Xiaofei Zheng

Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest

miRNAs regulate gene expression by inhibiting translation or by targeting messenger RNA (mRNA) for degradation in a post‐transcriptional fashion. In the present study, we show that ectopic expression of miR‐34a reduces both mRNA and protein levels of cyclin D1 (CCND1) and cyclin‐dependent kinase 6 (CDK6). We also demonstrate that miR‐34a targets the 3′‐untranslated mRNA region of CCND1 as well as CDK6, which in turn interferes with phosphorylation of retinoblastoma. In addition, we show that overexpression of miR‐34a induces a significant G1 cell‐cycle arrest in the A549 cell line. Taken together, our data suggest that the effects of miR‐34a on G1 cell cycle arrest are through the down‐regulation of CCND1 and CDK6, which is associated with other targets of miR‐34a either additively or synergistically.

miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to be involved in diverse biological processes by regulating gene expression. Numerous miRNAs have been identified in various species, and many more miRNAs remain to be detected. Generally, hundreds of mRNAs have been predicted to be potential targets of one miRNA, so it is a great challenge to identify the genuine miRNA targets. Here, we generated the cell lines depleted of Drosha protein and screened dozens of transcripts (including Cyclin D1) regulated potentially by miRNA-mediated RNA silencing pathway. On the basis of miRNA expressing library, we established a miRNA targets reverse screening method by using luciferase reporter assay. By this method, we found that the expression of Cyclin D1 (CCND1) was regulated by miR-16 family directly, and miR-16 induced G1 arrest in A549 cells partially by CCND1. Furthermore, several other cell cycle genes were revealed to be regulated by miR-16 family, including Cyclin D3 (CCND3), Cyclin E1 (CCNE1) and CDK6. Taken together, our data suggests that miR-16 family triggers an accumulation of cells in G0/G1 by silencing multiple cell cycle genes simultaneously, rather than the individual target.

miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells

Several studies have shown that miR-34a represses the expression of many genes and induces G1 arrest, apoptosis, and senescence. In the present study, we identified the role of miR-34a in the regulation of tumor cell scattering, migration, and invasion. Down-regulation of miR-34a expression was highly significant in 19 of 25 (76%) human hepatocellular carcinoma (HCC) tissues compared with adjacent normal tissues and associated with the metastasis and invasion of tumors. Furthermore, resected normal/tumor tissues of 25 HCC patients demonstrated an inverse correlation between miR-34a and c-Met-protein. In HepG2 cells, ectopic expression of miR-34a potently inhibited tumor cell migration and invasion in a c-Met-dependent manner. miR-34a directly targeted c-Met and reduced both mRNA and protein levels of c-Met; thus, decreased c-Met-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Taken together, these results provide evidence to show the suppression role of miR-34a in tumor migration and invasion through modulation of the c-Met signaling pathway.

Stress induces tRNA cleavage by angiogenin in mammalian cells.

tRNAs play a central role in protein translation, acting as the carrier of amino acids. By cloning microRNAs, we unexpectedly obtained some tRNA fragments generated by tRNA cleavage in the anticodon loop. These tRNA fragments are present in many cell lines and different mouse tissues. In addition, various stress conditions can induce this tRNA cleavage event in mammalian cells. More importantly, angiogenin (ANG), a member of RNase A superfamily, appears to be the nuclease which cleaves tRNAs into tRNA halves in vitro and in vivo. These results imply that angiogenin plays an important physiological role in cell stress response, except for the known function of inducing angiogenesis.

