Cardiac Disease Associated Genetic Variants in Yi Nationality in Regions with High Incidence of Yunnan Sudden Unexplained Death.

ABSTRACT: Objective To explore the association of cardiac disease associated genetic variants and the high incidence of Yunnan sudden unexplained death （YNSUD） in Yi nationality. Methods The genomic DNA was extracted from peripheral blood samples collected from 205 Yi villagers from YNSUD aggregative villages （inpatient group） and 197 healthy Yi villagers from neighboring villages （control group）. Fifty-two single nucleotide variants （SNVs） of 25 cardiac disease associated genes were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The SPSS 17.0 was used to analyze data. The pathogenicities of variants with differences between the two groups that have statistical significance were predicted by protein function prediction software PolyPhen-2 and SIFT. All villagers from inpatient group were given electrocardiogram （ECG） examination using a 12-lead electrocardiograph. Results The allele frequency and the genotype frequency of missense mutation DSG2 （rs2278792, c.2318G>A, p.R773K） of pathogenic genes of arrhythmogenic right ventricular cardiomyopathy （ARVC） in inpatient group was higher than that in control group （P<0.05）. Abnormal ECG changes were detected in 71 individuals （34.6%） in the inpatient group, among which 54 individuals carried R773K mutation, including clockwise （counterclockwise） rotation, left （right） axis deviation, ST segment and T wave alteration and heart-blocking. Conclusion Definite pathogenic mutations have not been found in the 52 cardiac disease genes associated SNVs detected in Yi nationality in regions with high incidence of YNSUD. The cause of high incidence of YNSUD in Yi nationality needs further study.

Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.

RNAi-mediated knockdown of relaxin decreases in vitro proliferation and invasiveness of osteosarcoma MG-63 cells by inhibition of MMP-9.

PURPOSE: The purpose of this study is to determine the role of relaxin knowdown by siRNA transfection in cellular growth and invasion of osteosarcoma MG-63 cells, and discusses the molecular mechanisms of this action. MATERIALS AND METHODS: The expression of relaxin in MG-63 cell was examined by western blot or RT-PCR. To evaluate the biological role of relaxin, proliferation assay (MTT) and invasion assay (BD Matrigel™), apoptosis assay (TUNEL and ELISA) and cell cycle analysis (flow cytometer) were performed after silencing relaxin using siRNA. MMP-9 expressions were analyzed using RT-PCR, western blot and zymography after silencing relaxin. RESULTS: Results showed that the downregulation of relaxin expression by siRNA in human osteosarcoma MG-63 cells significantly inhibited cell proliferation and invasion in vitro. Furthermore, relaxin knockdown led to cell arrest in the G1/G0 phase of the cell cycle, and eventual apoptosis enhancement in MG-63 cells. We provide evidence in our cell model that the relaxin siRNA down-regulated the expression of MMP-9 and the MMP-9 activity, suggesting that relaxin may promote the proliferation, invasion and metastasis of osteosarcoma cells by regulating the expression of MMP-9 and facilitating ECM degradation. CONCLUSIONS: Therefore, siRNA-directed knockdown of relaxin may represent a viable clinical therapy for osteosarcoma.

Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-ß1 into annulus fibrosus cells of rabbit degenerative intervertebral discs.

Intervertebral disc degeneration is a common condition that may lead to low back pain and radiculopathy. Understanding the pathophysiology and cellular and molecular events of degenerative disc disease has resulted in the proposal of a gene therapy approach to halt and reverse disc degeneration. We explored the feasibility of reversing intervertebral disc degeneration using human vascular endothelial growth factor165 (hVEGF165) and transforming growth factor-ß1 (TGF-ß1) gene therapy. hVEGF165 complementary DNA was obtained from pcDNA3(+)-hVEGF165 and cloned into adeno-associated virus (AAV)-pSNAV plasmids to construct the recombinant plasmid, AAV-pSNAV-hVEGF165. After identification through restriction enzyme digestion and DNA sequencing, the AAV-pSNAV-hVEGF165 was transfected into HEK293 cells and vascular endothelial cells. Protein expression of hVEGF165 was detected using a fluorescent immunohistochemical assay, and the effect of hVEGF165 on vascular endothelial cell proliferation was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Packaged AAV-hVEGF165 and AAV-TGF-ß1 were co-transfected into the annulus fibrosus cells of degenerative intervertebral discs. hVEGF165 and TGF-ß1 expression by annulus fibrosus cells and the effect of the co-transfection on the level of collagen type I protein expression by annulus fibrosus cells were detected with Western blot. The results of restriction enzyme digestion and DNA sequencing confirmed that AAV-pSNAV-hVEGF165 plasmids were constructed. The fluorescent immunohistochemical results confirmed hVEGF165 protein expression. The MTT results showed that the hVEGF165 protein promoted vascular endothelial cell proliferation. Biologically active AAV-hVEGF165 and AAV-TGF-ß1 were successfully constructed. Western blot confirmed hVEGF165 and TGF-ß1 expression in annulus fibrosus cells and demonstrated that the level of collagen type I protein expression was significantly higher in annulus fibrosus cells co-transfected with both AAV-hVEGF165 and AAV-TGF-ß1 compared with that in cells transfected with AAV-hVEGF165 or AAV-TGF-ß1 alone. hVEGF165 has a synergistic effect with TGF-ß1 that promotes the expression of collagen type I protein in annulus fibrosus cells from degenerative intervertebral discs.

Proteomic analysis of human bone marrow mesenchymal stem cells transduced with human telomerase reverse transcriptase gene during proliferation.

OBJECTIVES: Previous studies have reported immortalization and tumorigenicity of human mesenchymal stem cells (hMSCs) transduced with exogenous human telomerase reverse transcriptase (hTERT). We also have established a line of hMSCs transduced with hTERT (hTERT-hMSCs) and we have cultured these cells for 290 population doublings (PDs) during which they demonstrated a large proliferation potential but with no tumorigenicity. The aim of this study was to investigate the protein expression profile of hTERT-hMSCs with two-dimensional gel electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be able to analyse the effects of exogenous hTERT on protein expression in hMSCs. MATERIALS AND METHODS: We generated proteome maps of primary hMSCs and hTERT-hMSCs at PD 95 and PD 275. RESULTS: A total of 1543 +/- 145 protein spots in gels of primary MSCs at PD 12, 1611 +/- 186 protein spots in gels of hTERT-hMSCs at PD 95 and 1451 +/- 126 protein spots in gels of hTERT-hMSCs at 275 PD were detected. One hundred of these were successfully identified, including 20 which were differentially expressed. CONCLUSIONS: The results suggest that sustaining levels of prohibitin and p53 expression along with differential expression of proteins in hTERT-hMSCs provide an insight into lack of transforming activity of hTERT-hMSCs during cell proliferation.