Exosomes Derived from Hydroquinone-transformed Human Bronchial Epithelial Cells Inhibited Recipient Cell Apoptosis by transferring miR-221.

OBJECTIVE: Although benzene is a confirmed environmental carcinogen, the mechanism of its carcinogenicity remains largely unclear. The suggested oncogene, miR-221, is elevated and plays important roles in various tumors, but its role in benzene-induced carcinogenesis remains unknown. METHODS: In the present study, we constructed hydroquinone (HQ, a representative metabolite of benzene with biological activity)-transformed malignant cell line (16HBE-t) and analyzed the level of miR-221 in it with qRT-PCR. Exosomes from 16HBE-t cells incubated with or without an miR-221 inhibitor were isolated by ultracentrifugation, characterized by transmission electron microscopy and laser scanning confocal microscope, and then transfected into 16HBE cells. The effects of exosomal miR-221 on apoptosis induced by HQ in recipient cells were determined using flow cytometry. RESULTS: The amount of miR-221 in 16HBE-t was significantly increased compared with controls. When recipient cells ingested exosomes derived from 16HBE-t, miR-221 was increased, and apoptosis induced by HQ was inhibited. Blocking miR-221 in 16HBE-t using an inhibitor did not significantly alter miR-221 or apoptosis in recipient cells. CONCLUSION: Exosomal miR-221 secreted by 16HBE-t inhibits apoptosis induced by HQ in normal recipient cells.

[Epidemiological study on hyperuricemia and gout in Foshan areas, Guangdong province].

OBJECTIVE: To determine the prevalence rates and risk factors of hyperuricemia (HUA) and gout among residents aged over 20 years in Foshan areas. METHODS: A randomly stratified cluster sampling was conducted, and 7403 inhabitants were investigated on their prevalence rates of HUA and gout. RESULTS: (1) The prevalence of HUA was 15.09%, and the standardized rate was 15.27%, in which the prevalence in males was 19.90% and females was 10.54%. The prevalence of gout was 1.04% and the standardized rate was 1.08%, in which the prevalence in males was 1.73% and females was 0.39%. The prevalence of gout in patients with HUA was 6.89%. (2) Average serum uric acid was (336.4 ± 81.5) µmol/L, with (347.1 ± 88.6) µmol/L in males and (289.7 ± 78.6) µmol/L in females. The serum uric acid levels in male patients with HUA was higher than those in women. (3) Age, body mass index, systolic blood pressure, diastolic blood pressure, serum uric acid, blood sugar, triglyceride (TG), total cholesterol were significantly higher in patients with HUA and gout than in the normal group (P < 0.05 - 0.01). The incidence rates of patients with hyperuricemia and gout in the following indices as:overweight and obesity, high blood pressure, high blood sugar were significantly higher than those in the normal group (P < 0.05). Patients having gout in the following indices as age, TG, serum uric acid levels were significantly higher than the HUA group (P < 0.05). (4) Data from non-conditional logistic regression analysis showed that age, overweight, hypertension, diabetes, hyperlipidemia, use of diuretics, family history, alcohol uptake, eating seafood and drinking meat broth, post-menopausal women, and other factors were similar to those factors as patients with hyperuricemia. Tea, fresh vegetables, fruits seemed to be the protective factors. CONCLUSION: Both the prevalence rates of HUA and gout had significantly increased in Foshan areas in recent years. Restricting the intake of food with rich purine, alcohol intake as well as controlling obesity and blood pressure, improving the status of lipid metabolic disorder together with programs as hypertension control etc. were important measures in the strategies on prevention and treatment on hyperuricemia and gout.

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone.

OBJECTIVE: To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. METHODS: DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. RESULTS: MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. CONCLUSIONS: Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

[Construction of vector of multiple loci gene targeting in leghorn chicken based on BAC with Cre/lox P system].

Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not I/HindIII and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce I and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.

[hPARP1 genetic polymorphism in southern Chinese Han and Miao populations].

OBJECTIVE: To study hPARP1 genetic polymorphism in southern Chinese Han and Miao populations. METHODS: Blood samples from 187 and 210 southern healthy Han and Miao populations were collected. The mutations of exons 12,13,16 and 17 of hPARP1 gene were investigated by PCR-single-strand conformation polymorphism(SSCP). RESULTS: Fragments of 253 bp,313 bp,175 bp,362 bp within exons 12,13,16 and 17 respectively of hPARP1 gene were amplified by multiple PCR. An SSCP variant in exons 12,13,16 and 17 of PARP1 gene in 187 healthy Han and 210 healthy Miao individuals was identified. Seven single-base substitutions compared with the sequence of PARP1 gene were identified: a T to C transition in exon 12 (Phe548Ser), a G to T transition in exon 13 (Ala683Ser), a G to T transition in exon 16 (Asp798Tyr), and a A to G transition in exon 17 (His808Arg). CONCLUSION: There were polymorphism sites in exons 12,13,16,17 of hPARP1 gene in southern Chinese Han and Miao populations; these results may be useful for the establishment of PARP1 genotyping, and these newly described PARP1 alleles would be advantageous indicators for population studies.

[Vector-mediated RNA interference of DNA polymerase beta in human bronchial epithelial cells].

OBJECTIVE: To knock down the expression of polymerase beta gene in human bronchial epithelial cells with technology of vector-mediated RNA interference (RNAi), to provide research tool for the study on the functions and mechanisms of polymerase beta in repairing of DNA damaged by environmental chemical pollutants (ECPs). METHODS: Technology of molecular clone was used to construct the recombination vector of "pEGFP-C1-U6-dsRNA" for the polymerase beta RNAi. The recombinants were transfected into human bronchial epithelial cells with kit of liperfectamine 2000. The control groups included normal human bronchial epithelial cells and human bronchial epithelial cells transfected with "pEGFP-Cl1. Cells were screened by G418, then technology of fluorescence microscopy imaging was used to observe the result of transrfection. The expresison level of polymerase beta was detected by Western blotting. RESULTS: The expression level of polymerase beta in human bronchial epithelial cells transfected with the recombinant of "pEGFP-C1-U6-dsRNA" was about 17.3% of what in the normal cells. CONCLUSION: The RNAi of polymerase beta gene in human bronchial epithelial cells was successful.

[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].

OBJECTIVE: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs). METHODS: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. RESULTS: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. CONCLUSION: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.

[Construction of short hairpin RNA vector of inhibiting poly ADP-ribose polymerase activity].

OBJECTIVE: To construct expressing vector of short hairpin RNA (shRNA) in order to inhibit human PARP1 activity. METHODS: 2 pairs of 64 base oligos for hairpin RNA expression which targeted PARP1 gene were chemically synthesized and annealed then ligased with pSIREN-RetroQ vector with BamH II and EcoR I . Cut by EcoR t and Bgl II, shRNA and its upstream U6, which have 330 bp, were inserted into the same treated pEGFP-C1 vecter to construct GFP expression plasmids that inhibited hPARP1 protein shRNA plasmid (pEGFP-C1P). Oligos with a scrambled sequence were used as a negative control. RESULTS: Recombinant pEGFP-C1P1, pEGFP-C1P2 and pEGFP-C1N vectors was identified by digestion with EcoR I and Bgl II and confirmed by sequencing analysis with U6 primer. The results demonstrated that the 330 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct. CONCLUSION: pEGFP-C1-shRNA system has been constructed successfully. This will facilitate the study of PARP1's DNA repairing function.