Identification of human fetal liver miRNAs by a novel method

MicroRNAs (miRNAs) are short 20–25 nucleotides RNA molecules that have been shown to regulate gene expressions in a variety of eukaryotic systems. miRNAs are widespread in eukaryotes and several hundred of miRNAs have been identified, but still a lot of miRNAs have not been detected in various eukaryotic organisms. However, it is not an easy work to clone miRNAs by traditional methods. Here, we describe the identification of 27 miRNAs from a human fetal liver cDNA library by a novel cloning method. Low molecular weight RNA fraction (⩽200 nt) from fetal liver tissue was extracted, and polyadenylated by poly(A) polymerase. A 5′ RNA adaptor was ligated to poly(A)‐tailed RNA using T4 RNA ligase. After reverse transcription, the cDNA was amplified by PCR with two adaptor primers. The PCR product with a size about 109 bp was recovered and cloned into T vector. After sequencing, database searching, and expression profiling, 5 novel miRNAs were discovered among other 22 known miRNAs in human fetal liver. These finding indicate that a large diverse population of miRNAs may function to regulate gene expression in hepatocyte.

MicroRNA-101 regulates expression of the v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene in human hepatocellular carcinoma†

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of various biological processes. Although there is emerging evidence that some microRNAs can function as oncogenes or tumor suppressors, the specific role of miRNA in human hepatocellular carcinoma (HCC) is unclear at this point. In this study, we examined the microRNA expression profiles in a set of 20 human HCC specimens by miRNA microarray and quantitative real‐time polymerase chain reaction. The results showed that among the 20 HCC samples analyzed, microRNA‐101 was significantly down‐regulated twofold or more (twofold to 20‐fold) in 16 samples compared with the matching nontumoral liver tissues. Using both a luciferase reporter assay and Western blot analysis, we showed that microRNA‐101 repressed the expression of v‐fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene, a key component of the activator protein‐1 (AP‐1) transcription factor. Moreover, using a luciferase expression vector (pAP‐1‐Luc) driven by seven copies of an AP‐1 cis‐element, we observed that microRNA‐101 expression inhibited phorbol 12‐myristate 13‐acetate (PMA)–induced AP‐1 activity. In in vitro Matrigel invasion and Transwell migration assays, enhanced microRNA‐101 expression inhibited the invasion and migration of cultured HCC cells, respectively. These findings suggest that microRNA‐101 may play an important role in HCC. Conclusion: MicroRNA‐101, which is aberrantly expressed in HCC, could repress the expression of the FOS oncogene. (HEPATOLOGY 2009.)

MicroRNA-193b regulates proliferation, migration and invasion in human hepatocellular carcinoma cells.

BACKGROUND AND AIMS Recently, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. However, the functions of these miRNAs in HCC remain largely undefined. METHODS The expression profiles of miR-193b were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was also be used to screen the potential target genes of miR-193b. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-193b in hepatoma cells was examined further. RESULTS miR-193b was significantly down-regulated in most of the HCC tissues compared to the matching non-tumoural liver tissues. Furthermore, ectopic expression of miR-193b dramatically suppressed the ability of hepatoma cells to form colonies in vitro and to develop tumours in nude mice. CCND1 and ETS1 were revealed to be regulated by miR-193b directly. By regulating the expressions of these oncogenes, miR-193b induced cell cycle arrest and inhibited the invasion and migration of hepatoma cells. CONCLUSIONS miR-193b may function as a tumour suppressor in the development of HCC by acting on multiple tumourigenic pathways.

miR-183 inhibits TGF-β1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells

BackgroundIn recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined.MethodsThe expression profiles of miR-183 were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was used to screen the potential target genes of miR-183. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-183 in hepatoma cells was examined.ResultsAmong the 25 HCC samples analyzed, microRNA-183 was significantly up-regulated (twofold to 367-fold) in 17 samples compared with the matching nontumoral liver tissues. Programmed cell death 4 (PDCD4) was identified as the target gene of miR-183. Moreover, PDCD4 is a proapoptotic molecule involved in TGF-β1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-β1.ConclusionsWe conclude that miR-183 can inhibit apoptosis in human HCC cells by repressing the PDCD4 expression, and miR-183 may play an important role in HCC development.

A novel method to monitor the expression of microRNAs.

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18–25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.

Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes.