The psiTPTE22 gene has been designated as a TPTE pseudogene. Our study found that the 5' part of psiTPTE22 has no sequence similarity to TPTE and contains a 3.8-kb human endogenous retrovirus (HERV) element. Because of the HERV element, the 5' part of psiTPTE22 (psiTPTE22-HERV) expresses independently as a gene. Comparison between the DNA sequences of humans and chimps indicated that psiTPTE22-HERV is human specific. We identified 3 alternatively spliced transcript variants from psiTPTE22-HERV by a PCR-based strategy, which use the transcriptional termination signal contained in the HERV element. A 402-nt ORF was contained in the 2 longer transcripts. Western blotting using antibodies produced with chemically synthesized peptide confirmed that a 15-kDa protein was translated from this ORF. RT-PCR results indicated that the ORF-containing transcripts were mainly expressed in psiTPTE22-HERV-expressing samples. Real-time quantitative RTPCR results showed that expression of the 402-nt ORF was upregulated in normal tissues of kidney, liver, stomach, and lung but downregulated in corresponding tumor tissues. This gene is located near the centromere of chromosome 22 and has a high GC content around the promoter region. Bisulfite sequencing PCR results indicated that it is silenced in cancers by DNA methylation. The expression of psiTPTE22-HERV can be recovered in cancer cells using DNA methylation and histone deacetylase inhibitors. These results suggest psiTPTE22-HERV is regulated epigenetically by DNA methylation. Our study paved the way for further study on an interesting HERV-related human-specific gene, which is silenced in cancers by DNA methylation.

Objective To investigate the role of polo-like kinase-1(PLK1) on proliferation and telomerase activity of human colon cancer cells. Methods After SW480 colon cancer cell were transfected with small interfering RNA(siRNA)against PLK1,the real time RT-PCR and Western blot assay were used to examine PLK1 gene expression in all cancer cells.The proliferation and telomerase activity of cancer cells were determined by MTT and telomeric repeat amplification protocol enzyme-linked immunoadsordent assay(TRAP-ELISA),respectively. Results Expression of PLK1 of SW480 cancer cell transfected with siRNA down-regulated significantly.Transfection of PLK1 siRNA resulted significantly in inhibition of colon cancer cell in vitro.The results from TRAP showed that cancer cells exhibited marked apoptosis,in time-and dose-dependent pattern. Conclusions RNA interference PLK1 could inhibit proliferation through inducing suppressing telomerase activity of human colon cancer cell.

Up-regulation of human endogenous retroviruses (HERVs) is associated with many diseases, including cancer. In this study, an H family HERV (HERV-H)-related gene was identified and characterized. Its spliced transcript lacks protein-coding capacity and may belong to the emerging class of noncoding RNAs (ncRNAs). The 1.3-kb RNA consisting of four exons is transcribed from an Alu element upstream of a 5.0-kb structurally incomplete HERV-H element. RT-PCR and quantitative RT-PCR results indicated that expression of this HERV-related transcript was negatively associated with colon, stomach, and kidney cancers. Its expression was induced upon treatment with DNA methylation and histone deacetylation inhibitors. A BLAT search using long terminal repeats (LTRs) identified 50 other LTR homogenous HERV-H elements. Further analysis of these elements revealed that all are structurally incomplete and only five exert transcriptional activity. The results presented here recommend further investigation into a potentially functional HERV-H-related ncRNA.

Up-regulation of human endogenous retroviruses (HERVs) sometimes occurs in cancer. This study identifies a novel HERV (HERV-HX) in colon tumor tissues, which is an H family HERV (HERV-H) element located on chromosome Xp22.32. The full-length transcript sequence was identified and characterized. Quantitative RT-PCR indicated it is up-regulated in colon tumor samples. Using RT-PCR to analyze the transcription of the env-deleted HERV-HX and other env-intact HERV-H elements, it was demonstrated that transcription of HERV-H in colon cancer is not associated with the env gene. A 17-bp sequence was found within the 5′ U3 region of HERV-HX, and only 8-bp sequences existed on its homologous U3 regions in the corresponding loci. Promoter activity assays indicated that replacing the 17-bp sequence with an 8-bp sequence or deleting it reduced its activity, suggesting that the 17-bp sequence is important to the expression of HERV-HX.

A wide band-pass filter has been presented based on one-dimensional metallo-dielectric ternary photonic crystal. ZnS, Ag and MgF2 are used as the materials of the photonic crystal. The forbidden and allowed bands of the photonic crystal are determined by using the band-edge analysis. The result indicates that the filter has a wide pass band in the visible region and it can block ultraviolet and infrared light. Simulation of the transmission spectra shows that the filter has decent transmittance in the pass band at broad angle incidence ranging from 0° to 70°, which can meet the need of practical use.

Objective: To detect the mRNA levels of AK097082 and BC031001 encoded by psiTPTE22 gene in order to determine whether psiTPTE22 gene is the microarray-detected over-expressed gene in colon cancer and explore the role of psiTPTE22 in carcinogenesis.Methods: RT-PCR and real-time fluorescence quantitative PCR were used to detect the transcription of AK097082 and BC031001 encoded by psiTPTE22 gene in kidney,liver,stomach,lung,and colon cancer tissues and adjacent normal tissues as well as nine cancer cell lines and placenta tissues.Results: RT-PCR showed that only the mRNA of BC031001 encoded by psiTPTE22 was expressed in colon tissues.Real-time quantitative PCR assay and F test confirmed that there was no significant difference between colon cancer and adjacent normal tissues(P0.05).So psiTPTE22 was not the specific up-regulated gene in colon cancer detected by microarray.Real-time quantitative PCR assays also confirmed that BC031001 was over-expressed in kidney,liver,stomach,and lung normal tissues but had low expression in corresponding cancer tissues.BC031001 had no expression or weak expression in several cancer cell lines but had relatively high expression in placenta tissues.F tests found that the difference was significant in the expression level of BC031001 between kidney,liver,stomach,and lung adjacent normal tissues and the corresponding cancer tissues(P0.05).Conclusions: The mRNA expression of BC031001 encoded by psiTPTE22 gene has negative correlation with several kinds of cancer.Further studies are needed to reveal the mechanism underlying its down-regulation and its role in carcinogenesis.

OBJECTIVE:To study the impact of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of MG-63 and HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma.METHODS:One pair of siRNA targeting the cyclin A2 mRNA and a pair of nonsense siRNA were designed according the current criteria. SiRNA was chemically synthesized and purified. The siRNA was transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast (HSF) cells via oligofectamine. Cells transfected with nonsense siRNA served as the negative control and those only treated with PBS as the blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and colony-forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA treated MG-63 cells were examined.RESULTS:1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L cyclin A2-siRNA can reduced cyclin A2 mRNA and protein expression respectively by 9.43%, 56.35%, 79.17% and 84.30% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. After 48 h treatment with 10 nmol/L siRNA, MG-63 cells were arrested in G0/G1 phase and the proliferation of this tumor cell was suppressed by 39.06% 48 h after transfection. Furthermore, the treated MG-63 cells showed less colony-forming ability. Increasing the siRNA concentration to 50 nmol/L can further inhibit the proliferation of MG-63 cells by 54.94%. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 mRNA and protein expression in HSF reduced 58.13% 48 h after treatment by 50 nmol/L siRNA, these cells exhibited only a slight change in cell cycle, and no clear inhibition of proliferation and impaired plate colony-forming ability was observed.CONCLUSION:Cyclin A2 gene maybe served as a potential target for tumor therapy. RNA interference induces obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently downregulate the proliferation of MG-63 cells. There is few inhibitory effect on the proliferation by siRNAs for HSF cells. These results indicate that siRNAs against cyclin A2 could become a potential antiproliferative tool in future antitumor therapy.

OBJECTIVE To study the inhibitory effect of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of osteosarcoma MG-63 and human normal skin fibroblast HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma. METHODS Three pairs of siRNAs targeting cyclin A2 mRNA and a pair of nonsense siRNA were designed according to the current criteria. SiRNAs were chemically synthesized and purified. The siRNAs were transfected into MG-63 cells and HSF cells via oligofectamine. The cells transfected with nonsense siRNA served as negative control group and those only treated with PBS as blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and clone forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA-treated MG-63 cells were examined. RESULTS Although all three siRNAs could reduce the cyclin A2 expression, siRNA, appeared to be the most effective. After 48 h treatment with siRNA1, cyclin A2 mRNA and protein expression in MG-63 cells was significantly reduced by nearly 80% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. MG-63 cells treated with siRNA1 were arrested at G0/G1 phase by 80.1% and the proliferation of these tumor cells was suppressed 48 h after transfection. Furthermore, MG-63 cells showed a decreased colony forming ability after siRNA1 treatment. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 expression in HSF reduced by nearly 60% after treatment by siRNA1 for 48h, these cells exhibited only a slight change in cell cycling, and neither clear inhibition of proliferation nor impaired colony forming ability was observed. CONCLUSION Cyclin A2 is critical for proliferation of MG-63 cells. Cyclin A2-siRNAs can induce obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently down-regulate the proliferation of MG-63 cells. There is little effect on the proliferation of siRNA-treated HSF cells. Those results indicate that siRNAs against cyclin A2 may become a potential antiproliferative tool in future antitumor therapy.

