No abstract available.
RNA, Small Interfering

Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.
High-Throughput Screening Assays ; RNA Interference ; RNA, Small Interfering

Nuclear protein-1 (NUPR1) is a small nuclear protein that is responsive to various stress stimuli. Although NUPR1 has been associated with cancer development, its expression and roles in cholangiocarcinoma have not yet been described. In the present study, we found that NUPR1 was over-expressed in human cholangiocarcinoma tissues, using immunohistochemistry. The role of NUPR1 in cholangiocarcinoma was examined by its specific siRNA. NUPR1 siRNA decreased proliferation, migration and invasion of human cholangiocarcinoma cell lines (HuCCT1 and SNU1196 cells). From these results, we conclude that NUPR1 is over-expressed in cholangiocarcinoma and regulates the proliferation and motility of cancer cells.
Cell Line ; Cholangiocarcinoma ; Humans ; Immunohistochemistry ; Nuclear Proteins ; RNA, Small Interfering

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.
Autophagy* ; Carcinoma, Renal Cell ; Drug Resistance ; RNA, Small Interfering

OBJECTIVE: Human cervical cancer is caused by the high-risk types of human papillomavirus (HPV) such as HPV16, which possess the E6 and E7 oncogenes, whose expressions are a prerequisite for cancer development. We performed this study to compare the efficacy of antitumor activity by HPV siRNA which silences only E6 or both E6/E7. METHODS: We transfected siRNA 377 (HPV16 E6 siRNA), siRNA 3 (HPV16 E6 siRNA), and siRNA 198 (HPV16 E7 siRNA) into SiHa cell line (siRNA 377 silences only E6, and siRNA 3 and siRNA 198 silence both E6 and E7). We experimented cell counts and morphologic changes 24 and 48 hours after transfection and expressions of HPV16 E6/E7 mRNA by RT-PCR. RESULTS: siRNA 377, siRNA 3, and siRNA 198 suppressed the cell growth. siRNA 3 and siRNA 198 were more potent than siRNA 377 in cell growth suppression. siRNA 377 knocked down the expression of E6 mRNA, and both siRNA 3 and siRNA 198 knocked down the expression of E6/E7 mRNA. CONCLUSION: Our results suggest that simultaneous suppression of E6 and E7 was more potent than E6-specific suppression in cancer cell growth.
Cell Count ; Cell Line ; Humans ; Oncogenes ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms

Acute myeloid leukemia is a well characterized blood cancer in which the unnatural growth of immature white blood cell takes place, where several genes transcription is regulated by the micro RNAs (miRNAs). Argonaute (AGO) protein is a protein family that binds to the miRNAs and mRNA complex where a strong binding affinity is crucial for its RNA silencing function. By understanding pattern recognition between the miRNAs-mRNA complex and its binding affinity with AGO protein, one can decipher the regulation of a particular gene and develop suitable siRNA for the same in disease condition. In the current work, HOXA9 gene has been selected from literature, whose deregulation is well-established in acute myeloid leukemia. Four miRNAs (mir-145, mir-126, let-7a, and mir-196b) have been selected to target mRNA of HOXA9 (NCBI accession No. NM_152739.3). The binding interaction between mRNAs and mRNA of HOXA9 gene was studied computationally. From result, it was observed mir-145 has highest affinity for HOXA9 gene. Furthermore, the interaction between miRNAs-mRNA duplex of all chosen miRNAs are docked with AGO protein (PDB ID: 3F73, chain A) to study their interaction at molecular level through an in silico approach. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding of AGO-assisted miRNA based gene silencing mechanism in HOXA9 gene associated in acute myeloid leukemia computationally.
Computer Simulation ; Gene Silencing ; Humans ; Hydrogen Bonding ; Leukemia, Myeloid, Acute* ; Leukocytes ; MicroRNAs* ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering

