Objective To summarize the clinicopathological features,immunohistochemical characteristics,HOX transcript antisense RNA(HOTAIR)in situ hybridization status,treatment,and prognosis of myxopapillary ependymoma(MPE). Methods A total of 17 patients diagnosed with MPE based on pathological evidence in the Department of Pathology of Peking Union Medical College Hospital from November 2006 to July 2023 were selected,and the clinicopathological data of these patients were collected.Immunohistochemical staining for trimethylation at lysine 27 of histone H3 (H3K27me3),glial fibrillary acidic protein(GFAP),and epithelial membrane antigen(EMA)and alcian blue-periodic acid Schiff(AB-PAS)staining were performed in all the patients.Sixteen patients with spinal ependymomas were selected as the control group.Tissue microarrays were prepared from 17 MPE patients and the control group.HOTAIR ISH was performed and semi-quantitatively scored,and the scores of the two groups were compared by the Wilcoxon rank-sum test. Results The 17 MPE patients aged 14-64 years,with the mean age of(37.48±16.10)years and the male-to-female ratio of 0.7∶1.Their clinical manifestations mainly included lumbosacral and lower limb pains.Microscopically,tumor cells were arranged in a papillary pattern around fibrovascular axis,with abundant myxoid materials,and tumor cells were arranged in a loose meshwork in some patients.The immunohistochemical staining results showed that 17(100%),10(58.82%),and 8(47.06%)patients expressed GFAP,EMA,and D2-40,respectively,and 2(11.76%)patients lacked expression of H3K27me3.AB-PAS staining showed blue myxoid materials in all the 17(100%)patients.HOTAIR was expressed in both MPE and control groups,with higher semi-quantitative score in the MPE group than in the control group(P=0.004).Twelve patients were followed up,with a median follow-up period of 65.50 months,during which three patients showed recurrence.Conclusions MPE exhibits typical pathological features,and the combination with immunohistochemical staining for GFAP and EMA as well as AB-PAS staining facilitates diagnosis of this disease.A small number of patients loss the expression of H3K27me3.HOTAIR is highly expressed in MPE but lacks specificity,which limits its auxiliary diagnostic value.The overall prognosis of MPE is favorable,with a few patients experiencing recurrence.
Humans ; Ependymoma/metabolism* ; Male ; Adult ; Female ; Adolescent ; Middle Aged ; In Situ Hybridization ; Young Adult ; RNA, Antisense/genetics* ; Immunohistochemistry ; Prognosis

Natural antisense transcripts (NAT) and alternative polyadenylation (APA) of messenger RNA (mRNA) are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense (S-AS) gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA (pA) site in sense gene RNASEH2C, which generated longer 3' untranslated region (3'UTR) and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene's protein level in RNASEH2C-KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.
Cell Proliferation ; genetics ; Evolution, Molecular ; Gene Expression Regulation ; genetics ; HEK293 Cells ; Humans ; Polyadenylation ; genetics ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Ribonuclease H ; genetics ; Serine-Arginine Splicing Factors ; metabolism ; Transcription, Genetic ; Up-Regulation ; genetics

OBJECTIVETo detect the presence of p15 antisense RNA(p15AS) in acute myeloid leukemia(AML).

METHODSp15AS and p15 mRNA in two leukemia cell lines was detected with strand-specific primer RT-qPCR. To explore the connection between p15AS and AML, 43 patients with newly diagnosed AML and 21 patients with benign diseases (Iron deficiency anemia) as controls were enrolled. The expression level of p15AS in bone marrow cells derived from the patients and the controls were determined by strand-specific primer RT-qPCR, and its relationship with clinical features was analyzed.

RESULTSThe two AML lines displayed high p15AS and low p15 expression. Samples derived from the AML patients showed relatively increased expression of p15AS and down-regulated p15 expression in their bone cells. In contrast, the 21 controls showed high expression of p15 but relatively low expression of the p15AS. Compared with the normal controls, the expression levels of p15 protein were significantly lower among the AML patients (P<0.01). No relationships were detected between the level of p15AS and the patient's age, gender, FAB subtype, total white blood cell count, platelet count, proliferative degree of bone marrow cell and karyotype classification (P>0.05 for all comparisons).

CONCLUSIONHigh expression of p15 antisense RNA was frequently found among AML patients, and may play an important role in epigenetic silencing of p15.