Bulbus Fritillariae (BF) is the most commonly used antitussive herb in China. There are nine species of Fritillaria recorded as the drug BF in the Chinese Pharmacopoeia. Bulbus Fritillariae cirrhosae (BF cirrhosae) is a group that includes four species of BF; these four species come from wild sources with higher efficiency and lower toxicity compared to the other five species of BF. Due to reasons of carelessness and reduced costs, the other five species are often sold as BF cirrhosae. Analysis through appearance, microscopic and chemical techniques has limitations. Identifying botanical resources is a primary step in the standardization of herbal medicine. In the present article, the internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal DNA (nrDNA) of nine species and one variety of Fritillaria genus have been sequenced. A mutation site in the ITS1 region among BF cirrhosae and other species of BF has been found and can be recognized by the restriction endonuclease SmaI. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the nuclear ribosomal ITS1 region was used to differentiate BF cirrhosae from other species of BF and is a successful method in distinguishing the subgroups.

Lonicera japonica THUNB. is a commonly used anti-inflammatory herbal medicine. The therapeutic effectiveness of L. japonica depends significantly on its geographical origin. However, it is difficult to define criteria for confirming geographical authenticity using microscopic and chemical characteristics. In the present study, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA loci of L. japonica from different origins and related species was sequenced. The mutation site found in the ITS region from geo-authentic L. japonica can be recognized by the restriction endonuclease EcoN I. Since PCR products from geo-authentic L. japonica cannot be digested completely, a quantitative restriction fragment length polymorphism analysis method was developed. The cleavage rate of PCR products by EcoN I was determined to be more than 70% in all geo-authentic L. japonica and less than 20% in non-geo-authentic L. japonica and other species from genus Lonicera. The rate correlated remarkably with the geographical origin of L. japonica. Therefore, this method can be used to classify geo-authentic L. japonica.

目的 研究靶向人乳头状瘤病毒HPV-6b型E7基因的二聚体小干扰RNA(siRNA-HPV-6bE7)对靶基因表达的沉默作用.方法 建立并筛选稳定表达HPV-6bE7基因的B16和293T转染细胞株,用脂质体转染法将体外合成的siRNA-HPV-6bE7转染上述细胞株,采用实时荧光定量PCR分析靶基因HPV-6bE7的mRNA表达情况.结果 用不同浓度的siRNA-HPV-6bE7转染细胞48 h,50nmol/L浓度对B16细胞中靶基因表达的抑制作用最强(抑制率87.05%),1 nmol/L抑制作用较低(9.14%);而在293T细胞,10 nmol/L的siRNA对靶基因表达的抑制效应最大(78.87%),1 nmol/L仍有一定抑制作用(46.92%).50 nmol/L siRNA-HPV-6bE7转染B16细胞后,靶基因的mRNA表达在24 h内开始受抑制(32.47%),48 h作用最强(74.72%),96 h作用很低(8.91%);25 nmol/L和10 nmol/L的siRNA转染293T细胞后,均在24 h内起效(26.66%、20.31%),抑制作用至少能维持72 h(65.93%、35.23%).结论 siRNA-HPV-6bE7对B16和293T细胞外源性靶基因表达均有较强的特异性沉默作用,在不同细胞株达到最大抑制效应的siRNA浓度不同,但时效曲线的变化趋势基本一致,siRNA均在24 h内起效,48～72 h达到高峰,抑制作用至少能维持72 h.本研究结果为下一步在动物或临床进行siRNA干扰试验提供了实验依据.

人类内源性逆转录病毒(HERV)是几百万年前整合到人类基因组中的逆转录病毒的残余物,约占了整个基因组的8%.大部分HERV元件在进化过程中由于突变、缺失等的积累,已没有编码能力.但仍有少数由于正性选择的压力,完整的开放阅读框被保留了下来.它们在一些特定的组织或分化发育的特定阶段表面,可能具有重要的生理意义.而在某些疾病情况下的高表达水平提示其可能与疾病的发生发展相关.由于最初插入位点的关系或后来的转座作用,HERV元件相互之间以及对基因组中的其他基因都可能有影响.对HERV目前还了解甚少,该文将对HERV的生物学功能以及对HERV-K、-W、-H3个家族的研究概况作一简要综述。

