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Objective@#To investigate the effect of silencing troponin I3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes.@*Methods@#The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups: control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group). At 48 h, 72 h after transfection, the cells were collected, and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3, and Western blot was used to detect the protein expressions of Tnni3, Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry.@*Results@#At 48 h post-transfection with Tnni3-siRNA, H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27±0.05 vs. 1.00±0.00) and protein (0.18±0.03 vs. 1.00±0.00) compared with those transfected with NC-siRNA, and the differences were statistically significant (t=25.26, 47.40, all P<0.01). Apoptotic cells were observed in the NC-siRNA group and the Tnni3-siRNA group.At 72 h post-transfection, the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30±1.85)% vs. (0.33±0.15)%] compared with those transfected with NC-siRNA, an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39±0.13 vs. 1.00±0.00), and the differences were statistically significant (t=10.24, 5.19, all P<0.01). Compared with NC-siRNA-transfected H9C2 cells, a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h: 0.32±0.06 vs. 0.46±0.03; 72 h: 0.31±0.01 vs. 0.63±0.04; 96 h: 0.36±0.01 vs 0.75±0.04), and the differences were statistically significant (t=3.62, 13.45, 16.39, all P<0.01). At 72 h post-transfection with Tnni3-siRNA, the percentage of G1 phase, S phase and G2 phase cells was (71.25±3.82)%, (18.28±2.78)% and (9.94±1.09)%, respectively.There was a significant increase in the proportion of G2 phase cells [(9.94±1.09)% vs. (4.54±0.99)%] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA, an increased expression of Cyclin A1 protein (1.89±0.09 vs.1.00±0.00) and a decreased expression of Cyclin B1 protein (0.47±0.06 vs.1.00±0.00) were observed in Tnni3-siRNA-transfected H9C2 cells, respectively, and the differences were statistically significant (t=6.35, 17.12, 15.32, all P<0.01).@*Conclusions@#Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis, suppress cell proliferation, and led to G2 cell cycle arrest.
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Objective To investigate the effect of silencing troponin Ⅰ3 (Tnni3) gene expression on biological property of rat embryonic H9C2 cardiomyocytes.Methods The rat embryonic H9C2 cardiomyocytes were cultured and divided into 2 groups:control group transfected with negative control small interfering RNA (NC-siRNA group) and experimental group transfected with Tnni3 small interfering RNA (Tnni3-siRNA group).At 48 h,72 h after transfection,the cells were collected,and real time quantitative polymerase chain reaction (qPCR)was used to detect the mRNA expressions of Tnni3 and Caspase-3,and Western blot was used to detect the protein expressions of Tnni3,Cyclin A1 and Cyclin B1.Annexin V-fluorescein isothiocyanate(FITC) apoptosis detection kit was used to analyze cell apoptosis.Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) solution and cell cycle was detected by flow cytometry.Results At 48 h post-transfection with Tnni3-siRNA,H9C2 cells exhibited a significant decrease in Tnni3 mRNA (0.27 ± 0.05 vs.1.00 ± 0.00) and protein (0.18 ± 0.03 vs.1.00 ± 0.00) compared with those transfected with NC-siRNA,and the differences were statistically significant (t =25.26,47.40,all P ＜ 0.01).Apoptotic cells were observed in the NC-siRNA group and the Tnai3-siRNA group.At 72 h post-transfection,the percentage of apoptotic cells significantly increased in H9C2 cells transfected with Tnni3-siRNA [(11.30 ± 1.85) ％ vs.(0.33 ± 0.15) ％] compared with those transfected with NC-siRNA,an increased expression of Caspase-3 mRNA was also observed in Tnni3-siRNA-transfected H9C2 cells (1.39 ±0.13 vs.1.00 ±0.00),and the differences were statistically significant (t =10.24,5.19,all P ＜ 0.01).Compared with NC-siRNA-transfected H9C2 cells,a time-dependent reduction in cell proliferation was observed in Tnni3-siRNA-transfected H9C2 cells (48 h:0.32 ± 0.06 vs.0.46 ± 0.03;72 h:0.31 ± 0.01 vs.0.63 ±0.04;96 h:0.36 ± 0.01 vs 0.75 ± 0.04),and the differences were statistically significant (t =3.62,13.45,16.39,all P ＜ 0.01).At 72 h post-transfection with Tnni3-siRNA,the percentage of G1 phase,S phase and G2 phase cells was (71.25 ± 3.82) ％,(18.28 ± 2.78) ％ and (9.94 ± 1.09) ％,respectively.There was a significant increase in the proportion of G2 phase cells [(9.94 ± 1.09) ％ vs.(4.54 ±0.99) ％] in H9C2 cells transfected with Tnni3-siRNA compared with those transfected with NC-siRNA,an increased expression of Cyclin A1 protein (1.89 ±0.09 vs.1.00 ±0.00) and a decreased expression of Cyclin B1 protein (0.47 ± 0.06 vs.1.00 ± 0.00) were observed in Tnni3-siRNA-transfected H9C2 cells,respectively,and the differences were statistically significant (t =6.35,17.12,15.32,all P＜0.01).Conclusions Silencing Tnni3 gene expression in rat embryonic H9C2 cardiomyocytes can induce cell apoptosis,suppress cell proliferation,and led to G2 cell cycle arrest.
