[Effects of Histone Methyltransferase Inhibitors on the Survival, Apoptosis and Cell Cycle of Raji Cells].

OBJECTIVE: To determine the effects of three histone methylase inhibitors UNC1999, DZNep and GSK343 on the survival, apoptosis and cell cycle of non-hodgkin's lymphoma Raji cells. METHODS: PCR amplified 16 and 18 exons of enhancer of zeste homolog 2 ( EZH2) gene were detected. The expression of EZH2 in normal adult lymphocytes and Raji cells was detected by Western blot. The Raji cells were treated by UNC1999, DZNep and GSK343, followed by CCK-8 assays analyzing cell survival, flow cytometry detecting cell apoptosis and cell cycle, and Western blot detecting the expressions of EZH2 and H3K27 me3. RESULTS: The Sanger sequencing results showed that the Raji cells did not carry Y641 and A677 mutation sites of EZH2. The Western blot results showed high expressions of EZH2 in the Raji cells. The results of CCK-8 showed that UNC1999, DZNep and GSK343 inhibited cell survival, and the weakest effect was from DZNep. The flow cytometric assay showed that UNC1999, DZNep and GSK343 promoted apoptosis of the Raji cells, and the effect of UNC1999 was stronger than that of GSK343 and DZNep. The cell cycle was arrested at phase G 1/G 0 after treatment of the Raji cells with the three inhibitors, with UNC1999 triggering the most significant changes. The Western blot showed that UNC1999 and GSK343 inhibited the histone methylase activity of EZH2 and significantly reduced the expression of H3K27 me3. CONCLUSION: EZH2 inhibitors can inhibit cell survival, promote cell apoptosis and arrest cell cycle at phase G 1/G 0 of Raji cells through reducing the expression of H3K27me3. UNC1999 has a stronger effect than GSK343 and DZNep.

Arg124Cys mutation of the TGFBI gene in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

AIM: To analyze mutations in transforming growth factor beta-induced (TGFBI) gene in a Chinese pedigree with Reis-Bücklers corneal dystrophy (RBCD, also known as GCD3). METHODS: In a five-generation Chinese family, eight members were identified with RBCD and the rest were unaffected. All members of the family underwent complete ophthalmologic examinations. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all the affected members of the pedigree, but not in the unaffected members. CONCLUSION: R124C which was a known mutation for lattice corneal dystrophy type I, segregated with the RBCD in this pedigree. This elucidated the correlation between genotype and phenotype in a Chinese family of RBCD.

Beclin 1-mediated macroautophagy involves regulation of caspase-9 expression in cervical cancer HeLa cells.

OBJECTIVE: To investigate the role of Beclin 1 in HeLa cells and to obtain further insight into the relationship between autophagy and apoptosis. METHODS: Beclin 1 silencing was achieved using RNA interference. The expression of gene was measured using quantitative real time RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. The ultrastructural analysis was under the electron microscope. RESULTS: In pSUPER-Bec transfectants (Beclin 1 gene partially silenced) the expression of mRNA and protein of Beclin 1 were significantly suppressed in comparison to pSUPER-non (scramble RNA control) or untreated cells in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in pSUPER-Bec transfectants compared with pSUPER-non transfectants. Meanwhile pcDNA3.1-Bec transfectants (Beclin 1 gene overexpressed) showed reduction of cell proliferation but augmentation of cell programmed death compared with vector vehicle. The autophagy-promoting activity of beclin 1 in HeLa cells is associated with inhibition of HeLa cellular proliferation, in vivo tumorigenesis in nude mice. The expression pattern of caspase-9 was extraordinarily similar to that of Beclin 1in siRNA against Beclin 1 transfectants and constructive expression of Beclin 1transfectants. CONCLUSION: siRNA against Beclin 1 transfectants promoted the cell proliferation but overexpression of Beclin 1 promoted the autophagy cell death, and in the process of autophagy triggered by Beclin 1 expression followed accordingly the regulation of the expression of caspase-9. We conjecture that the autophagy gene Beclin 1 may be the critical molecular switch that plays an important role in fine tuning the autophagy and apoptosis through caspase-9, and defection of autophagy or apoptosis may be an important mechanism in tumorigenesis.

[Expression of glucocorticoid receptor alpha and beta mRNA in orbital tissues in thyroid associated ophthalmopathy].

