| Objective Explore the mechanism of microRNA-152 resistance to progesterone in endometrial carcinoma,search the early diagnosis of endometrial cancer and new markers related to targeted therapy.Methods 1.Immunohistochemical staining was used to analyze DNMT1 and PRB expression levels in endometrial cancer tissues,endometrial hyperplasia without atypia and normal endometrial tissue.2.Real-time PCR was used to detect miR-152 and its target DNMT1 expression levels in endometrial cancer tissues(progesterone receptor high expression and progesterone receptor low expression tissues),endometrial hyperplasia without atypia and normal endometrial tissue.3.Two human endometrial cancer cell lines Ishikawa(PR+,high expression of progesterone receptors)and KLE(PR-,low expression of progesterone receptors)with different progesterone receptor expression levels were selected.And Real-time PCR was used to detect miR-152,DNMT1 and PRB expression levels in human endometrial cancer cell lines.4.MiR-152 mimic and inhibitor,mimic NC and inhibitor NC were transiently transfected into endometrial cancer cell lines Ishikawa and KLE by liposome method.72 h after transfection,RNA was extracted,and the expression of miR-152,DNMT1,and PRB was detected by Real-time PCR after reverse transcription.5. MiR-152 mimic and its control mimic NC were transiently transfected into endometrial carcinoma cell line KLE.CCK-8 assay was used to detect cell proliferation at 0,48 and 72 h after transfection.Results 1. Immunohistochemical staining showed that the expression of DNMT1 in endometrial carcinoma was significantly higher than that in normal endometrial tissues and endometrial hyperplasia without atypia(P<0.0001).There was no significant difference in the expression of DNMT1 between normal endometrial tissue and endometrial hyperplasia without atypia(P=0.218).The expression of PR in endometrial cancer tissue was significantly lower than that in normal endometrium and endometrial hyperplasia without dysplasia(P<0.0001).However,there was no significant difference in the expression of PR in normal endometrial tissues and endometrial hyperplasia without atypia(P=0.202).The expression of DNMT1 in PR-negative endometrial carcinoma tissues was significantly higher than that in PR-positive endometrial carcinoma tissues.The difference was statistically significant(P=0.002).2. Real-time PCR results showed that compared with normal endometrial tissue,miR-152 expression in endometrial cancer tissue was significantly reduced,the difference was statistically significant(P<0.0001).Compared with PRB(+)endometrial carcinoma tissues,miR-152 expression levels were lower in PRB(-)endometrial carcinoma tissues and the difference was statistically significant(P=0.0479).Compared with normal endometrial tissue,the expression level of DNMT1 in endometrial cancer tissues was significantly higher,and the difference was statistically significant(P<0.0001).Compared with PRB(+)endometrial cancer tissues,the expression level of DNMT1 in PRB(-)endometrial cancer tissues was higher,and the difference was statistically significant(P=0.0389).3. Real-time PCR results showed that miR-152 was lower in PR(-)KLE cells than PR(+)Ishikawa cells in endometrial carcinoma.Compared with PR(+)Ishikawa cells,the expression level of DNMT1 in PR(-)KLE cells was significantly increased,while the expression of PRB was significantly decreased,with statistically significant differences(P<0.0001).4.Real-time PCR results showed that after transfection of miR-152 mimic,the average expression of miR-152 was significantly increased in Ishikawa and KLE cells,while after miR-152 inhibitor was transfected,the average expression of miR-152 was significantly decreased.The differences were statistically significant(P<0.0001).4. Real-time PCR results showed that after transfection of miR-152 mimic,the average expression of DNMT1 in both Ishikawa and KLE cell lines decreased,and the differences were statistically significant(Ishikawa: P=0.0004;KLE: P=0.0001).Compared with endometrial cancer PR(+)Ishikawa cells,the expression level of DNMT1 in PR(-)KLE cells of endometrial cancer decreased less,the difference was statistically significant(P=0.0026).After transfected with miR-152 inhibitor,the average expression of DNMT1 was increased,and the difference was statistically significant(Ishikawa: P=0.0002;KLE: P<0.0001).Compared with(+)Ishikawa cells,the expression level of DNMT1 in PR(-)KLE cells of endometrial cancer increased significantly,and the difference was statistically significant(P=0.0192).6. Real-time PCR results showed that after transfection of miR-152 mimic,the average expression of PRB in Ishikawa and KLE cell lines increased,the difference was statistically significant(P<0.0001).However,compared with PR(+)Ishikawa cells of endometrial cancer,PRB expression in PR(-)KLE cells of endometrial cancer increased less,and the difference was statistically significant(P=0.0002).After transfection with miR-152 inhibitor,the average expression of PRB was decreased,and the difference was statistically significant(P<0.0001).However,compared with PR(+)Ishikawa cells of endometrial carcinoma,the expression level of PRB in PR(-)KLE cells of endometrial cancer decreased significantly,and the difference was statistically significant(P=0.0047).7.The results of CCK-8 assay showed that after transfection of miR-152 mimic for 48 h,KLE cells grew slowly and cell proliferation was inhibited(P=0.0024).After 72 h of miR-152 mimic transfection,cell proliferation was inhibited more.The differences were statistically significant(P=0.001).Conclusions 1.miR-152 is down-regulated in endometrial cancer tissues and plays a role in tumor suppressor genes in the development of endometrial cancer.2.Down-regulation of miR-152 and up-regulation of its target DNMT1 play a catalytic role in the down-regulation or deletion of PRB in endometrial cancer.There may be a miR-152-DNMT1-PRB regulatory pathway that plays a role in progesterone resistance in endometrial cancer.3.miR-152 may serve as a new biological indicator of endometrial cancer progesterone resistance to targeted therapy. |
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