BACKGROUND & OBJECTIVE We have identified a novel gene HERV-H-X from colon cancer tissue recently. This research was to analyze the deletion of the env region of HERV-H-X, to compare its expression with that of the env open reading frames (ORFs) in other HERV-H proviruses, and to study its differential expression in colon cancer and normal colon tissues. METHODS Multiple alignments were performed for HERV-H-X and 3 other HERV-H proviruses (HERV-H/env62, HERV-H/env60, and HERV-H/env59) containing intact env ORF. HERV-H-X-specific polymerase chain reaction (PCR) and env ORF-common PCR for HERV-H/env62, env60 and env59 were conducted to study their expression in 8 pairs of colon cancer and adjacent normal tissues. Real-time quantitative PCR was conducted to study the differential expression of HERV-H-X in 17 pairs of colon cancer and adjacent normal tissues. RESULTS The missing part in the env region of HERV-H-X corresponded to env ORFs in HERV-H/env62, HERV-H/env60 and HERV-H/env59. HERV-H-X was only detected in colon cancer tissues, while env ORFs were detected both in cancer and normal tissues. The expression of HERV-H-X was up-regulated in colon cancer tissues by 24.9 folds of that in normal tissues (P<0.01). CONCLUSION HERV-H-X is highly expressed in colon cancer, but its up-regulation is not related to env gene as it lacks an env ORF.

To study knockdown effect of small interfering RNA (siRNA) to PLK1 (Polo-like kinase 1) mRNA in colorectal cancer cell line SW480 and its mitosis and growth was changed. Ten special siRNA molecules were designed targeting different sites of PLK1 mRNA sequence and chemically synthesized. The siRNA molecules were transfected into SW480 by Oligofectamine. The gene mRNA level was assayed by Real-Time PCR. The changes of PLK1 protein, SW480 cell cycle and survival percentage was checked by Western-blot, Flow cytometry and Cell counter assays respectively. All 10 siRNA molecules knocked PLK1 mRNA down in different level. Of them P1, P4 and P9 showed over 80% knockdown efficiency and the others had more than 20% knockdown efficiency to PLK1 mRNA. The best knockdown effect over 95% of all groups was at 25 nmol/L of a mixture with P1, P4 and P9 siRNA equally. In this situation the protein was very less and the cells were blocked at G2 phase of cell cycle. After 72 h cell survival percentages were consistent with PKL1 mRNA level change by siRNA gradient concentration. The results showed that siRNA targeting PLK1 mRNA had effectively knocked PLK1 mRNA down in SW480 cell line. And a blended siRNAs held the best knockdown effect. The cell was blocked on the mitosis and growth.

AIM: To investigate the effects of polo-like kinase-1 (PLK1) antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis and cell cycle of human colon cancer cell line SW480.METHODS: After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan.RESULTS: The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index (AI) induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G(2)/M compared with control groups.CONCLUSION: PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.

Small interfering RNA (siRNA) technology is a powerful tool to knockdown gene expression in mammalian cells including genes of viral origin. To study the inhibition of hepatitis B virus (HBV) by combined siRNAs in vitro, HepG2 2.2.15, a human hepatoblastoma cell that constitutively produces HBV particles, was transfected with 12 siRNAs that were designed to target different regions on HBV genome. Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture media, and HBV replication was determined by real-time quantitative PCR. The results showed that HBV replication could be inhibited by treatment with these siRNAs in a dose-dependent manner. The expression of HBsAg was inhibited by 80% and 90% with 80 nmol/L single siRNA treatment and 20 nmol/L combined siRNAs treatment, respectively. HBeAg was inhibited by about 60% with single siRNA treatment, and the inhibition didn't increase much in the case of combined siRNAs treatment. HBV replication was inhibited by 90% with 80 nmol/L single siRNA treatment or 20 nmol/L combined siRNAs treatment. These data provide solid evidence for developing new approaches in HBV treatment with combined siRNAs.

We identified a novel gene ST13 from a subtractive cDNA library of normal intestinal mucosa in 1993, more studies showed that ST13 was a co-chaperone of Hsp70s. Recently we detected the ST13 gene expression in tumor tissue and adjacent normal tissue of the same colorectal cancer patient and investigated if the ST13 gene expression might have any prognostic value. Analysis was performed at molecular level by reverse transcription-PCR using real-time detection method. We measured two genes simultaneously, ST13 as the target gene and glyceraldehydes-3-phosphate dehydrogenase as a reference gene, in primary colorectal tumor specimens and tumor-adjacent normal mucosa specimens from 50 colorectal cancer patients. The expression levels of the ST13 gene were significantly decreased in primary tumors compared with adjacent mucosa (P<0.05). But there were no significant differences in the expression of ST13 as compared with different Dukes' stage, tumor differentiation grade, invasion depth, lymph node metastasis and disease-specific survival.

OBJECTIVES:To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro.METHODS:(1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry.RESULTS:Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment.CONCLUSION:Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.