OBJECTIVE: To evaluate the expression of glucocorticoid receptor alpha (GRalpha) and beta (GRbeta) messenger RNA (mRNA) in orbital tissues from thyroid associated ophthalmopathy (TAO). METHODS: Samples of extraocular muscle and orbital fat were obtained from 17 patients with TAO and 10 healthy individuals. Total RNA was extracted and reversely transcripted into cDNA. The expression of GRalpha and GRbeta mRNA was detected by means of fluorescent quantitative polymerase chain reaction (PCR). RESULTS: Expression of GRalpha mRNA was much higher than GRbeta mRNA in all extraocular muscle and orbital fat biopsies. The relative copy of GRalpha was 40.15 +/- 11.37 in TAO patients and 20.64 +/- 7.07 in the controls. GRalpha: GRbeta mRNA ratio of these two groups was 77.76 +/- 18.77 and 148.34 +/- 23.86, respectively. There was significant difference between these two groups (P < 0.05). No significant difference was noted between extraocular muscle and orbital fat biopsies, between glucocorticoid-treated and non-treated patients or among hyperthyroidism, hypothyroidism and euthyroidism (P > 0.05). CONCLUSIONS: The increased expression of GRalpha mRNA and decreased GRalpha: GRbeta ratio in orbital tissues may play an important role in the pathogenesis of TAO and the effects of glucocorticoid treatment.

[Effect of adenovirus-mediated mda-7/IL-24 gene infection on apoptosis of drug-resistant human ovarian cancer cell lines OVCAR-3 and OVCAR-8/TR].

BACKGROUND & OBJECTIVE: Melanoma differentiation associated gene-7 (mda-7/IL-24) has double functions: specifically induces tumor cell apoptosis and modulates immune responses. Therefore, it is a strong candidate for human cancer gene therapy. This study was to evaluate the effect of adenovirus-mediated mda-7/IL-24 infection on the apoptosis of drug-resistant ovarian cancer cell lines OVCAR-3 and OVCAR-8/TR. METHODS: Adenovirus-mediated mda-7/IL-24 (Ad.mda-7/IL-24) was constructed using AdEasy 1 system. OVCAR-3 and OVCAR-8/TR cells were infected by Ad.mda-7/IL-24. The expression of MDA-7/IL-24 protein was detected by Western blot. Cell apoptosis was detected by flow cytometry with Hoechst33258 staining. Cell cycle distribution was detected by flow cytometry. RESULTS: The recombinant Ad.mda-7/IL-24 was confirmed by DNA sequencing and electrophoresis. The expression of MDA-7/IL-24 protein was detected in the cells after infection. Within 72 h after Ad.mda-7/IL-24 infection, the maximal apoptosis rates of OVCAR-3 and OVCAR-8/TR cells were 14.1% and 32.4%, respectively, significantly higher than empty vector group and uninfected group. CONCLUSIONS: The recombinant Ad.mda-7/IL-24 was successfully constructed. It can induce apoptosis in drug-resistant ovarian cancer OVCAR-3 and OVCAR-8/TR cells.

[Construction of shRNA expression vectors for autophagy gene Beclin 1 and effect of the vectors on transfected HeLa cells in vitro].

OBJECTIVE: Autophagy gene Beclin 1 plays an important role in several types of human cancer. In this study, RNA interference (RNAi) technique was employed to determine the effect of inhibiting Beclin 1 on the growth of tumor cells. METHODS: According to the encoding sequence of mRNA of Beclin 1, the target site for the RNAi technique was designed and the vector for shRNA (short hairpin RNA) expression in tumor cells was constructed. The HeLa cell line was transfected with the sfRNA to inhibit the expression of Beclin 1. RESULTS: The constructed vector significantly inhibited the expression of the mRNA and protein of Beclin 1 in the HeLa cells. The growth of the transfected cells was promoted, and less apoptosis cells were identified in these cells. CONCLUSIONS: The shRNA expression vector can effectively inhibit the expression of Beclin 1 in the HeLa cells, and promote the growth of HeLa cells.

[Correlation of autophagy gene Beclin1 to tumorigenesis and development of epithelial ovarian cancer].

