MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN

MicroRNAs (miRNAs) regulate gene expression by binding to target sites and initiating translational repression and/or mRNA degradation. In our previous study, we have shown that expression of serum microRNA (miR)-21 is correlated with TNM stage and lymph node metastasis and might be an independent prognostic factor for NSCLC patients. However, the roles of miR-21 overexpression in NSCLC development are still unclear. The purpose of this study is to investigate the effect of miR-21 and determine whether miR-21 can be a therapeutic target for human NSCLC. Taqman real-time quantitative RT-PCR assay was performed to detect miR-21 expression in NSCLC cell lines and tissues. Next, the effects of miR-21 expression on NSCLC cell characteristics including growth, invasion, and chemo- or radioresistance were also determined. Results showed that miR-21 is commonly upregulated in NSCLC cell lines and tissues with important functional consequences. In addition, we found that anti-miR-21 could significantly inhibit growth, migration and invasion, and reverse chemo- or radioresistance of NSCLC cells, while miR-21 mimics could increase growth, promote migration and invasion, and enhance chemo- or radioresistance of NSCLC cells. Meanwhile, miR-21 mimics could inhibit expression of PTEN mRNA and protein and the luciferase activity of a PTEN 3??-untranslated region (UTR)-based reporter construct in A549 cells, while anti-miR-21 could increase expression of PTEN mRNA and protein and the luciferase activity of a PTEN 3??-UTR-based reporter construct in A549 cells. Furthermore, overexpression of PTEN could mimic the same effects of anti-miR-21 in NSCLC cells, and siRNA-mediated downregulation of PTEN could rescue the effects on NSCLC cells induced by anti-miR-21. Taken together, these results provide evidence to show the promotion role of miR-21 in NSCLC development through modulation of the PTEN signaling pathway.     

Conversion of pancreatic cells to hepatocytes

Transdifferentiation is the name used to describe the conversion of one differentiated cell type to another. During development, the liver and pancreas arise from the same region of the endoderm and cells from the two organs can transdifferentiate in the adult under different experimental procedures. We have produced two in vitro models for the transdifferentiation of pancreatic cells to hepatocytes. The first utilizes a pancreatic exocrine cell line AR42J-B13 and the second comprises cultures of mouse embryonic pancreas. We have analysed the pancreatic hepatocytes and they express a range of liver markers including albumin, transferrin and transthyretin. We also present evidence for (i) the molecular mechanism which regulates the conversion between pancreas and liver and (ii) the cellular basis of the switch in phenotype.     

Induction and regulation of acute phase proteins in transdifferentiated hepatocytes

Acute phase proteins (APPs) are predominantly synthesized in the liver and play an important role in restoring homeostasis. In the present study, we set out to answer two questions using transdifferentiated hepatocytes induced from pancreatic cells as a model for studying the acute phase response. Firstly, do transdifferentiated hepatocytes express acute phase proteins following culture with glucocorticoid and cytokines? Secondly, what is the molecular basis of the induction of acute phase proteins in transdifferentiated hepatocytes? Hepatic transdifferentiation was induced in 11.5-day mouse embryonic pancreas or the pancreatic cell line AR42J-B13 (B13) by culture with dexamethasone. We found that acute phase proteins [alpha2-macroglobulin (MG), haptoglobin (Hp)] were induced in both systems following culture with dexamethasone. The combined treatment of dexamethasone and oncostatin M (OSM) enhanced the expression of the acute phase proteins in B13 cells and the mechanism of the up-regulation by the cytokine is probably mediated by phosphorylation of STAT3 and STAT1. In addition, ectopic expression of either C/EBPbeta or C/EBPalpha in B13 cells induced haptoglobin expression and culture with oncostatin M was sufficient to enhance the expression of haptoglobin in C/EBPbeta transfected cells from 18% to 43%. The results of the present study indicate transdifferentiated hepatocytes have the potential to be a useful model to study liver function in vitro.     

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.     

MicroRNAs differentially expressed in postnatal aortic development downregulate elastin via 3' UTR and coding-sequence binding sites

Elastin production is characteristically turned off during the maturation of elastin-rich organs such as the aorta. MicroRNAs (miRNAs) are small regulatory RNAs that down-regulate target mRNAs by binding to miRNA regulatory elements (MREs) typically located in the 3' UTR. Here we show a striking up-regulation of miR-29 and miR-15 family miRNAs during murine aortic development with commensurate down-regulation of targets including elastin and other extracellular matrix (ECM) genes. There were a total of 14 MREs for miR-29 in the coding sequences (CDS) and 3' UTR of elastin, which was highly significant, and up to 22 miR-29 MREs were found in the CDS of multiple ECM genes including several collagens. This overrepresentation was conserved throughout mammalian evolution. Luciferase reporter assays showed synergistic effects of miR-29 and miR-15 family miRNAs on 3' UTR and coding-sequence elastin constructs. Our results demonstrate that multiple miR-29 and miR-15 family MREs are characteristic for some ECM genes and suggest that miR-29 and miR-15 family miRNAs are involved in the down-regulation of elastin in the adult aorta.     

miR-129-5p调节小鼠乳腺上皮细胞内Igf-1的表达

MicroRNAs(miRNAs)是一类大约22个核苷酸的非编码RNA.它能通过调控生长因子表达,引发肿瘤形成、细胞增殖和组织器官发育.本文通过构建miR-129-5p靶基因序列的双荧光素酶报告载体分析了miR-129-5p与靶基因之间的关系,应用脂质体转染技术和实时荧光定量技术观察了miR-129-5p在小鼠乳腺上皮细胞中的表达量及其变化,通过CASY细胞活力仪检测转染后的细胞增殖与活力变化,采用Western 印迹方法检Igf-1的变化.结果表明：miR-129-5p在小鼠乳腺青春期表达最高,成功构建了Igf-1基因 3′UTR荧光素酶报告载体, miR-129-5p抑制其荧光素酶活性（P <0.01）,转染抑制子后miR-129-5p表达降低（P < 0.01）,胰岛素样生长因子（Igf-1)表达增强（P <0.05）,细胞增殖和活力增强（P <0.01）,结果提示：miR-129-5p可能通过抑制靶基因蛋白Igf 1的表达,进而抑制小鼠乳腺上皮细胞增殖和活力.     

