Zfx The Reporter

Emerging reports demonstrated that long non-coding RNAs (lncRNAs) play a role in the pathogenesis and metastasis of cancers. However, the biological functions and underlying mechanisms of LncRNA CEBPA-AS1 in acute myeloid leukemia (AML) remain largely elusive. The level of CEBPA-AS1 was examined in AML clinical tissues and cell lines via fluorescence in situ hybridization (FISH) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In vivo and in vitro functional tests were applied to identify the pro-oncogenic role of CEBPA-AS1 in AML development. The overexpressed CEBPA-AS1 was linked to poor survival in AML patients. Moreover, the relationships among CEBPA-AS1, Zinc Finger Protein X-Linked (ZFX), and miR-24-3p were predicted by bioinformatics and validated by RNA immunoprecipitation (RIP) and luciferase reporter assays. Our findings unveiled that transcription factor ZFX particularly interacted with the promoter of CEBPA-AS1 and activated CEBPA-AS1 transcription. Downregulation of CEBPA-AS1 inhibited the proliferation and invasion while promoted apoptosis of AML cells in in vitro, as well as in vivo, xenograft tumor growth was modified. However, overexpression of CEBPA-AS1 observed the opposite effects. Furthermore, CEBPA-AS1 acted as a competitive endogenous RNA (ceRNA) of miR-24-3p to attenuate the repressive effects of miR-24-3p on its downstream target CTBP2. Taken together, this study emphasized the pro-oncogenic role of CEBPA-AS1 in AML and illustrated its connections with the upstream transcription factor ZFX and the downstream regulative axis miR-24-3p/CTBP2, providing important insights to the cancerogenic process in AML.

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(E): Analyses of c-Myc promoter activity using luciferase reporter assay in GSCs in response to ZFX ectopic expression. DNA fragments containing indicated regions of human c-Myc promoter were cloned into the luciferase reporter system. GSCs were transduced with the luciferase report system and Flag-ZFX or vector control. The relative luciferase activity were measured and quantified. The c-Myc promoter fragment lacking the P2 region lost the response to ZFX ectopic expression. **, p0.05.

(F and G): Luciferase reporter assay showing mutations in the conserved ZFX binding site (G) on P2 region of c-Myc promoter abolished the promoter activity in response to ZFX overexpression. WT: wild type; MT: mutated. **, p0.05.

Similar to many transcription factors, the overexpression of ZFX cDNA from a heterologous promoter is toxic to cells (B.R., unpublished data). To overexpress murine Zfx under its native regulation, the entire genomic Zfx locus has been introduced into mESC as a bacterial artificial chromosome (BAC) transgene [13]. We have developed a method for stable BAC introduction into hESC to serve as reporter transgenes [17]. We adapted this approach to introduce additional copies of the human ZFX locus as BAC transgene in the H9 hESC. Several nucleofected H9 clones showed increased ZFX mRNA expression by quantitative RT-PCR (Figure 2A). We chose two clones (ZFXOver1 and ZFXOver2) with the highest ZFX expression levels and compared them to the parental H9 line, a control clone (ZFXNormal) with normal ZFX expression levels from the same experiment, as well as independent H9 GFP and YFP-expressing BAC transgenic reporter lines from our earlier studies (see Table 1 for a comprehensive description of controls used in this study) [17], [20]. We verified that the BAC reporter control clones expressed normal amounts of ZFX (data not shown).

In the current study we demonstrate a role for the transcription factor ZFX in modulating the self-renewal of hESC using gain- and loss-of-function approaches. ZFX reduction caused a loss of self-renewal while BAC-mediated ZFX overexpression increased the clonogenicity and decreased spontaneous differentiation of hESCs. Importantly, ZFX-overexpressing clones retained their ability to undergo differentiation in response to appropriate stimuli. The use of BAC transgenesis was critical to circumvent general toxic effects of ZFX overexpression observed using heterologous promoters. Gene expression driven by the endogenous gene locus in a BAC provides advantages over heterologous promoters, such as native gene regulation, reduced position effect [23] and copy number-dependent expression [24]. Until now, BACs were used to direct reporter gene expression [17] or as vehicles for homologous recombination in hESC [25]. We believe this study is the first to demonstrate their utility as vectors for functional gene expression in hESCs.

MiR-142-3p could be sponged by circ_0020123. (A) The binding sites and the constructed mutate sites of circ_0020123 in miR-142-3p were shown. Dual-luciferase reporter assay (B) and RIP assay (C) were used to confirm the interaction between circ_0020123 and miR-142-3p. (D) QRT-PCR was used to measure miR-142-3p expression in NSCLC tumor tissues (Tumor, n = 30) and normal non-cancerous tissues (Normal, n = 30). (E) The expression of miR-142-3p in NSCLC cells (H1299, H1581 and A549) and 16HBE cells was detected using qRT-PCR. (F) MiR-142-3p expression was examined by qRT-PCR in H1299 and A549 cells transfected with si-NC, si-circ_0020123#1 or si-circ_0020123#2. (G) Pearson correlation analysis was performed to measure the correlation between circ_0020123 and miR-142-3p in NSCLC tumor tissues. **P < 0.01, ***P < 0.001.

ZFX was a target of miR-142-3p. (A) The sequences of WT-ZFX 3ʹUTR and MUT-ZFX 3ʹUTR were presented. (B) The interaction between ZFX and miR-142-3p was verified using dual-luciferase reporter assay. (C) The ZFX mRNA level in NSCLC tumor tissues (Tumor, n = 30) and normal non-cancerous tissues (Normal, n = 30) was detected by qRT-PCR. (D) WB analysis was used to measure the protein level of ZFX in NSCLC cells (H1299, H1581 and A549) and 16HBE cells. (E) After transfected with miR-NC, miR-142-3p, in-miR-NC or in-miR-142-3p into H1299 and A549 cells, the ZFX protein level was determined by WB analysis. (F) The correlation between miR-142-3p and ZFX in NSCLC tumor tissues was evaluated using Pearson correlation analysis. ***P < 0.001.

We test expression levels of HOXA-AS2, miRNA-302c-3p, the transcription factor zinc finger X-chromosomal protein (ZFX), and the chitinase-like protein YKL-40 in endometrial carcinoma by qRT-PCR and western blotting. Luciferase reporter and qRT-PCR assays were conducted to identify potential binding sites of HOXA-AS2 to miRNA-302c-3p. Cell cycle, migration and invasion ability of endometrial cancer cells were investigated using flow-cytometric analysis, CCK-8 and transwell assays, respectively.

MicroRNA 144 (miR-144), a small non-coding RNA, is frequently dysregulated in human several tumour progression,but its role and the underlying mechanisms in hepatocellular carcinoma (HCC) is poorly investigated. In thepresent study, the expression of miR-144 was firstly analysed in datasets derived from GSE21362 and TCGA, andthen detected in HCC tissues and cell lines by quantitative RT-PCR (qRT-PCR) analysis. MiR-144 was shown to besignificantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfectedinto HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays.Gain of function assay revealed miR-144 remarkably inhibited cell proliferation, migration and invasion. In addition,bioinformatical analysis and luciferase reporter assay identified ZFX as a novel target of miR-144 in HCC cells, asconfirmed by qRT-PCR and Western blot. Furthermore, ZFX was found to be significantly up-regulated usingOncomine database analysis. Loss of function assay further indicated knockdown of ZFX had similar effects ofmiR-144-mediated HCC cell proliferation and invasion. Therefore, miR-144 has been demonstrated to act as a tumoursuppressor in HCC cell growth and motility by directly targeting ZFX, which implicates its potential applications inthe development of HCC treatment.

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