Mofang Liu

Repression of the miR‐17‐92 cluster by p53 has an important function in hypoxia‐induced apoptosis

We here report that miR‐17‐92 cluster is a novel target for p53‐mediated transcriptional repression under hypoxia. We found the expression levels of miR‐17‐92 cluster were reduced in hypoxia‐treated cells containing wild‐type p53, but were unchanged in hypoxia‐treated p53‐deficient cells. The repression of miR‐17‐92 cluster under hypoxia is independent of c‐Myc. Luciferase reporter assays mapped the region responding to p53‐mediated repression to a p53‐binding site in the proximal region of the miR‐17‐92 promoter. Chromatin immunoprecipitation (ChIP), Re‐ChIP and gel retardation assays revealed that the binding sites for p53‐ and the TATA‐binding protein (TBP) overlap within the miR‐17‐92 promoter; these proteins were found to compete for binding. Finally, we show that pri‐miR‐17‐92 expression correlated well with p53 status in colorectal carcinomas. Over‐express miR‐17‐92 cluster markedly inhibits hypoxia‐induced apoptosis, whereas blocked miR‐17‐5p and miR‐20a sensitize the cells to hypoxia‐induced apoptosis. These data indicated that p53‐mediated repression of miR‐17‐92 expression likely has an important function in hypoxia‐induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

A novel miR‐155/miR‐143 cascade controls glycolysis by regulating hexokinase 2 in breast cancer cells

Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro‐tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro‐inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation‐induced miR‐155 functions as an important mediator in this process. We further show that miR‐155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR‐155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPβ (a transcriptional activator for mir‐143), miR‐155 represses mir‐143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post‐transcriptional level. The miR‐155‐mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR‐155/miR‐143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.

Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis

MicroRNA 21 (miR-21) is overexpressed in virtually all types of carcinomas and various types of hematological malignancies. To determine whether miR-21 promotes tumor development in vivo, we knocked out the miR-21 allele in mice. In response to the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate mouse skin carcinogenesis protocol, miR-21-null mice showed a significant reduction in papilloma formation compared with wild-type mice. We revealed that cellular apoptosis was elevated and cell proliferation was decreased in mice deficient of miR-21 compared to wild-type animals. In addition, we found that a large number of validated or predicted miR-21 target genes were up-regulated in miR-21-null keratinocytes, which are precursor cells to skin papillomas. Specifically, up-regulation of Spry1, Pten, and Pdcd4 when miR-21 was ablated coincided with reduced phosphorylation of ERK, AKT, and JNK, three major downstream effectors of Ras activation that plays a predominant role in DMBA-initiated skin carcinogenesis. These results provide in vivo evidence that miR-21 exerts its oncogenic function through negatively regulating its target genes.

Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis.

Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

A statin‐regulated microRNA represses human c‐Myc expression and function

c‐Myc dysregulation is one of the most common abnormalities found in human cancer. MicroRNAs (miRNAs) are functionally intertwined with the c‐Myc network as multiple miRNAs are regulated by c‐Myc, while others directly suppress c‐Myc expression. In this work, we identified miR‐33b as a primate‐specific negative regulator of c‐Myc. The human miR‐33b gene is located at 17p11.2, a genomic locus frequently lost in medulloblastomas, of which a subset displays c‐Myc overproduction. Through a small‐scale screening with drugs approved by the US Food and Drug Administration (FDA), we found that lovastatin upregulated miR‐33b expression, reduced cell proliferation and impaired c‐Myc expression and function in miR‐33b‐positive medulloblastoma cells. In addition, a low dose of lovastatin treatment at a level comparable to approved human oral use reduced tumour growth in mice orthotopically xenografted with cells carrying miR‐33b, but not with cells lacking miR‐33b. This work presents a highly promising therapeutic option, using drug repurposing and a miRNA as a biomarker, against cancers that overexpress c‐Myc.

