Abstract 509: Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma
Authors: Jing Wen, Xiuying Xie, Jianhua Fu
Journal: Cancer Research (2018)

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies of the gastrointestinal tract. miR-424 was one of the most significantly upregulated miRNAs in the ESCCs compared with normal esophageal tissues (NEs) identified by our previous miRNA arrays. The effects of miR-424 in tumor development and progression are conflicting according to previous reports till now. Here, we characterized the role of miR-424 in ESCC cell cycle progression and proliferation regulation. Expression of miR-424 was determined using qRT-PCR analysis. Cell proliferation assay by CCK-8 and the xenograft tumor model were used to determine ESCC proliferation in vitro and in vivo. Cell cycle distribution was analyzed by flow cytometry after synchronizing and releasing cells from G0/G1 phase, the onset of S-phase, or mitosis. Targets of miR-424 were validated using luciferase assays. The presence of E2F1-binding sites in the putative promoter of miR-424 gene was investigated using Chromatin immunoprecipitation (ChIP). As a result, miR-424 expression was elevated in ESCC specimens than in their normal counterparts. High miR-424 expression was a predictor of poor survival for Stage II-III ESCC patients undergoing surgery. Consistent with clinical data, knockdown miR-424 function in ESCC cells remarkably suppressed ESCC proliferation in vitro and tumor formation in mice model, while opposite results were observed after overexpressing miR-424. Cell cycle analyses revealed that miR-424 knockdown negatively regulates both G1/S and G2/M transition in ESCC cells, which were mediated by two newly identified miR-424 targets PRKCD and WEE1, respectively. During G1/S transition, increased level of PRKCD protein in KYSE410-miRZip-424 cells resulted in increased activation of PRKCD and downstream p38MAPK and JNK, which enhanced the stability of p21Cip1 protein. p21Cip1 proceeded to block CDK2 activation during G1/S transition. On the other hand, level of CDC2 phosphorylation at Tyr15, the inactive state of CDC2, was increased as a result of increased WEE1 expression by miR-424 knockdown when cells were at the G2/M border. Further evaluation of miR-424 expression in various cell cycle phases showed that miR-424 expression increased along with pri-miR-424 and pre-miR-424 when cell cycle progressed from G0/G1- to S-phase in ESCC cells. ChIP results demonstrated that G1/S transcriptional activating regulator E2F1 was enriched in regions of the putative miR-424 promoter containing the predicted E2F1-binding sites. Here, our results proved that miR-424 functions as an onco-miRNA in ESCCs. Activation of E2F1 when ESCC cells at the G1/S border induced the transcription of miR-424, which in turn targeted the expression of key genes to promote cell cycle progression and cell proliferation of ESCCs. Citation Format: Jing Wen, Xiuying Xie, Jianhua Fu. Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 509.