Melanin is a pigment produced from tyrosine in melanocytes. Although melanin has a protective role against UVB radiation-induced damage, it is also associated with the development of melanoma and darker skin tone. Tyrosinase is a key enzyme in melanin synthesis, which regulates the rate-limiting step during conversion of tyrosine into DOPA and dopaquinone. To develop effective RNA interference therapeutics, we designed a melanin siRNA pool by applying multiple prediction programs to reduce human tyrosinase levels. First, 272 siRNAs passed the target accessibility evaluation using the RNAxs program. Then we selected 34 siRNA sequences with ΔG ≥−34.6 kcal/mol, i-Score value ≥65, and siRNA scales score ≤30. siRNAs were designed as 19-bp RNA duplexes with an asymmetric 3′ overhang at the 3′ end of the antisense strand. We tested if these siRNAs effectively reduced tyrosinase gene expression using qRT-PCR and found that 17 siRNA sequences were more effective than commercially available siRNA. Three siRNAs further tested showed an effective visual color change in MNT-1 human cells without cytotoxic effects, indicating these sequences are anti-melanogenic. Our study revealed that human tyrosinase siRNAs could be efficiently designed using multiple prediction algorithms.
Dihydroxyphenylalanine ; Gene Expression ; Humans ; Melanins ; Melanocytes ; Melanoma ; Monophenol Monooxygenase* ; RNA ; RNA Interference ; RNA, Small Interfering* ; Skin Pigmentation ; Tyrosine ; Weights and Measures

OBJECTIVE: This study was to identify small inhibitory RNAs (siRNAs) that are effective in inhibiting growth of cervical cancer cell lines harboring human papilloma virus (HPV) and to examine how siRNAs interact with interferon beta (IFN-beta) and thimerosal. METHODS: The HPV18-positive HeLa and C-4I cell lines were used. Four types of siRNAs were designed according to their target (both E6 and E7 vs. E6 only) and sizes (21- vs. 27-nucleotides); Ex-18E6/21, Ex-18E6/27, Sp-18E6/21, and Sp-18E6/27. Each siRNA-transfected cells were cultured with or without IFN-b and thimerosal and their viability was measured. RESULTS: The viabilities of HPV18-positive tumor cells were reduced by 21- and 27-nucleotide siRNAs in proportion to the siRNA concentrations. Of the two types of siRNAs, the 27-nucleotide siRNA constructs showed greater inhibitory efficacy. Sp-18E6 siRNAs, which selectively downregulates E6 protein only, were more effective than the E6- and E7-targeting Ex-18E6 siRNAs. siRNAs and IFN-beta showed the synergistic effect to inhibit HeLa cell survival and the effect was proportional to both siRNA and IFN-beta concentrations. Thimerosal in the presence of siRNA exerted a dose-dependent inhibition of C-4I cell survival. Finally, co-treatment with siRNA, IFN-beta, and thimerosal induced the most profound decrease in the viability of both cell lines. CONCLUSION: Long (27-nucleotides) siRNAs targeting E6-E7 mRNAs effectively reduce the viability of HPV18-positive cervical cancer cells and show the synergistic effect in combination with IFN-b and thimerosal. It is necessary to find the rational design of siRNAs and effective co-factors to eradicate particular cervical cancer.
Cell Line ; Cell Survival ; HeLa Cells ; Humans ; Interferon-beta ; Papilloma* ; RNA* ; RNA, Messenger ; RNA, Small Interfering ; Thimerosal ; Uterine Cervical Neoplasms*

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.
Eosinophils ; Mutagenesis ; RNA, Small Interfering ; T-Lymphocytes ; Th2 Cells ; Transcription Factors ; Transcriptional Activation ; Transfection

OBJECTIVE: To evaluate the effects of both exogenous and endogenous osteopontin on normal and malignant ovarian epithelial cell growth, and on paclitaxel chemo-resistance. METHODS: The ovarian cancer cell line OV429, which showed low level of endogenous osteopontin and paclitaxel sensitive cell line OV420, which showed high level of endogenous osteopontin, and a normal ovarian epithelial (HOSE: Human ovarian surface epithelial) cells were treated with purified osteopontin. Furthermore, OV420 was treated with osteopontin siRNA alone or in combination with paclitaxel. Proliferation rates and cell cycle progression of treated cells were determined by the tetrazolium colorimetric (XTT) assay and FACS analysis, respectively. RESULTS: Exogenous osteopontin increased the proliferation rate of OV429 and OV420 but had negligible effect on normal HOSE. Ovarian cancer cell lines treated with siRNA showed significantly reduced the growth rates (P<0.05), and they were arrested in G2/M phase of the cell cycle. Furthermore, OV420 treated with paclitaxel in the presence of osteopontin siRNA showed significantly decreased the survival rate. CONCLUSION: Osteopontin promote cell growth in malignant but not in normal ovarian epithelial cells, and may confer paclitaxel-resistance by adhesion to each cell and minimized the cell surface which exposure to chemo-agents.
Cell Cycle ; Cell Line ; Epithelial Cells* ; Humans ; Osteopontin* ; Ovarian Neoplasms ; Paclitaxel ; RNA, Small Interfering ; Survival Rate