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ObjectiveTo investigate the association between R405Q polymorphism of GLI1 gene and tetralogy of fallot(TOF).Methods In the case-control study,the R405Q polymorphism of GLI1 gene in 112 children with TOF and 200 healthy controls were detected with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).The distribution of genotype and allele frequency at R405Q polymorphism site were analyzed and to investigate its relationship with the risk of TOF.ResultsThe distribution of genotype frequency at R405Q polymorphism site was not different between TOF group and the healthy control group(x2 =5.317 ,P = 0.07) .However, the distribution of allele frequency at R405Q polymorphism site was significantly different between TOF group and the healthy control group (x2 = 6.790, P = 0.009) , and the relative risk for TOF in A allele carriers was higher than that in G allele carriers (OR = 1.561,95％ CI 1.116 ～ 2.185) Conclusion The R405Q polymorphism of GLI1 gene is associated with TOF and people with A allele have higher risk with TOF.GLI1 gene might be the genetic susceptibility gene of TOF.
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Objective In the candidate region 12q13 of simple congenital heart defect(CHD),two single nucleotide polymorphisms(SNPs)in the coding-region of GLI1 gene,rs11553626 and rs2228226,were chosen to investigate their distribution in 180 simple CHD patients and 200 normal controls which were collected in the first affliated hospital,China Medical University and The General Hospital of Shenyang Military Area,in order to determine the relationship between GLI1 gene and simple CHD.Methods Genotypes of these two SNPs were analyzed in 180 simple CHD patients and 200 normal controls by Denatured High Performance Liquid Chromatography(DHPLC)and sequencing from Jan.2000 to Jun.2005.?~2 test was applied to analyze the genotype frequency and allele frequency between CHD groups and control groups.Results No polymorphisms were found at rs11553626 while G/C polymorphisms were found at rs2228226.Remarkable significance was observed at rs2228226 in the allele frequency distribution(?~2=8.956,P
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<p><b>OBJECTIVE</b>To identify and characterize laryngeal cancer related novel genes located on chromosome 6q25.</p><p><b>METHODS</b>Electric hybridization was performed in human genome database using EST (expression sequence tag) as probe. Novel genes were deduced by software from positive DNA clones and their cDNAs were amplified by RT-PCR using primers designed according to the sequence of the putative genes.</p><p><b>RESULTS</b>A novel gene was cloned successfully. The full length of this gene was about 21 kb. It contained two exons and produced a 1006 bp transcript coding a protein with 235 amino acid residues. It's 5'flanking sequence contained two binding sites of oncoprotein c-Myc, thus it was named MTLC (c-Myc target from laryngeal cancer cells). Homologous assay showed that MTLC exhibited little overall homology to known human proteins but it exhibited good overall homology to mouse MT-MC1 protein with an identity of 78%. The primary structure of MTLC protein contained a nuclear location signal motif, but it did not have other conserved domains. The results of subcellular location experiment showed that MTLC expressed in nuclei of human hepatocellular carcinoma cell line Bel7402 cells, while a wide distribution of MTLC in various tissues was demonstrated by Northern blotting.</p><p><b>CONCLUSION</b>MTLC may play an important role as a target gene of c-Myc and as a transcription factor in keeping the normal physiological process of cells.</p>