:To investigate the methylation level of transcription factor activator protein 2e(TFAP2E)in gastric cancer tissues and adjacent normal gastric tissues,and to study the relationship between TFAP2E methylation and the fluorouracil efficacy.Methods:The methylation rate of TFAP2E in 112 gastric cancer tissues and 76 normal gastric mucosa samples was measured by methylation sensitive high-resolution melting(MS-HRM)and compared the frequency of TFAP2E methylation in the two groups.The optimal cut-off value of TFAP2E methylation level was determined by receiver operating characteristic(ROC)curve,and the relationship between TFAP2E methylation status and clinicopathological characteristics of gastric cancer patients was analyzed.Among 112 cases of gastric cancer,36 cases were stage Ⅳ patients who received fluorouracil-based chemotherapy,and all of them had a clear evaluation of chemotherapy efficacy.Immunohistochemistry was performed on gastric cancer tissues of these patients to detect the expression of TFAP2E and analyze the correlation between the expression level and methylation status of TFAP2E and the fluorouracil efficacy.Results:1.The incidence of TFAP2E methylation was 96.4%(108/112)in gastric cancer tissues while 69.7%(53/76)in adjacent normal gastric mucosa tissues.The incidence of TFAP2E methylation in the former was significantly higher than that in the latter(x2=16.258,P<0.001);2.By using ROC curve and combining the specific values of TFAP2E methylation rate in gastric cancer tissue and its adjacent tissues,40.25%was determined as the optimal cut-off value of TFAP2E methylation rate,that is,methylation rate≥40%was determined as TFAP2E hypermethylation,while methylation rate<40%was determined as TFAP2E hypomethylation.3.The univariate correlation analysis showed that there was no correlation between TFAP2E methylation level and gender,age,clinical stage,lymph node involvement,degree of differentiation,lesion location and distant metastasis(P>0.05);4.In gastric cancer patients with stage IV treated with fluorouracil drugs,the chemotherapy response was not related to age,gender,differentiation degree,lesion location,clinical stage,lymph node involvement and distant metastasis(P>0.05),but related to TFAP2E methylation level(χ2=4.702;P=0.03).Further,binary logistic regression analysis was used to suggest that TFAP2E hypermethylation was associated with fluorouracil resistance in gastric cancer patients(OR=6.333,P=0.046);5.In gastric cancer tissues,TFAP2E methylation status was negatively correlated with TFAP2E protein expression level.Conclusion:The frequency of TFAP2E methylation in gastric cancer tissues was significantly higher than that in adjacent normal gastric mucosa tissues;In advanced gastric cancer tissues,TFAP2E hypermethylation is associated with fluorouracil resistance.Part Ⅱ The role of exosomal miRNAs in 5-fluorouracil resistance induced by TFAP2E hypermethylationObjective:To detect the methylation level of transcription factor activator protein2e(TFAP2E)in gastric cancer cells MGC803 and its drug-resistant cells MGC803/5-Fu,to analyze the expression difference of exosomal miRNAs between the two groups,and to explore the molecular mechanism of TFAP2E hypermethylation in gastric cancer cells to 5-fluorouracil resistance.Methods:The drug-resistant gastric cancer cells MGC803/5-Fu was constructed,and the methylation level of TFAP2E in gastric cancer cells MGC803 and MGC803/5-Fu were detected by methylation sensitive high-resolution melting(MS-HRM).The real-time fluorescent quantitative PCR and Western Blot were performed to detect the mRNA and protein expression of TFAP2E,respectively.The ultra-high speed centrifugation was used to extract exosomes from MGC803 and MGC803/5-Fu cells.The transmission electron microscopy was used to observe the morphology and size of exosomes,and the expression of exosomal markers was assessed by Western blot.The miRNAs sequencing technology was performed to detect miRNAs expression profiles of exosomes from gastric cancer cell line MGC803 and its drug-resistant cell line MGC803/5-Fu,and screen out differentially expressed miRNAs.Exosomes from MGC803/5-Fu cells(5-Fu/exo)were extracted and labeled with PKH26 row staining,the uptake process of MGC803 was observed by laser confocal microscopy.After successful uptake,CCK8 assay was used to determine the sensitivity changes of MGC803 to 5-Fu,and qRT-PCR was performed to detect the expression changes of differential miRNAs.In addition,after transfection of TFAP2E into drug-resistant cells MGC803/5-Fu,the changes of TFAP2E methylation level and expression,the changes of differential miRNAs expression and the alterations of 5-Fu sensitivity were compared before and after transfection.Results:1.Compared with MGC803 cells,the methylation level of TFAP2E in MGC803/5-Fu cells was higher,the expression level of TFAP2E was lower(P<0.05),and MGC803 cells were more sensitive to 5-Fu than MGC803/5-Fu cells(P<0.05);2.Exosomes were 30-100 nm in size and exhibited a bilayer membrane structure under the transmission electron microscopy,and the expression of exosomal specific protein was significantly increased(P<0.01);3.The miRNAs sequencing results showed that miR-106a-5p and miR421 were most significantly differentially expressed,and the expression levels of the above two miRNAs in MGC803/5-Fu were 38.433-fold and 24.133-fold higher than in MGC803,respectively;4.After the successful uptake of MGC803/5-Fu exosome(5-Fu/Exo)by MGC803,the expression levels of miR-106a-5p and miR-421 were significantly upregulated compared with that before uptake(P<0.01,P<0.01),but the sensitivity of 5-Fu was significantly decreased compared with that before uptake(P<0.01).There were no differences in TFAP2E methylation status and expression of TFAP2E(P>0.05);5.After MGC803/5-Fu cells were successfully transfected with TFAP2E,the expression levels of TFAP2E mRNA and protein were significantly higher than those before transfection(P<0.01,P<0.01),the expression levels of miR-106a-5p and miR-421 were significantly decreased(P<0.01,P<0.01),and 5-Fu sensitivity was higher than that before transfection(P<0.01),but TFAP2E methylation status had no significant change.Conclusion:TFAP2E hypermethylation is associated with 5-Fu resistance in gastric cancer cells.Hypermethylation of TFAP2E leads to the down regulation of its protein level,which up regulates the expression levels of miR-106a-5p and miR-421 in exosomes,and ultimately leads to the 5-Fu resistance of gastric cancer cells.PartⅢ The mechanism study of exosomal miR-106a-5p/EGR2 regulating TFAP2E hypermethylation associated fluorouracil resistance in gastric cancerObjective:To study the expression of miR-106a-5p in exosomes from gastric cancer cells and serum exosomes from gastric cancer patients,and to further explore the possible molecular mechanism of fluorouracil resistance induced by TFAP2E hypermethylation through the prediction of its downstream target genes.Methods:Serum samples from gastric cancer patients with stage Ⅳ were collected before the first chemotherapy,and serum exosomes were extracted.The transmission electron microscopy was used to identify the morphology and size of exosomes,and Western Blot was performed to detect the expression of serum exosomal specific proteins.The miRNAs expression profiles of serum exosomes in the chemosensitive group and chemoresistant group of gastric cancer patients were detected by miRNAs sequencing technology,and differentially expressed miRNAs were selected and intersected with the miRNAs selected from gastric cancer cells,and qRT-PCR was used to verify the intersected miRNAs.The downstream target genes of the verified miRNAs were predicted by Bioinformatics tools.In addition,GO and KEGG pathway analysis were performed.The protein interaction analysis was performed on TFAP2E,and the genes corresponding to the interaction proteins were intersected with the target genes predicted by the above miRNAs,and downstream targets with interaction relationship with TFAP2E were screened out and verified in gastric cancer tissues and cells by qRT-PCR and Western Blot.The expression of miR-I06a-5p in serum and EGR2 in gastric cancer tissues were detected by qRT-PCR,and the correlation among miR-106a-5p expression,EGR2 expression and TFAP2E methylation rate was analyzed.Results:1.Under the transmission electron microscopy,serum exosomes were vesicular,about 50-150 nm in diameter,with high electron density at the edge of vesicles and obvious granularity.The expression of exosomal specific proteins was significantly increased(P<0.01).2.The miRNAs sequencing analysis was performed on exosomes extracted from serum samples:a total of 692 miRNAs with>2-fold differential were screened,among which,468 were upregulated miRNAs,224 were downregulated miRNAs.The 468 upregulated miRNAs were intersected with the differentially upregulated miRNAs screened in the second part of this study,and the differentially upregulated 6 miRNAs in both the serum exosomes from chemoresistant patients and the exosomes from drug-resistant cells were screened out:miR-296-5p,miR-200C-3p,miR-202-3p,miR-421,miR-133a-5p,and miR-106-5p.3.The downstream target genes predicted from the above 6 miRNAs were analyzed by GO and KEGG pathways,suggesting that the target genes were enriched in cancer-related pathways.4.The results of the TFAP2E protein interaction analysis were intersected with the results of the above six miRNAs target genes,suggesting that FOXJ3,EGR2 and E2F3 may interact with TFAP2E,and that EGR2 and E2F3 were all downstream target genes of miR-106a-5p.5.The mRNA and protein expression of EGR2 in the tissues of chemoresistant patients were significantly lower than those of chemosensitive patients(P<0.05,P<0.05,respectively),whereas there was no differance of E2F3 expression between chemoresistant and chemosensitive tissues(P>0.05),and both the expression of EGR2 and E2F3 in MGC803/5-Fu cells were significantly lower than those in MGC803 cells(P<0.01).6.The expression of miR-106a-5p,EGR2 and the methylation rate of TFAP2E were statistically analyzed:the contour map showed that the extremely low expression of EGR2 was located in the region with high methylation rate of TFAP2E and high miR-106a-5p expression,and the extremely high expression of EGR2 was located in the region with low methylation rate of TFAP2E and low miR-106a-5p expression.The matrix showed that miR106a-5p was more correlated with EGR2 expression.The drug-sensitive group and drugresistant group were shown by scatter plot:the expression level of miR-106a-5p was positively correlated with the methylation rate of TFAP2E in both groups,and the expression level of miR-106a-5p was negatively correlated with the expression level of EGR2 in both groups.The results of bubble plot showed that the expression of EGR2 in drug-resistant group was significantly lower than that in sensitive group.Conclusion:TFAP2E hypermethylation may lead to fluorouracil drugs resistance in gastric cancer by up regulating miR-106a-5p and inhibiting the expression of target gene EGR2... |
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