Adult ; Aged ; Bone Marrow Cells ; metabolism ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Epigenesis, Genetic ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Antisense ; genetics ; metabolism ; Up-Regulation ; Young Adult

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.
DNA Primers ; Down-Regulation ; Gene Expression ; Intramolecular Transferases ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; RNA, Antisense ; Saccharomyces cerevisiae ; enzymology ; genetics ; Squalene ; analogs & derivatives ; metabolism ; Transformation, Genetic

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

OBJECTIVETo observe the effects of miR-224 antisense oligonucleotide (ASO) on the proliferation and apoptosis of gastric cancer cells in vitro and vivo.

METHODSThe expression of miR-224 in the cancer tissues and their adjacent tissues in 120 gastric cancer patients were detected by real-time quantitative PCR. The biological effects of miR-224 ASO on human gastric cancer SGC7901 cells was assessed by MTT assay, clone formation assay, flow cytometry and in vivo experiment in nude mice.

RESULTSCompared with the control group (0.50 ± 0.07), miR-224 ASO significantly reduced the miR-224 mRNA expression in the cancer patients (0.09 ± 0.01, P < 0.05). MTT assay results showed that the survival rate of gastric cells at 24 h, 48 h and 72 h was 53.6%, 59.1% and 70.1% in the miR-224 ASO group, and 12.3%, 17.4% and 24.7%, respectively, in the control group (P < 0.05 for all). Clone formation assay revealed that clone formation rate in the miR-224 ASO group was (5.33 ± 0.74)%, significantly lower than the (33.33 ± 8.38)% in the control group (P < 0.05). Flow cytometry indicated that the apoptotic index was (15.68 ± 1.46)% in the miR-224 ASO group and (3.36 ± 0.88)% in the control group (P < 0.01). In addition, the expressions of Bcl2 mRNA and protein were 1.05 ± 0.04 and 0.21 ± 0.03 in the miR-224 ASO group, significantly lower than that in the control group (4.87 ± 0.96 and 0.88 ± 0.09, P < 0.01). The in vivo study further showed that the tumor volume in the experimental group is significantly smaller than that in the control group (P = 0.01).

CONCLUSIONSMiR-224 is overexpressed in human gastric cancer. Reducing the expression of miR-224 can effectively inhibit the growth and promote apoptosis of gastric cancer cells. miR-224 may become a new target for the regulation of gene expression in gastric cancer.

Adult ; Aged ; Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Neoplasm Transplantation ; Oligonucleotides, Antisense ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Tumor Burden

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phβApr-neol cells (P < 0.01). CONCLUSION These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.
Animals ; DNA, Antisense ; therapeutic use ; DNA, Complementary ; Heparan Sulfate Proteoglycans ; genetics ; Heterografts ; Humans ; Laryngeal Neoplasms ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo study the effect of phospholamban antisense RNA (asPLB) on sarcoplasmic reticulum Ca2+-ATPase activity and cardiac function in rats with diabetes mellitus (DM) mediated by recombinant adeno-associated virus (rAAV) vector.

METHODSSix weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely: DM-rAAV-asPLB group, DM-saline group and DM group (control group). The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum (SR) Ca2+-ATPase and left ventricular function were measured.

RESULTSThe PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2+-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2+-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group.

CONCLUSIONrAAV-asPLB can down-regulate PLB protein expression and up-regulate PLB phosphorylation and SR Ca2+-ATPase activity, thus contributing to the improvement of in vivo left ventricular function.

Animals ; Calcium-Binding Proteins ; genetics ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Male ; Phosphorylation ; RNA, Antisense ; administration & dosage ; Rats ; Rats, Wistar ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Ventricular Function, Left ; drug effects

Tumor-associated macrophages (TAMs) mostly exhibit M2-like (alternatively activated) properties and play positive roles in angiogenesis and tumorigenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic factor. During tumor development, TAMs secrete VEGF and other factors to promote angiogenesis; thus, anti-treatment against TAMs and VEGF can repress cancer development, which has been demonstrated in clinical trials and on an experimental level. In the present work, we show that miR-150 is an oncomir because of its promotional effect on VEGF. MiR-150 targets TAMs to up-regulate their secretion of VEGF in vitro. With the utilization of cell-derived vesicles, named microvesicles (MVs), we transferred antisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates miR-150 and VEGF levels in vivo and attenuates angiogenesis. Therefore, we proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel, natural, microvesicle-based method for the transfer of nucleic acids.
Animals ; Carcinogenesis ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Exosomes ; HEK293 Cells ; Heterografts ; Humans ; Macrophages ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs ; genetics ; metabolism ; Neoplasm Transplantation ; RNA, Antisense ; administration & dosage ; genetics ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; metabolism