Objective To study the effect of Golgi protein 73(GP73) on inflammation, and to reveal the effect of GP73 on tumorigenesis and metastasis.Methods The transcriptional activity of NF-κB and the expression of IL-1β, IL-6 and TNF-αwith GP73 overexpression or knockdown were detected to illuminate the role of GP73 in inflammation.According to the TCGA database, the correlation between the transcriptional activity of GP73 and the expression of NF-κB, IL-1β, IL-6 and TNF-αwas analyzed to determine the role of GP73 in tumor inflammation.Results Correlative analysis showed that there was a positive correlation between the expression of GP73 with NF-κB, IL-1β, IL-6 and TNF-α.The transcriptional activity of NF-κB was upregulated by GP73 overexpression, but downregulated by GP73 knockdown.The expression of IL-1β, IL-6 and TNF-αwas upregulated by GP73 overexpression.Ammonium pyrrolidinedithiocarbamate ( PDTC ) was in-volved in inflammation reaction induced by GP73.Conclusion GP73 is possibly involved in inflammation and promotes tu-morigenesis and metastasis.

Objective To knock out the GP73 gene in H22 cells originating in mice using CRISPR/Cas9 gene editing system and construct H22 GP73 gene knockout stable strain for identification of its functions .Methods Two pairs of sgRNAs that could specifically identify the upstream and downstream of GP 73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using pX 459 .After enzyme digestion and sequencing , two pairs of recombinant plasmids were co-transfected into H22 cells before puromycin was used to screen positive cells and monoclonal cells which stably knocked out GP 73 gene were developed .The knockout effect was measured by Western blotting.Cell Titer 96? AQueous One Solution Assay was used to detect the effect on cell reproductive capacity when the GP73 was knocked out .The transferability was detected through wound healing test .Results The result of Western blotting suggested that GP73 protein was undetected in the construction of H22 GP73 knockout gene stable strain after transfection.The transfer and reproduction slowed down .Conclusion H22 GP73 gene knockout stable strain can be successfully built using CRISPR/Cas9 gene editing system ,thus facilitating studies on the function of GP 73 in hepatocarcinogenesis .

Objective To analyze the genetic characteristics of the complete genome of a human echovirus 30 (Echo30) KM/A363/09 strain isolated in Yunnan, China in 2009.Methods Primers specif-ic for Echo30 were designed .The extracted RNA was amplified by using RT-PCR.Seven fragments covering the complete viral genome were sequenced and the complete sequences were aligned with other sequences of enterovirus reference strains downloaded from Genbank . By using Mega5.1, Geneious, RDP3 and SimPlot3.5.1 softwares, the phylogenetic and recombination analysis were carried out .Results The com-plete nucleotide sequence of KM/A363/09 isolate was 7425 bp in length, encoding 2194 amino acids.KM/A363/09 isolate was highly similar with Bastianni prototype strain showing the homology of 81.2%in nucle-otide and 95.8%in amino acid.The eight Echo30 isolates shared 81.2%-88.6%homologies in nucleotide sequences and 95.8%-97.8%in amino acid sequences .Phylogenetic analysis showed that the KM/A363/09 strain belonged to one clade of Echo 30 in China.The genetic recombination of KM/A363/09 isolate was detected in the non-structural region .Conclusion KM/A363/09 isolate belongs to one clade of Echo 30 in China indicating that the evolution of Echo 30 has occurred in China .

Objective To investigate the effect of BTB/POZ ( broad complex, tramtrack and bric a brac/poxviruses and zinc finger) on proliferation of breast cancer cell lines.Methods The eukaryotic expression plasmid pCMV-HA-BPOZ was constructed by cloning from cDNA of human genome.Western blotting was used to detect the expression of pCMV-HA-BPOZ.Cells growth assay was used to detect the effect of BPOZ on proliferation and colony formation assay was used to detect the effect of BPOZ on cell growth ability.Results Western blotting showed that HA-BPOZ was efficiently expressed in cells.Moreover, the growth ability and proliferation of cells were significantly inhibited in BPOZ overexpressed cells compared with the control cells (both P <0.05).Conclusionp CMV-HA-BPOZ plasmid is constructed.BPOZ can restrain breast cancer cell lines MCF7 and ZR-75-1 cells from proliferating and growing.The results of our study can con-tribute to the study of functions of BPOZ in breast cancer.

