MicroRNAs (miRNAs) recruit the RNA-induced silencing complex (RISC) to repress the translation of target mRNAs. While the 5' 7-methylguanosine cap of target mRNAs has been well known to be important for miRNA repression, the underlying mechanism is not clear. Here we show that TNRC6A interacts with eIF4E2, a homologue of eIF4E that can bind to the cap but cannot interact with eIF4G to initiate translation, to inhibit the translation of target mRNAs. Downregulation of eIF4E2 relieved miRNA repression of reporter expression. Moreover, eIF4E2 downregulation increased the protein levels of endogenous IMP1, PTEN and PDCD4, whose expression are repressed by endogenous miRNAs. We further provide evidence showing that miRNA enhances eIF4E2 association with the target mRNA. We propose that miRNAs recruit eIF4E2 to compete with eIF4E to repress mRNA translation.
Autoantigens ; metabolism ; Cell Line ; Eukaryotic Initiation Factor-4E ; metabolism ; Gene Silencing ; Humans ; MicroRNAs ; genetics ; Protein Transport ; RNA, Messenger ; biosynthesis ; genetics ; RNA-Binding Proteins ; metabolism

Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Golgi Apparatus ; genetics ; metabolism ; Golgi Matrix Proteins ; Insulin-Secreting Cells ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proinsulin ; genetics ; metabolism ; Rats ; rab1 GTP-Binding Proteins ; genetics ; metabolism

OBJECTIVETo observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.

METHODSLv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs. Transfection efficiency was observed by fluorescence microscopy. Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR. Proliferation and apoptosis were detected by MTT and flow cytometry, respectively. The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 * 10(7) HepG2 cells into 30 BALB/c nude mice. The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.

RESULTSThe Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin. Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P less than 0.05). None of the treatments affected proliferation or apoptosis of the L02 cells (P more than 0.05). The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P less than 0.05). Tumor growth was significantly inhibited by the combination (P less than 0.05). In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells. Cell morphology of major organs such as liver, spleen and kidney were similar to the control group. The combination also produced the most significant effect on tumor microvascular density (P less than 0.05) and the highest apoptosis index (P less than 0.05).

CONCLUSIONThe HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells. Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.

Animals ; Apoptosis ; Autoantigens ; genetics ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Collagen Type IV ; genetics ; Down-Regulation ; Flow Cytometry ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Hep G2 Cells ; Humans ; Lentivirus ; Liver Neoplasms ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Telomerase ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.
Animals ; Autoantigens ; metabolism ; Cells, Cultured ; Flow Cytometry ; Gene Knockdown Techniques ; Genetic Vectors ; Hematopoietic Stem Cells ; cytology ; Humans ; Lentivirus ; genetics ; Mice ; Plasmids ; Ribonucleoproteins ; metabolism ; Transfection

Glucosamine, a naturally occurring amino monosaccharide, has been reported to play a role in the regulation of apoptosis more than half century. However the effect of glucosamine on tumor cells and the involved molecular mechanisms have not been thoroughly investigated. Glucosamine enters the hexosamine biosynthetic pathway (HBP) downstream of the rate-limiting step catalyzed by the GFAT (glutamine:fluctose-6-phosphate amidotransferase), providing UDP-GlcNAc substrates for O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification. Considering that O-GlcNAc modification of proteasome subunits inhibits its activity, we examined whether glucosamine induces growth inhibition via affecting proteasomal activity. In the present study, we found glucosamine inhibited proteasomal activity and the proliferation of ALVA41 prostate cancer cells. The inhibition of proteasomal activity results in the accumulation of ubiquitinated proteins, followed by induction of apoptosis. In addition, we demonstrated that glucosamine downregulated proteasome activator PA28gamma and overexpression of PA28gamma rescued the proteasomal activity and growth inhibition mediated by glucosamine. We further demonstrated that inhibition of O-GlcNAc abrogated PA28gamma suppression induced by glucosamine. These findings suggest that glucosamine may inhibit growth of ALVA41 cancer cells through downregulation of PA28gamma and inhibition of proteasomal activity via O-GlcNAc modification.
Acetylglucosamine/chemistry/metabolism ; Alloxan/pharmacology ; Apoptosis/*drug effects ; Autoantigens/genetics/*metabolism ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Gene Expression Regulation, Neoplastic ; Glucosamine/*pharmacology ; Humans ; Male ; Phosphorylation ; Prostatic Neoplasms/*enzymology ; Proteasome Endopeptidase Complex/*antagonists & inhibitors/genetics/metabolism ; RNA, Small Interfering/genetics ; Ubiquitinated Proteins/metabolism