piRNA-Triggered MIWI Ubiquitination and Removal by APC/C in Late Spermatogenesis

The PIWI/PIWI-interacting RNA (piRNA) machinery has been well documented to maintain genome integrity by silencing transposons in animal germ cells. Recent studies have advanced our understanding of the biogenesis and function of this machinery; however, its metabolism has remained largely unexplored. Here, we show that murine PIWI (MIWI) is degraded through the APC/C-26S proteasome pathway and that piRNAs play an indispensable role in this process by enhancing MIWI interaction with an APC/C substrate-binding subunit. Interestingly, piRNA-triggered MIWI destruction occurs in late spermatids, which in turn leads to piRNA elimination, suggesting a feedforward mechanism for coordinated removal of the MIWI/piRNA machinery at a specific developmental stage. Importantly, the proper removal of MIWI/piRNA is essential for sperm maturation. Together, our results reveal a role for piRNAs in regulating the clearance of the MIWI/piRNA machinery via the ubiquitin-proteosome pathway and demonstrate the critical importance of proper temporal regulation of MIWI/piRNA in male germ cell development.

IL-1β-Mediated Repression of microRNA-101 Is Crucial for Inflammation-Promoted Lung Tumorigenesis

Inflammatory stimuli clearly contribute to lung cancer development and progression, but the underlying pathogenic mechanisms are not fully understood. We found that the proinflammatory cytokine IL-1β is dramatically elevated in the serum of patients with non-small cell lung cancer (NSCLC). In vitro studies showed that IL-1β promoted the proliferation and migration of NSCLC cells. Mechanistically, IL-1β acted through the COX2-HIF1α pathway to repress the expression of microRNA-101 (miR-101), a microRNA with an established role in tumor suppression. Lin28B was identified as critical effector target of miR-101 with its repression of Lin28B, a critical aspect of tumor suppression. Overall, IL-1β upregulated Lin28B by downregulating miR-101. Interestingly, cyclooxygenase-2 inhibition by aspirin or celecoxib abrogated IL-1β-mediated repression of miR-101 and IL-1β-mediated activation of Lin28B along with their stimulatory effects on NSCLC cell proliferation and migration. Together, our findings defined an IL-1β-miR-101-Lin28B pathway as a novel regulatory axis of pathogenic inflammatory signaling in NSCLC.

Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2‐aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi‐step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half‐life of around 1 s, which is followed by two slower steps occurring with a half‐life in the range of minutes at 25°C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi‐step process. cAMP·CRP, which activates transcription at galP1, enhanced the rate‐limiting step.

Suppression of miR‐199a maturation by HuR is crucial for hypoxia‐induced glycolytic switch in hepatocellular carcinoma

Glucose metabolic reprogramming is a hallmark of cancer. Cancer cells rapidly adjust their energy source from oxidative phosphorylation to glycolytic metabolism in order to efficiently proliferate in a hypoxic environment, but the mechanism underlying this switch is still incompletely understood. Here, we report that hypoxia potently induces the RNA‐binding protein HuR to specifically bind primary miR‐199a transcript to block miR‐199a maturation in hepatocellular carcinoma (HCC) cells. We demonstrate that this hypoxia‐suppressed miR‐199a plays a decisive role in limiting glycolysis in HCC cells by targeting hexokinase‐2 (Hk2) and pyruvate kinase‐M2 (Pkm2). Furthermore, systemically delivered cholesterol‐modified agomiR‐199a inhibits [18F]‐fluorodeoxyglucose uptake and attenuates tumor growth in HCC tumor‐bearing mice. These data reveal a novel mechanism of reprogramming of cancer energy metabolism in which HuR suppresses miR‐199a maturation to link hypoxia to the Warburg effect and suggest a promising therapeutic strategy that targets miR‐199a to interrupt cancerous aerobic glycolysis.

Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos.

Expression, modification, and activity of microRNAs are dynamically regulated in early mouse embryos unveiled by sensitive sequencing. Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3′ mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos.