| ObjectiveAnalysis of the circular RNA transcriptome in the grade 3 endometrial cancer,analysis and mining the grade 3 endometrial cancer adjacent tissues and cancer tissues differentially expressed circ RNAs And related downstream targets,providing a potential biological treatment markers for the grade 3 endometrial cancer.and to clarify the development mechanism of the grade 3 endometrial cancer to provide new ideas.Methods1.Use circRNAs Microarray hybridization sequencing two grade 3 endometrial adenocarcinoma carcinoma adjacent tissues and cancer tissues of circ RNAs transcriptome analysis,and looking for differential circ RNAs(includes 4 circ RNAs with the most significant difference in up-regulation and significantly associated with the tumor pathway,3 circ RNAs with the most significant difference in down-regulation and significantly associated with the tumor pathway).2.The above 7 circ RNAs were detected by RTq PCR in 30 cases of endometrioid adenocarcinoma and adjacent tissues(including 15 cases of G3 EEC cancer tissues and adjacent tissues and 15 cases of the grade 2 endometrioid endometrial carcinoma or the grade 1 endometrioid endometrial carcinoma cancer tissues and adjacent tissues),and the results were consistent with the sequencing results.The above 7 circ RNAs were detected by RTq PCR in the G3 EEC cell line Ishikawa(ISK)and G1 EEC cell line KLE cells.Select the circ RNA with the same sequence and the most obvious difference as the target circ RNA for further analysis,and analyze thecorrelation between the circRNA and the clinical pathological parameters of 30 patients.3.Target micro RNAs: To further explore the relationship between micro RNAs and m RNAs downstream of this circ RNA,Based on mi Randa,Target Scan,Pic Tar Database,We predict micro RNAs that can match circ RNAs sequences,KEGG analysis of micro RNAs,screening for the most significant micro RNAs in the tumor pathway and validation.Spearman correlation analysis was performed between circ RNA and micro RNAs in the above G3 EEC and G1/G2 EEC.4.Target m RNAs: Based on mi Rmap,micro T,mi Randa,Pic Tar and Target Scan databases,meaningful micro RNAs were targeted to predict m RNAs,and RTq PCR was used to verify predicted m RNAs at the above cell and tissue levels.Spearman correlation analysis was performed between micro RNAs and m RNAs in the above G3 EEC and G1/G2 EEC.5.To further investigate the role of CCL22 and CCR4 in G3 EEC,immunohistochemical staining was used to detect the expression of CCL22 and CCR4 in 30 G3 EEC cancer tissues,30 G1/G2 EEC cancer tissues and 30 normal endometrial tissues.The relationship between it and clinical pathological parameters was analyzed.The effects of CCL22 and CCR4 on the survival rate of G3 EEC and G1/G2 EEC were analyzed.Results1.circ RNAs microarray hybridization sequencing results: 62167 circ RNAs differentially expressed in grade 3 endometrial adenocarcinoma carcinoma adjacent tissues and cancer tissues(the most prominent circ RNAs in the up-regulation and significantly associated with the tumor pathway include hsa＿circ＿0005692,hsa＿circ＿0039569,hsa＿circ＿0001523,and hsa＿circ＿0001610.The most significant circ RNAs in the down-regulation and significantly associated with the tumor pathway include hsa＿circ＿0000437,hsa＿circ＿0001776,and hsa＿circ＿0009043).2.The results of RTq PCR detection of differential circ RNAs: At the tissue level,hsa＿circ＿0005692,hsa＿circ＿0039569 and hsa＿circ＿0001610 were highly expressed in G3 EEC relative to G1/G2 EEC(P<0.05)and consistent with sequencing results,hsa＿circ＿0000437 and hsa＿circ＿0001776 were lowly expressed in G3 EEC relative to G1/G2 EEC(P<0.05)and consistent with sequencing results,the expressions of hsa＿circ＿0001523 and hsa＿circ＿0009043 are inconsistent with the sequencing results.At the cellular level,hsa＿circ＿0005692,hsa＿circ＿0039569 and hsa＿circ＿0001610 were found to be more highly expressed in the poorly differentiated endometrial cancer cell line KLE(P<0.05),and hsa＿circ＿0000437 and hsa＿circ＿0001776 were lower