OBJECTIVETo analyze the biochemical and pathological changes in mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet combined with low-dose streptozotocin (STZ) injections.

METHODSC57BL/6J mice were divided randomly into normal control group (NC group), high-fat diet group (HC group) and high-fat diet plus STZ group (HC+STZ group). The mice were fed on normal chow or a high-fat diet for 1 month before two introperitoneal injections of STZ (40 mg/kg) or citrate buffer with an interval of 24 h as appropriate. Fasting blood glucose (FBG) was detected every week for 4 weeks, and oral glucose tolerance test (OGTT) was performed one month after the injections, after which the biochemical profiles, islet and liver were evaluated by immunohistochemical and pathological analysis.

RESULTSIn HC+STZ group, FBG was above the cutoff value (13.89 mmol/L) in 75% of the mice at 1 week after STZ injections and in all the mice at two weeks except for the death of 1 mouse, with a success rate of modeling of 91.3%. FBG in HC group, though slightly higher than that in NC group, remained normal (6.8 mmol/L). The body weight in HC+STZ and HC groups was significantly higher than that in NC group after feeding but without obvious increases after the injections (P<0.01). Blood glucose in HC+STZ group at 0.5 to 2 h after OGTT and the area under curve (AUC) were higher than those in NC and HC groups (P<0.01); the AUC in HC group was a also higher than that in NC group (P<0.05). Plasma creatinine was significantly higher in HC+STZ group than in NC (P<0.01) and HC (P<0.05) groups. Insulin secretion by the islets decreased obviously in HC+STZ and HC group. The mice in HC+STZ group showed atrophy, fibrosis, and vacuolization in the islets with mild fatty liver but no visible renal pathologies.

CONCLUSIONHigh-fat diet and low-dose STZ injections can induce T2DM in mice with very similar biochemical and pathological changes to human T2DM and with such complications as fatty liver.

Animals ; Blood Glucose ; Body Weight ; Diabetes Mellitus, Type 2 ; physiopathology ; Diet, High-Fat ; Fatty Liver ; physiopathology ; Glucose Tolerance Test ; Insulin ; Insulin Resistance ; Islets of Langerhans ; pathology ; Kidney ; pathology ; Mice ; Mice, Inbred C57BL ; Streptozocin

OBJECTIVETo identify the complete genome sequence of an echovirus 20 isolate (KM/EV20/2010) and understand its genetic variation and evolution characteristics.

METHODSSeven overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were aligned with other echovirus 20 isolates. Phylogenetic and pairwise alignment analyses based on genome and complete VP1 regions were conducted with software Mega 4.1, RDP3 and SimPlot 3.5.1.

RESULTSThe genome of the echovirus strain was 7 395 nucleotides in length, and contained a 744-nt non-translated region (NTR) at the 5' end and a 96-nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion. Based on the complete genome sequence alignments, the echovirus strain showed 80.1% nucleotide and 96.7% amino acid homology to echovirus 20 prototype JV-1 strain. The phylogenetic trees constructed on the genome and complete VP1 regions all indicated that the echovirus strain was not in one cluster with echovirus 20 prototype JV-1 strain, while had a closer relationship with echovirus 30 prototype Bastianni. Genotyping results from phylogenetic analysis showed that echovirus 20 has six genotypes. The strain used in this study belonged to genotype IV. The nucleotide divergence was 9.4%-21.7% among the 6 genotypes. The possible putative recombination was detected in its non-coding sequence of the echovirus 20 strain used in this study.

CONCLUSIONBased on the bioinformatics analysis. The echovirus 20 strains isolated in China could be divided into six genotypes, and the echovirus 20 in this study belonged to genotype IV.

Amino Acid Sequence ; Base Sequence ; China ; Computational Biology ; Enterovirus B, Human ; genetics ; isolation & purification ; Genome, Viral ; Genomics ; Genotype ; Humans ; Open Reading Frames ; Phylogeny ; Sequence Alignment