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of co-transfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Co-transfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.
Adenoviridae ; genetics ; Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Cell Survival ; Cytomegalovirus ; genetics ; Genetic Vectors ; Glioma ; diagnostic imaging ; genetics ; metabolism ; pathology ; Half-Life ; Humans ; Iodide Peroxidase ; genetics ; metabolism ; Iodine Radioisotopes ; metabolism ; Iron-Binding Proteins ; genetics ; metabolism ; Mice ; Mice, Nude ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; metabolism ; Symporters ; genetics ; metabolism ; Technetium ; Telomerase ; genetics ; Tomography, Emission-Computed, Single-Photon ; Transfection

OBJECTIVETo investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell.

METHODS(1) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4.1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)], blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 µg protein), CASK antibody precipitation group (100 µg protein), IgG antibody group (100 µg protein), Western blot group (20 µg protein). Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn), KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/L PBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group (transfected with pcDNA-Achn vector), and blank control group (treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10.777, 6.112, P values all below 0.05). The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3.1 group (with t value respectively 5.367, 6.053, 9.831, P values all below 0.05). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group (with t value respectively 5.481, 9.517, P values all below 0.05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [(15.6 ± 0.5)%] as compared with that in LPS induction group [(32.8 ± 2.6)%, t = 10.083, P < 0.05], and that in cotransfection group showed further inhibition [(7.0 ± 2.0)%, t = 9.827, P < 0.01]. (4) Apoptosis rate in Achn inhibition group [(45.6 ± 10.9)%] was higher than that in blank control group [(13.2 ± 4.3) %, t = 7.043, P < 0.05]; while that in Achn induction group [(5.3 ± 2.9)%] was lower than that in blank control group (t = 6.499, P < 0.05).

CONCLUSIONSAchn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.

Apoptosis ; Autoantigens ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Endothelial Cells ; cytology ; Guanylate Kinases ; metabolism ; Humans ; Ribonucleoproteins ; genetics ; metabolism ; Transfection

BACKGROUNDTumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein. The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.

METHODSThe eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN. Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting. Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry. To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.

RESULTSDNA sequence confirmed that pcDNA-tumstatin was successfully constructed. RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively. After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase. And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.

CONCLUSIONSOverexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

Autoantigens ; genetics ; metabolism ; Blotting, Western ; Cell Cycle ; genetics ; physiology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Collagen Type IV ; genetics ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Flow Cytometry ; Humans ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo screen the proteins interacting with FXR1P for functional investigation of FXR1P.

METHODSThe yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis.

RESULTSThe recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1.

CONCLUSIONSThese results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.

Autoantigens ; genetics ; metabolism ; Estradiol Dehydrogenases ; genetics ; metabolism ; Ferritins ; genetics ; metabolism ; Gene Library ; Humans ; Membrane Proteins ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo investigate the mRNA expressions of RASSF1A, Galectin-3 and TPO in papillary thyroid carcinoma and some other thyroid benign lesions, and evaluate their diagnostic significance.

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of RASSF1A, galectin-3 and TPO in the samples from 73 cases, including 23 cases with papillary thyroid cancer, 16 with nodular goiter, 29 with thyroid adenoma and 5 with Hashimoto's disease.

RESULTSA statistically significant difference in the mRNA expression of RASSF1A, Galectin-3 and TPO was observed between papillary thyroid carcinoma and follicular benign lesions (P<0.05). However, there was no significant difference among various kinds of benign lesions (P>0.05). A negative correlation of the expression of RASSF1A and Galectin-3 mRNA was found between thyroid benign lesions and malignant ones (P = 0.000). While the mRNA expression of RASSF1A and TPO was positively correlated between benign and malignant lesions (P = 0.028).

CONCLUSIONLoss of expression of RASSF1A and TPO mRNA but high expression of Galectin-3 mRNA in papillary thyroid carcinoma are common. Therefore, the products of these three genes may be closely related to the development of thyroid papillary carcinoma, and may be used as useful markers in differential diagnosis of papillary thyroid carcinoma from the benign lesions. The results are more reliable if this detection method is used in combination with other techniques.

Adolescent ; Adult ; Aged ; Autoantigens ; genetics ; metabolism ; Biomarkers, Tumor ; metabolism ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Galectin 3 ; genetics ; metabolism ; Goiter, Nodular ; genetics ; metabolism ; pathology ; Hashimoto Disease ; genetics ; metabolism ; pathology ; Humans ; Iodide Peroxidase ; genetics ; metabolism ; Iron-Binding Proteins ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult