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已有研究证明,抑癌子PTEN(phosphatase and tensin homolog)缺失或表达下调可引起子宫内膜癌变;在喉鳞状细胞癌中miR-1297可下调PTEN的表达。然而,miR-1297下调PTEN的机制,以及是否miR-1297靶向抑制PTEN参与子宫内膜癌变尚未见报道。本研究证明,miR-1297靶向抑制PTEN基因的表达参与子宫内膜癌变。收集临床病理检查确诊为子宫内膜腺癌的癌组织及癌旁组织18例。实时定量PCR(RT-q PCR)及Western印迹结果显示,与癌旁子宫内膜比较,子宫内膜癌组织miR-1297高表达,而PTEN则呈低表达或缺失。内膜癌和癌旁内膜组织中,miR-1297与PTEN表达水平的散点图分析揭示,miR-1297的表达水平与PTEN的表达水平呈明显的负相关(内膜癌:miR-1297与PTEN的相关系数r=-0.6928,P=0.0438;癌旁组织:相关系数r=-0.4085,P<0.0001)。报告酶活性测定显示,与对照比较,p GL3-PTEN-3’-UTR-1/2/3加miR-1297模拟物共转染后,荧光素酶活性在转染的Ishikawa和ECC-1细胞中分别降低了约21%(P=0.0196)和17%(P=0.0306);相反,转染miR-1297抑制物后,两种细胞的荧光素酶活性分别升高约59%(Ishikawa:P=0.0014)和50%(ECC-1:P=0.0025)。结果提示,miR-1297可通过特异识别PTEN mRNA的3’-UTR,抑制PTEN的表达。基因转染结合Western印迹结果证明,在原代子宫内膜细胞中,过表达miR-1297模拟物导致PTEN蛋白表达下调,而其下游底物分子——磷酸化的AKT(p-AKT)表达上调;而过表达miR-1297抑制物后,PTEN表达上调,p-AKT表达下调。Connexin V/PI双染结合流式分析证明,与对照细胞(3%细胞凋亡)比较,在子宫内膜癌细胞系Ishikawa中,敲减miR-1297表达后细胞调亡明显增加(至10%),这可能与抑制miR-1297后PTEN的表达上调有关。上述结果提示,miR-1297在子宫内膜癌高表达,而PTEN低表达。上述结果还提示,miR-1297作为一个促癌子可通过特异识别PTEN mRNA的3’-UTR,抑制PTEN的表达,促进AKT磷酸化/激活,从而促进内膜癌的发生,这可能是子宫内膜癌变的发生原因之一。
Studies have shown that the deletion or down-regulation of PTEN (phosphatase and tensin homolog) can cause endometrial carcinogenesis, and miR-1297 can down-regulate the expression of PTEN in laryngeal squamous cell carcinoma. However, the mechanisms by which miR-1297 down-regulates PTEN and whether miR-1297 targets PTEN to inhibit endometrial cancer have not been reported yet. This study demonstrates that miR-1297 targeting inhibits PTEN gene expression in endometrial cancer. Clinicopathological examination confirmed the diagnosis of endometrial adenocarcinoma of the cancer tissue and adjacent tissues in 18 cases. Real-time quantitative PCR (RT-qPCR) and Western blotting showed that miR-1297 was highly expressed in endometrial carcinoma tissues compared with adjacent non-cancerous endometrial tissues, while PTEN was lowly expressed or absent. Scatterplot analysis of the expression levels of miR-1297 and PTEN in endometrial and para-cancerous endometrium revealed a significant negative correlation between miR-1297 expression and PTEN expression (endometrial cancer: miR-1297 and The correlation coefficient of PTEN r = -0.6928, P = 0.0438; adjacent tissue: correlation coefficient r = -0.4085, P <0.0001). The reported enzymatic activity assays showed luciferase activity was significantly increased in transfection of Ishikawa and ECC-1 cells after co-transfection with pGL3-PTEN-3’-UTR-1/2/3 plus miR-1297 mimics (P = 0.0196) and 17% (P = 0.0306), respectively; in contrast, the luciferase activity of both cells increased by approximately 59%, respectively, after transfection with the miR-1297 inhibitor (Ishikawa: P = 0.0014) and 50% (ECC-1: P = 0.0025). The results suggest that miR-1297 inhibits PTEN expression by specifically recognizing the 3’-UTR of PTEN mRNA. Gene transfection combined with Western blotting results demonstrated that overexpression of miR-1297 mimics resulted in down-regulation of PTEN protein and upregulation of its downstream substrate molecule, phosphorylated AKT (p-AKT), in primary endometrial cells; However, after over-expression of miR-1297 inhibitor, PTEN expression was up-regulated and p-AKT expression was down-regulated. Connexin V / PI double staining combined with flow cytometry demonstrated that apoptosis was significantly increased (to 10%) in knockdown of miR-1297 expression in the endometrial cancer cell line Ishikawa compared to control cells (3% apoptosis) ), Which may be related to the upregulation of PTEN after miR-1297 was inhibited. The above results suggest that miR-1297 is highly expressed in endometrial cancer, while PTEN is low expressed. The above results also suggest that miR-1297 as a pro-cancer promoter can promote the carcinogenesis of endometrial cancer by specifically recognizing the 3’-UTR of PTEN mRNA, inhibiting the expression of PTEN, and promoting phosphorylation / activation of AKT, which may be intrauterine Membranous cancer one of the reasons.