miR-513a-3p sensitizes human lung adenocarcinoma cells to chemotherapy by targeting GSTP1.

Cisplatin is a classic chemotherapy agent used for treating human non-small cell lung cancer (NSCLC). However, cisplatin resistance is a challenge against successful clinical use. Glutathione S-transferase P1 (GSTP1) has been reported to contribute to cisplatin resistance in many studies. MicroRNAs (miRNAs) are short non-coding RNAs that are 21-25 nucleotides in length. They play a role in post-transcriptional gene regulation by inducing repression and/or mRNA degradation. Recent studies have shown that miRNAs are responsible for cisplatin resistance. This study aims to determine whether deregulated miRNAs can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1. Real-time RT-PCR revealed that GSTP1 mRNA expression was 2.7 ± 0.38 folds (p=0.039) upregulated in A549/CDDP cells, compared with the parental A549 cells, while miR-513a-3p expression was 0.34 ± 0.03 folds (p=0.023) downregulated. Luciferase activity assay proved that GSTP1 was a target gene of miR-513a-3p, which was confirmed by Western blot analysis. Furthermore, CCK-8 assay showed that overexpression of miR-513a-3p could enhance cisplatin-induced apoptosis in human lung adenocarcinoma cell lines, A549/CDDP and SPC-A-1. In conclusion, our data demonstrated that miR-513a-3p can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1.

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA.

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.

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-beta1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells.

BACKGROUND: In 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. METHODS: The 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. RESULTS: Among 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-beta1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-beta1. CONCLUSIONS: We 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.

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

UNLABELLED: 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.

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.

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.

Survivin knockdown combined with apoptin overexpression inhibits cell growth significantly.

The initiation and progression of tumor is regulated by multiple genes. Survivin belongs to the inhibitor of apoptosis protein (IAP) family and is overexpressed in most types of human tumors. Apoptin, originally identified from chicken anemia virus (CAV), can specifically induce apoptosis of human tumor cells rather than normal cells. In this study, survivin expression was silenced by microRNA (miRNA)-mediated RNA interference (RNAi); meanwhile, the engineered miRNA vector was also designed to express apoptin gene. The apoptosis and cell growth were then examined by flow cytometry and MTT assay. The miRNA-mediated knockdown of survivin in combination with apoptin overexpression significantly induced apoptosis and inhibited cell growth. Importantly, the combined strategy was more effective on inducing apoptosis and inhibiting cell growth than either survivin downregulation or apoptin overexpression alone. Taken together, the combined strategy offers potential advantages in control of tumorigenesis, and thus deserves further research as a preferred approach in cancer gene therapy.

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.

[Characterization of the changes in protease of Deinococcus radiodurans following irradiation].

The activity of protease from Deinococcus radiodurans R1 (DRR1) recovered from UV light and gamma-ray irradiation were characterized by SDS-PAGE zymography or FITC-labeled casein as substrate. It was demonstrated that proteases with large molecular weight were abundantly and constitutively expressed in DRR1, which consistent with the hypothesis that proteases were among the PHX (predicted highly expressed) proteins in DRR1. A broad range of gelatinolytic protease with molecular mass more than 140 kD and caseinolytic protease with molecular mass more than 120 kD were present in DRR1 cell. The prominent gelatinolytic protease of 174 kD was also active when denatured by SDS and displayed different operation dynamics during the repair process post UV-irradiation and gamma-irradiation, the protease showed its highest activity during gamma-irradiation and in the late recovery stage post-irradiation. Some proteases were demonstrated to be induced specifically by irradiation which suggests that these proteases may regulate breakdown of proteins responsible in sequential pathway, and an elaborate and fine tuning protease system may exist in D. radiodurans during the repair process post irradiation. The expression of protease was also influenced by the nutrition of the culture. A rod-shaped and radiation resistant bacterium RR533.2 isolated from soil in Beijing, China was revealed to display similar zymography pattern using gelatin or casein as substrate.

Genotype and allele frequency of the variable number tandem repeat polymorphism in the VNTR6-1st of the of human telomerase reverse transcriptase (hTERT) gene in Chinese population.

The variable number of tandem repeat (VNTR) polymorphism in the intron 6 of the human telomerase reverse transcriptase(hTERT) was investigated. Seven alleles of 18, 20, 21, 22, 23, 26 and 35 repeats of the 36 bp VNTR6-1st and 14 genotypes were identified among 210 unrelated healthy Chinese individuals (Han people) living in Beijing. The 20, 22 and 35 repeats were the most frequent alleles which accounted for 94.76% of the total alleles observed. The allele frequency of the Chinese population showed no difference with that of the Korea population living in Pusan except for allele of 35 repeats. But the 5' flanking region of VNTR6-1st polymorphic sequence of the Chinese population included a 53-bp insert compared with that of korea population except for some alleles of 22 repeats.

[Isolation and characterization of a novel radiation-resistant rod-shaped bacterium].

A novel radiation-resistant bacterium was isolated from soil of lake bank in Beijing. The bacterium produced orange-pigmented colonies and formed rod-shaped cells that stained gram negative alike the Deinobacter grandis previously described by Japan's scientist. It was found with electron microscopy that the isolate is of 0.6 microm - 1.6 microm size and has a 30-40 nm thickness of cell wall, being slightly larger and thicker than the Deinobacter grandis. There was a difference in the concentration and molecule weight of catalase between the isolate and the Deincoccus radiodurans R1. The deoxyribonucleic acid guanine plus cytosine (G + C) base ratio was 70.7 mol%. 16S RNA gene sequencing also showed that this rod-shaped bacterium possessed a high homology with the Deinobacter grandis, suggesting that it might be classified into the genus Deinobacter and constitute a new species in this genus.

[A short tandem repeat polymorphism in the inducible nitric oxide synthase gene in Chinese population].

Polymorphism of the (CCTTT)n repeat, a short tandem repeats (STR) located in promoter region of the inducible nitric oxide synthase (iNOS) gene, was analyzed in a total of 316 Chinese healthy subjects. Twelve alleles and forty-nine genotypes were identified. Three alleles that had not been reported previously, namely 17-repeats, 18-repeats and 19-repeats, were found in Chinese population. Mendelian inheritance of the allele in a family composed of three generations was determined. Some of the observed allele frequency of this STR locus in Chinese differed significantly from that of Caucasians in England.

Deinococcus radiodurans CatB Gene Cloning and Expression in Escherichia coli.

A 1 611 bp length complete coding sequence of the catalase (Cat) was obtained through bioimformatical analysis of the database of D.radiodurans genome, and then was amplified from D. radiodurans genomic DNA by polymerase chain reaction. The amplified gene was cloned into pKK223-3 vector and transformed into E.coli UM2, a Cat-deficient mutant. Staining of non-denaturing polyacrylamide gels for Cat activity demonstrated that pKK223-3 insert encoded a Cat that co-migrated with the CatB found in D.radiodurans cell lysates. Expression of CatB gene from D.radiodurans in E.coli UM2 restored the resistance to H(2)O(2) at low concentrations.