miR-483-5p promotes growth, migration and invasion of hepatocellular carcinoma cells by up-regulating IGF2 gene transcription / 中国病理生理杂志

AIM:To investigate the effect of miR-483-5p on P3 promoter-driven mRNA(P3 mRNA)expres-sion of human insulin-like growth factor 2(IGF2)gene and its role in the development of hepatocellular carcinoma (HCC).METHODS: The expression levels of miR-483-5p and P3 mRNA were analyzed by real-time PCR in human HCC cell lines Huh7,Hep3B,Bel-7402,HepG2 and SMMC-7721,normal human liver cell line HL-7702,83 cases of hu-man HCC tissues and their matched adjacent nontumorous tissues(MANT), and 22 cases of normal adult liver tissues (NALT).The association between P3 mRNA level and miR-483-5p level was evaluated by Pearson correlation analysis. The full-length sequences of 5'UTR of P3 mRNA containing wild-type and mutant miR-483-5p-binding sequences were cloned into pGL3 promoter vector, which were termed pGL3-P3-5'UTR-WT and pGL3-P3-5'UTR-MUT, respectively. These luciferase reporter constructs were transfected into HeLa,293T and Huh7 cells together with miR-483-5p mimic, miR-483-5p inhibitor or scrambled control,and the luciferase activity was measured using dual-luciferase reporter system. The miR-483-5p mimic,miR-483 inhibitor and scrambled control were also transfected into Huh 7 cells and Hep3B cells, and P3 mRNA level was detected by real-time PCR.The expression levels of miR-483-5p in the nuclear and cytoplasmic fractions of Hep3B cells and Huh7 cells were detected by real-time PCR.The effect of miR-483-5p on P3 mRNA transcrip-tion was evaluated by nuclear run-on assay.The effect of miR-483-5p on the stability of P3 mRNA was analyzed by RNA stability assay.Furthermore,the effects of miR-483-5p on the viability, apoptosis, migration and invasion of Huh7 cells were investigated.RESULTS:Significamtly high levels of miR-483-5p and P3 mRNA were detected in the 5 human HCC cell lines and the human HCC tissues as compared with the human normal liver cell line HL-7702, and the MANT and NALT,respectively.Linear correlation analysis revealed that P 3 mRNA level was positively correlated to miR-483-5p level in the 5 human HCC cell lines and the human HCC tissues.miR-483-5p directly recognized the P3 mRNA 5'UTR to pro-mote gene expression.Overexpression of miR-483-5p resulted in a significant increase in P3 mRNA expression in a dose-dependent manner in the Huh7 cells and Hep3B cells.The mature miR-483-5p was present in both cytoplasm and nucleus of Hep3B cells and Huh7 cells.miR-483-5p induced nascent P3 mRNA transcription in the nucleus of Huh7 cells.miR-483-5p did not alter P3 mRNA stability in Huh7 cells.Furthermore,miR-483-5p led to increased viability,apoptosis inhi-bition,and enhanced migration and invasion abilities in the Huh 7 cells.CONCLUSION:High expression of miR-483-5p promotes the growth,migration and invasion of HCC cells in part through up-regulating P3 mRNA transcription,and is con-sequently involved in the development of HCC.

The construction of IGF-II P4 promoter-driven tk expression vector / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.</p><p><b>METHODS</b>Recombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis.</p><p><b>RESULTS</b>Identification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively.</p><p><b>CONCLUSION</b>The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.</p>

Mutation analysis of the MMACHC gene in a pedigree with methylmalonic aciduria / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutation of the methylmalonic aciduria (cobalamin deficiency) CblC type, with homocystinuria (MMACHC) gene in a pedigree with methylmalonic aciduria.</p><p><b>METHODS</b>The MMACHC gene mutation was detected using polymerase chain reaction (PCR) and DNA sequencing. The MMACHC gene of 50 healthy people was also sequenced as control.</p><p><b>RESULTS</b>A new mutation of 146_154 del CCTTCCTGG was found in the patient and his father, and was absent in the controls.</p><p><b>CONCLUSION</b>A new mutation (146_154 del CCTTCCTGG) in the MMACHC gene was detected in a Chinese family with methylmalonic aciduria.</p>

Affinity maturation of a single-chain antibody against hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To obtain a single-chain antibody with high affinity against hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>A second single-chain antibody mutant library was established using an error-prone PCR and a phage display. Single-chain antibodies with high affinity for hepatocellular carcinoma were selected using ELISA.</p><p><b>RESULTS</b>The content of the second single-chain antibody mutant library was about 4.5 x 10(7). Two selected mutants, M90 and M116, were obtained after 3 rounds of panning and ELISA. Immunoassay showed that both M90 and M116 could bind to human HCC cells. The relative affinity of M90 was 1.7-fold higher than that of the original antibody, and M116 was 2-fold higher than that of the original antibody.</p><p><b>CONCLUSION</b>Error-prone PCR is an effective and simple method for affinity maturation of antibodies isolated from a phage antibody library.</p>

The Purification and the Immunological Activity Study of LexA in Pseudomonas aeruginosa / 中国生物工程杂志

Objective To clone lexA gene,express and purify the repressor LexA from Pseudomonas aeruginosa(PA),prepare the polyclonal antibody against PC-1 protein in rabbits,detect immunological activity of LexA protein.Methods The genomic DNA was extracted from the PAO1,the gene fragment encoding the mature LexA was amplied by PCR.It was linked the vector pET32a(+)and expressed in the E.coli BL21(DE3).The expressed protein was purified by two steps of Ni2+ chelate affinity chromatography and gel filtration chromatography respectively.The purified LexA protein immune the rabbits by injection and prepare the polyclonal antibody against PC-1 protein.The immunological activity of expressed and purified LexA protein was detected by ELISA,and Western blot.Results The expressed fused protein was found in insoluble form,accounted for 45% of the total bacteria protein.The final purity was 98.97%,which was determined by the HPLC.The expressed and purified LexA protein had satisfactory immunological activity.

Relationship between alterations of p16INK4a and p14ARF genes of CDKN2A locus and gastric carcinogenesis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between alterations of p16INK4a and p14ARF genes and gastric carcinogenesis.</p><p><b>METHODS</b>Tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. Homozygous deletion, mutation, methylation of the CpG islands, mRNA expression of p16INK4a and p14ARF genes were assessed by polymerase chain reaction (PCR), PCR-single strand conformation polymorphism (SSCP), PCR based methylation assay, and reverse transcription (RT)-PCR.</p><p><b>RESULTS</b>(1) The overall homozygous deletion rate of p16INK4a and p14ARF was 35.4% (17/48), and no homozygous deletion was examined in all the gastric tissues neighboring tumor. (2) There was no point mutation of p16INK4a and p14ARF in 31 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. (3) Methylation of the CpG islands of p16INK4a and p14ARF was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring cancers with a significant difference (P <0.01). (4) The rate of p16INK4a mRNA loss was 47.9% (23/48) in gastric cancer, and the cases of combined methylation of exons 1alpha and 2 had a higher loss rate (100%, 6/6) of p16INK4a mRNA than those of methylation form the other exons (11.8%, 2/17) (P <0.01). The loss rate of p14ARF mRNA was 45.8% (22/48) in gastric cancer, and patients with combined methylation of exons 1beta and 2 had a higher loss rate (100%, 3/3) of p14ARF mRNA than those of the methylation from the other exons (15%, 3/20) (P < 0.05). (5) The combined loss of p16INK4a and p14ARF mRNAs was examined in 1 (5.6%) of 18 cases of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 cases of poorly and not-differentiated carcinomas with significant difference (P <0.05).</p><p><b>CONCLUSION</b>p16INK4a and p14ARF genes were frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which might have played an important role in the development of gastric cancer.</p>