BACKGROUND & OBJECTIVE: Some studies have showed that autophagy suppression may result in malignant transformation, and the inactivation of autophagy gene Beclin1 induces malignancy. This study was to investigate the role of Beclin1 in the tumorigenesis and development of epithelial ovarian carcinoma, and to explore the effect of Beclin1 overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro. METHODS: The expression of Beclin1 in 25 specimens of normal ovarian tissue, 25 specimens of benign ovarian neoplasia, 19 specimens of borderline ovarian tissue, and 69 specimens of epithelial ovarian carcinoma was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3.1/Beclin1 was constructed and transfected into SKOV3 cellsû plasmid pcDNA3.1 was used as control. The effect of Beclin1 overexpression on the proliferation of SKOV3 cells was evaluated by MTT assay. Cell apoptosis was measured by flow cytometry (FCM). RESULTS: The expression of Beclin1 was high in normal and benign ovarian neoplasia tissues, and there was no significant difference between the 2 groups (P>0.05). Reduced Beclin1 expression was observed in borderline lesions, and the lowest level was detected in ovarian carcinoma tissues (P<0.05). The inhibition rate was significantly higher in pcDNA3.1/Beclin1-SKOV3 cells than in pcDNA3.1-SKOV3 cells [(68.75+/-5.10)% vs. (10.91+/-4.20)%, P<0.05]. At 72 h after transfection, the apoptosis rate was significantly higher in pcDNA3.1/Beclin1-SKOV3 cells than in pcDNA3.1-SKOV3 cells and SKOV3 cells [(19.07+/-0.65)% vs. (4.30+/-0.50)% and (3.87+/-0.84)%, P<0.05]. CONCLUSIONS: Beclin1 expression is down-regulated in epithelial ovarian cancer tissues, which may relate to tumorigenesis and development of epithelial ovarian cancer. Beclin1 overexpression can inhibit proliferation and induce apoptosis of SKOV3 cells.

[Expression of PPAR-gamma in human hilar bile duct carcinoma cell line QBC939 and the effects of PPAR-Y activated by its ligand on the growth of QBC939].

OBJECTIVE: To explore the expression of Peroxisome Proliferator-activated Receptor-gamma (PPAR-gamma) in QBC939 and the effects of PPAR-gamma activated by its ligand pioglitazone on the growth of human bile duct carcinoma cell line. METHODS: QBC939 cells were cultured and treated with different concentration of pioglitazone; the expression of PPAR-gamma mRNA was detected by RT-PCR; the effects of PPAR-gamma activated by its ligand on cell proliferation were examined by cell count under light microscope; the influences of activated PPAR-gamma on cell cycle were examined by flow cytometry, and the apoptosis of cancer cells induced by PPAR-gamma ligand pioglitazone was detected by flow cytometry and TUNEL methods. RESULTS: PPAR-gamma was expressed in human hilar bile duct carcinoma cell line QBC939. And after PPAR-gamma was activated by its ligand pioglitazone, it significantly inhibited cell proliferation, produced G2/M phase arrest and induced apoptosis of QBC939. CONCLUSIONS: PPAR-gamma, after being activated by its ligand pioglitazone, can inhibit the cell growth of QBC939 remarkably through suppression of cell proliferation, increase in proportion of G2/M phase cells and induction of apoptosis, so PPAR-gamma may be a molecular therapeutical target against the human bile duct carcinoma.

[Gene mutation analysis of coagulation factor VIII from a female patient with hemophilia A].

Hemophilia A affects male, whereas females are carriers and generally spared from this disease. However, we here reported a 65-year-old female with Hemophilia A while screening the gene mutation of coagulation factor VIII. The female went to hospital because of tripping to lead her right chest to be injured with subcutaneous hematoma. She had historically a hemorrhagic diathesis. The physical examination discovered her hip limited to bend and move, but no discrepancy length between her two legs. The initial laboratory tests showed that the activated partial thromboplastin time (APTT) was 61. 3 seconds (20-40 seconds), and the APTT corrected by mixing with normal plasma was 41.3 s, but the levels of PT, FIB and TT were normal. The plain radiographs revealed the hip joints to suffer from the acetabular dysplasia and osteoarthritis. The level of FVIII:C was 2%, F IX:C 200%, vWF:Ag 120%, vWF:Rcof 100%, vWF:CBA 128%, and the F VIII binding assay to vWF was normal. The primers for exon 14 of F VIII gene were designed according to the NM - 000132 gene sequence. DNA was abstracted from the patient blood. PCR were carried out and the DNA sequence was followed. A new mutation of 4111A-->C was discovered, which caused the amino acid sequence changed (T 1314 P). The mutation of T 1314 P may be the cause of this female patient to get the hemophilia A. This mutation was a novel one which has never been reported before.