ABSTRACT
Objective The purpose of this study was to investigate the expression of human mitochondrial transcription termi-nation factor-3 ( hMTERF3) in non-small cell lung cancer ( NSCLS) and to analyze its clinicopathological significance. Methods The paraffin block samples used in this study included 65 cases of NSCLC and 32 cases of normal alveolar epithelial tissues. We determined the expressions of hMTERF3 in NSCLC and normal alveolar epithelial tis-sues by immunohistochemistry, calculate the survival rate using the Kaplan-Meier method, and analyzed the risk factors affecting the prognosis of NSCLC using the Cox Proportional Hazard Model. Results In the 65 cases of NSCLC, 31 ( 47. 69%) showed positive expression of hMTERF3. The total survival time was significantly shor-ter in the patients with a high than in those with a low hMTERF3 ex-pression ([30.39±3.35] vs [57.61±7.12] mo, P<0.05). The riskfactors affecting the prognosis of NSCLC included positive expression of hMTERF3 (HR=3.302, 95% CI:1.598-6.905) and lymph node metastasis (HR=4.052, 95% CI: 1.212-12.398). Conclusion hMTERF3 is overexpressed in NSCLC. Highly expressed hMTERF3 and lymph node metastasis reduce the survival time of NSCLC patients, suggesting that hMTERF3 may be a potential bio-marker for the prognosis of NSCLC.
ABSTRACT
Objective Human mitochondrial transcription termination factor 3 ( MTERF3 ) is a negative regulator of mito?chondrial gene expression and energy metabolism. This study was to construct a prokaryotic expression system for MTERF3 in Esche?richia coli ( E. coli ) and prepare its mouse?anti?human polyclonal antibody. Methods The complete open reading frame ( ORF) of human MTERF3 cDNA was amplified by RT?PCR and subcloned into prokaryotic expression vector pET28b. Then the recombinant plasmid pET28b?MTERF3 was transformed into competent E.coli BL21(DE3) and IPTG induced the expression of 6×his fusion protein. The recom?binant human MTERF3 protein was purified through Ni2+?NTA agar?ose gel column affinity chromatography and the purified recombinant protein was used as immunogen to immunize the BALB/c mice to pre?pare its specific polyclonal antibody. The titer and specificity of the antibody were analyzed by ELISA, Western blot and cellular immuno?fluorescence, respectively. Results The recombinant human MTERF3 protein was successfully expressed in E. coil and the mouse?anti?human MTERF3 polyclonal antibody with high quality was successfully prepared. ELISA showed that the titer of the antibody was 1:105 . Western blot and immunofluorescence detection revealed that the mouse?anti?human MTERF3 antibody could recognize the native MTERF3 antigen specifically. Conclusion Human MTERF3 expressed in the prokaryotic system has strong immunogenicity and the polyclonal antibody obtained from immunizing mice has high titer and specificity. The prokaryotic expression of human MTERF3 and the preparation of its antibody lay the foundation for further function research of human MTERF3.
ABSTRACT
Objective The proteins encoded by mitochondrial transcription termination factor 1 ( MTERF1) plays important roles in regulating the mitochondrial gene expression and oxidative phosphorylation .This study was to investigate the characteristics and regulation mechanisms of the human MTERF1 gene with bioinformatics tools . Methods Using online bioinformatics software and phylogenetic foot-printing, we analyzed the promoter distribution , GpG island, transcription factors , and binding sites of the human MTERF1 gene. Results The human MTERF1 gene was located on 7q21.2, with a full length of 7845 bp, consisting of 4 exons and 3 introns.There were at least 2 promoters in the 5′region of the gene.The core promoter of the MTERF1 gene was located between 1878 and 2447 bp, which played a key role in its transcription .An 812 bp CpG island was observed in the gene promoter region .In addi-tion, 26 transcription factor binding sites were found in the conserved promoter region of human and mouse homologous MTERF1 genes. Conclusion Gene promoter-related online bioinformatics software can improve the efficiency of human MTERF1 gene promoter resear-ches and provide significant information for the prediction of gene pro-moter function.