Dataset Information

The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis.

ABSTRACT:

Background

Non-small cell lung carcinoma (NSCLC) is a major worldwide health threat due to its low cure rate and high lethality. Emerging evidence suggests that epidermal growth factor receptor (EGFR) plays vital roles in cancer initiation and progression, and is considered an important cancer-driving protein. However, how EGFR expression is regulated during NSCLC development remains to be fully elucidated.

Methods

In NSCLC clinical samples, EGFR protein levels were measured by western blotting and qRT-PCR, respectively. Combining microRNA (miRNA) target prediction software and the pulldown assay, we predicted microRNAs (miRNAs) that targeted EGFR. Next, three NSCLC cell lines, A549 (p53 WT), H322 (p53 mutant), and H1299 (p53 null), were used to demonstrate the direct targeting of EGFR by miR-193a. In addition, we investigated the biological effects of EGFR inhibition by miR-193a in vitro using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU), transwell, and apoptosis assays. Then, using ChIP and luciferase assays, we demonstrated that miR-193a was directly activated by p53 at the transcriptional level and that p53-induced-miR-193a and EGFR form a double-negative feedback loop.

Results

We found that EGFR mRNA and protein were upregulated in NSCLC. We predicted that EGFR was a target of miR-193a and validated that miR-193a bound directly to the 3'-UTR of the EGFR mRNA. Moreover, miR-193a inhibited NSCLC proliferation and invasion, and promotes NSCLC apoptosis by directly downregulating EGFR. Then, we demonstrated that p53 directly activated miR-193a transcription, whereas EGFR functioned as a transcriptional repressor to negatively control miR-193a expression, forming a feedback loop. The loop promoted NSCLC cell proliferation and migration and accelerated tumor growth in xenograft mice.

Conclusions

This study highlights a double-negative feedback loop in NSCLC. The feedback loop is crucial because overexpressing EGFR strongly accelerated tumor growth, while miR-193a restoration blocked tumor growth in vivo. Our findings are in line with the emerging opinion that miRNAs and protein regulators form regulatory networks in critical biological processes and that their dysregulation can lead to cellular dysfunction. In conclusion, this study provides important insights into the molecular mechanisms of NSCLC progression and may help inform the development of new therapeutics for managing NSCLC.

SUBMITTER: Wang W

PROVIDER: S-EPMC6535738 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis.

Wang Wei W Shen Xia-Bo XB Jia Wei W Huang Da-Bing DB Wang Yong Y Pan Yue-Yin YY

Therapeutic advances in medical oncology 20190523

<h4>Background</h4>Non-small cell lung carcinoma (NSCLC) is a major worldwide health threat due to its low cure rate and high lethality. Emerging evidence suggests that epidermal growth factor receptor (EGFR) plays vital roles in cancer initiation and progression, and is considered an important cancer-driving protein. However, how EGFR expression is regulated during NSCLC development remains to be fully elucidated.<h4>Methods</h4>In NSCLC clinical samples, EGFR protein levels were measured by we ...[more]

PMID: 31205511

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Project description:Though androgen deprivation therapy is the standard treatment for prostate cancer (PCa), most patients would inevitably progress to castration-resistant prostate cancer (CRPC) which is the main cause of PCa death. Therefore, the identification of novel molecular mechanism regulating cancer progression and achievement of new insight into target therapy would be necessary for improving the benefits of PCa patients. This study aims to study the function and regulatory mechanism of HOTAIR/EZH2/miR-193a feedback loop in PCa progression.MSKCC and TCGA datasets were used to identify miR-193a expression profile in PCa. Cell Counting Kit-8 (CCK-8) assays, colony formation, invasion, migration, flow cytometry, a xenograft model and Gene Set Enrichment Analysis were used to detect and analyze the biological function of miR-193a. Then, we assessed the role of HOTAIR and EZH2 in regulation of miR-193a expression by using plasmid, lentivirus and small interfering RNA (siRNA). Luciferase reporter assays and chromatin immunoprecipitation assays were performed to detect the transcriptional activation of miR-193a by EZH2 and HOTAIR. Further, qRT-PCR and luciferase reporter assays were conducted to examine the regulatory role of miR-193a controlling the HOTAIR expression in PCa. Finally, the correlation between HOTAIR, EZH2 and miR-193a expression were analyzed using In situ hybridization and immunohistochemistry.We found that miR-193a was significantly downregulated in metastatic PCa through mining MSKCC and TCGA datasets. In vitro studies revealed that miR-193a inhibited PCa cell growth, suppressed migration and invasion, and promoted apoptosis; in vivo results demonstrated that overexpression of miR-193a mediated by lentivirus dramatically reduced PCa xenograft tumor growth. Importantly, we found EZH2 coupled with HOTAIR to repress miR-193a expression through trimethylation of H3K27 at miR-193a promoter in PC3 and DU145 cells. Interestingly, further evidence illustrated that miR-193a directly targets HOTAIR showing as significantly reduced HOTAIR level in miR-193a overexpressed cells and tissues. The expression level of miR-193a was inversely associated with that of HOTAIR and EZH2 in PCa.This study firstly demonstrated that miR-193a acted as tumor suppressor in CRPC and the autoregulatory feedback loop of HOTAIR/EZH2/miR-193a served an important mechanism in PCa development. Targeting this aberrantly activated feedback loop may provide a potential therapeutic strategy.

Project description:BackgroundGlioblastoma is the most common primary malignant intracranial tumor with poor clinical prognosis in adults. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) function as important regulators in cancer progression, including glioblastoma. Here, we identified a new lncRNA LPP antisense RNA-2 (LPP-AS2) and investigated its function and mechanism in the development of glioma.MethodsHigh-throughput RNA sequencing was performed to discriminate differentially expressed lncRNAs and mRNAs between glioma tissues and normal brain tissues. Expression of LPP-AS2, epidermal growth factor receptor (EGFR) and miR-7-5p in glioma tissues and cell lines was detected by real-time quantitative PCR (RT-qPCR), and the functions of lncRNA LPP-AS2 in glioma were assessed by in vivo and in vitro assays. Insight into the underlying mechanism of competitive endogenous RNAs (ceRNAs) was obtained via bioinformatic analysis, dual luciferase reporter assays, RNA pulldown assays, RNA immunoprecipitation (RIP) and rescue experiments.ResultsThe results of high-throughput RNA-seq indicated lncRNA LPP-AS2 was upregulated in glioma tissues and further confirmed by RT-qPCR. Higher LPP-AS2 expression was related to a poor prognosis in glioma patients. Based on functional studies, LPP-AS2 depletion inhibited glioma cell proliferation, invasion and promoted apoptosis in vitro and restrained tumor growth in vivo, overexpression of LPP-AS2 resulted in the opposite effects. In addition, LPP-AS2 and EGFR were observed in co-expression networks. LPP-AS2 was found to function as a ceRNA to regulate EGFR expression by sponging miR-7-5p in glioma cells. The result of chromatin immunoprecipitation (ChIP) assays validated that c-MYC binds directly to the promoter region of LPP-AS2. As a downstream protein of EGFR, c-MYC was modulated by LPP-AS2 and in turn enhanced LPP-AS2 expression. Thus, lncRNA LPP-AS2 promoted glioma tumorigenesis via a miR-7-5p/EGFR/PI3K/AKT/c-MYC feedback loop.ConclusionsOur study elucidated that LPP-AS2 acted as an oncogene through a novel molecular pathway in glioma and might be a potential therapeutic approach for glioma diagnosis, therapy and prognosis.

Dataset Information

The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis.

Background

Methods

Results

Conclusions

Publications

The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis.

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