The effects of MOTILIPERM on cisplatin induced testicular toxicity in Sprague–Dawley rats

Genome-wide miRNA expression profile has identified microRNA (miR)-96 as one of upregulated miRNAs in clinical bladder cancer (BC) tissues compared to normal bladder tissues. The aim of this study was to confirm the expression pattern of miR-96 in BC tissues and to investigate its involvement in carcinogenesis. Quantitative real-time PCR was performed to detect the expression levels of miR-96 in 60 BC and 40 normal control tissues. Bioinformatics prediction combined with luciferase reporter assay were used to verify whether the cyclin-dependent kinase inhibitor CDKN1A was a potential target gene of miR-96. Cell counting kit-8 and apoptosis assays were further performed to evaluate the effects of miR-96-CDKN1A axis on cell proliferation and apoptosis of BC cell lines. We validated that miR-96 was significantly increased in both human BC tissues and cell lines. According to the data of miRTarBase, CDKN1A might be a candidate target gene of miR-96. In addition, luciferase reporter and Western blot assays respectively demonstrated that miR-96 could bind to the putative seed region in CDKN1A mRNA 3′UTR, and significantly reduce the expression level of CDKN1A protein. Moreover, we found that the inhibition of miR-96 expression remarkably decreased cell proliferation and promoted cell apoptosis of BC cell lines, which was consistent with the findings observed following the introduction of CDKN1A cDNA without 3′UTR restored miR-96. Our data reveal that miR-96 may function as an onco-miRNA in BC. Upregulation of miR-96 may contribute to aggressive malignancy partly through suppressing CDKN1A protein expression in BC cells.     

Anti-miR-197 inhibits migration in HCC cells by targeting KAI 1/CD82

Aim

To investigate the metastatic effects and mechanisms of miR-197 in hepatocellular carcinoma (HCC).

Methods and results

The levels of miR-197 increased in HCC cells and tissues compared with a normal hepatic cell line (LO2) and adjacent nontumorous liver tissues, respectively. miR-197 expression negatively correlated with CD82 mRNA expression in these cell lines and tissues. Dual luciferase reporter assay and Western blot confirmed a direct interaction between miR-197 and CD82 3′UTR sequences. After miR-197 was silenced in HCC cells, CD82 expression increased. In the presence of human hepatocyte growth factor (HGF), cells silenced for anti-miR-197 exhibited elongated cellular tails and diminished lamellipodia due to reductions in both ROCK activity and the levels of Rac 1 protein. Downregulation of miR-197 along with the upregulation of CD82 in HCC cells resulted in the inhibition of HCC migration and invasion in vitro and in vivo.

Conclusion

Taken together, these data suggest that anti-miR-197 suppresses HCC migration and invasion by targeting CD82. The regulation of the miR-197/CD82 axis could be a novel therapeutic target in future HCC effective therapy.     

miR-21 targets the tumor suppressor RhoB and regulates proliferation, invasion and apoptosis in colorectal cancer cells

It has become increasingly clear that microRNAs play an important role in many human diseases including cancer. Here, we show that expression of miR-21 in HEK293 and several colorectal cancer cells was found inversely correlated with ras homolog gene family, member B (RhoB) expression. miR-21 expression significantly suppressed RhoB 3' UTR luciferase-reporter activity, but the inhibitory effect was lost when the putative target sites were mutated. Exogenous miR-21 over-expression mimicked the effect of RhoB knockdown in promoting proliferation and invasion and inhibiting apoptosis, whereas anti-miR-21 or RhoB expression yielded opposite effects, in colorectal cancer cells. These results suggest that miR-21 is a regulator of RhoB expression and RhoB could be a useful target in exploring the potential therapeutic benefits of miR-21 mediated tumor cell behaviors in colorectal cancer.     

MicroRNA expression profile in RAW264.7 cells in response to Brucella melitensis infection

MicroRNA (miRNA) is small non-coding RNA with approximate 22 nt in length. Recent studies indicate that miRNAs play significant roles in pathogen-host interactions. Brucella organisms are Gram-negative facultative intracellular bacteria that cause Brucellosis. Brucella strains infect macrophages and establish chronic infection by altering host life activities including apoptosis and autophagy. Here, we report a comprehensive analysis of miRNA expression profiles in mock- and Brucella-infected RAW264.7 cells using high-throughput sequencing approach. In total, 344 unique miRNAs were co-expressed in the two libraries, in which 57 miRNAs were differentially expressed. Eight differentially expressed miRNAs with high abundance were subjected to further analysis. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in apoptosis, autophagy and immune response. In particular, a total of 25 target genes are involved in regulating apoptosis and autophagy, indicating that these miRNAs may play important regulatory roles in the Brucella-host interactions. Furthermore, the interactions of miR-1981 and its target genes, Bcl-2 and Bid, were validated by luciferase assay. The results show that miR-1981 mimic up-regulated the luciferase activity of psiCHECK-2 Bcl-2 3' UTR, but the luciferase activity of psiCHECK-2 Bid 3' UTR was not changed significantly. Taken together, these data provide valuable framework on Brucella induced miRNA expression in RAW264.7 cells, and suggest that Brucella may establish chronic infection by regulating miRNA expression profile.