in the poorly differentiated endometrial cancer cell line KLE(P< 0.05).3.The hsa＿circ＿0005692,the most obvious difference and associated with the tumor pathway,was selected for further analysis.The relationship between hsa＿circ＿0005692 and clinical pathological parameters was analyzed.The expression level of hsa＿circ＿0005692 was not correlated with age,lymph node metastasis,FIGO stage and myometrial invasion(P>0.05),and was significantly correlated with tumor differentiation(P=0.001).4.Micro RNAs predicted to match the hsa＿circ＿0039569 sequence: hsa-mi R-449c-5p and hsa-mi R-34b-5p match hsa＿circ＿0005692 sequence and are associated with tumor-associated pathways.At the tissue level,RTq PCR analysis of hsa-mi R-449c-5p and hsa-mi R-34b-5p revealed that the expression levels of hsa-mi R-449c-5p and hsa-mi R-34b-5p were significantly lower in G3 EEC cancer tissues than in G1 EEC/G2 EEC cancer tissues(P< 0.05),the expression levels of hsa-mi R-449c-5p and hsa-mi R-34b-5p were significantly lower in G3 EEC cancer tissues than in adjacent tissues at the tissue level(P< 0.05).The expression of hsa-mi R-449c-5p and hsa-mi R-34b-5p was lower in G1 EEC/G2 EEC cancer tissues than in adjacent tissues(P<0.05).At the cellular level,RT-PCR detection in ISK and KLE revealed that hsa-mi R-449c-5p and hsa-mi R-34b-5p had lower expression in poorly differentiated endometrial cancer cell line KLE(P<0.05).The results of Spearman correlation analysis between hsa＿circ＿0005692 and hsa-mi R-449c-5p/hsa-mi R-34b-5p in G3 EC and G1/G2 EC showed: hsa＿circ＿0005692 and hsa-mi R-449c-5p/hsa-mi R-34b-5p has a significant negative correlation in G3 EC,and hsa＿circ＿0005692 has nosignificant negative correlation with hsa-mi R-449c-5p/hsa-mi R-34b-5p in G1/G2 EC.Therefore,hsa-mi R-449c-5p and hsa-mi R-34b-5p were selected for further study.5.Targeted prediction of hsa-mi R-449c-5p and hsa-mi R-34b-5p: CCL22/CCR4 matches hsa-mi R-449c-5p/hsa-mi R-34b-5p sequences and is associated with tumor-associated pathways.At the tissue level,RTq PCR analysis of CCL22 and CCR4 revealed that the expression levels of CCL22 and CCR4 were significantly higher in G3 EEC cancer tissues than in adjacent tissues(P< 0.05).The expression of CCL22 and CCR4 was higher in G3 EEC cancer tissues than those in G1 EEC/G2 EEC cancer tissues(P<0.05).At the cellular level,RT-PCR detection in ISK and KLE revealed that CCL22 and CCR4 had higher expression in poorly differentiated endometrial cancer cell line KLE(P<0.05).Spearman correlation analysis of hsa-mi R-449c-5p/hsa-mi R-34b-5p and CCR4 in G3 EEC and G1/G2 EEC,respectively: there is a significant negative correlation between hsa-mi R-449c-5p/hsa-mi R-34b-5p and CCR4 in G3 EEC,and there is a significant positive correlation between CCR4 and CCL22 in G3 EEC.6.Immunohistochemical staining results: the expression of CCL22 and CCR4 in G3 EEC cancer tissues was higher than that in G1 EEC/G2 EEC cancer tissues and normal endometrial tissue(P<0.05),the expression of CCL22 and CCR4 in G1 EEC/G2 EEC cancer tissues was higher than normal endometrial tissue(P<0.05).7.The relationship between CCL22/CCR4 and clinical pathological parameters showed that the expression level of CCL22 and CCR4 was not associated with age,lymph node metastasis,FIGO stage and myometrial invasion(P>0.05),and was significantly associated with tumor differentiation(P<0.05).Survival curve showed that the 5-year survival rate of G3 EEC patients with positive CCL22 and CCR4 expression was low.Conclusions1.hsa＿circ＿0005692 may play an important role in the development of G3 EEC and may become a potential biotherapeutic target for G3 EEC.2.The hsa＿circ＿0005692-hsa-mi R-449c-5p/hsa-mi R-34b-5p-CCR4/CCL22 axis may exist in grade 3 endometrial adenocarcinoma carcinoma and play an important role in its development.3.CCL22 and its receptor CCR4 are associated with endometrial cancer differentiation and prognosis,and may become potential biotherapeutic targets for G3 EEC. |
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