Genetic variation in a miR-335 binding site in BIRC5 alters susceptibility to lung cancer in Chinese Han populations.

Polymorphisms in 3' untranslated region (UTR) of cancer-related genes might affect their regulation by microRNAs (miRNAs) and thereby contribute to carcinogenesis. In this study, we screened single nucleotide polymorphisms (SNPs) in 3' UTR of cancer-related genes and investigated their effects on risk of lung cancer. First, we genotyped seven SNPs in a Chinese Han population with 600 lung cancer patients and 600 matched healthy controls and found that compared with the TT genotype of rs2239680 in 3' UTR of baculoviral IAP repeat containing 5 (BIRC5), C allele was associated with a significantly increased risk of lung cancer and advanced pathologic stage, with the odds ratio for participants carrying the CT or CC genotype being 1.50 [95% confidence interval (CI) 1.20-1.89, P<0.01] and 2.29 (95% CI 1.64-3.18, P<0.01), respectively. These results were further replicated and confirmed in another independent population with 1000 lung cancer cases and 1000 matched healthy controls. In support of the postulation that the 3' UTR SNP may directly affect miRNA-binding site, reporter gene assays indicated BIRC5 was a direct target of miR-335, and the rs2239680 T>C change resulted in altered regulation of BIRC5 expression. Moreover, BIRC5 was over expressed in lung cancer tissues compared with the normal lung tissues, and the protein levels of BIRC5 correlated with SNP genotypes in normal lung tissues. Our findings defined a 3' UTR SNP in human BIRC5 oncogene that may increase individual susceptibility to lung cancer probably by attenuating the interaction between miR-335 and BIRC5.

Inhibition of xenogeneic response in porcine endothelium using RNA interference.

BACKGROUND: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. METHODS: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RESULTS: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. CONCLUSIONS: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.

[The role RNA interference of alpha1, 3GT plays in resistance to complement mediated cytotoxicity of porcine endothelial cells].

OBJECTIVE: To evaluate whether RNA interference can protect porcine endothelial cells from complement mediated cytotoxicity. METHODS: Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected with alpha1,3-galactosyltransferase (alpha1, 3-GT) specific siRNAs. Cells transfected with mismatch SiRNA was used as negative controls. Forty-eight hours later the cells were collected. The expression of alpha1, 3-GT mRNA was examined by RT-PCR. The expression of alpha-Gal was examined by flow cytometry. PED cells ere labeled with (51)Cr and mixed with normal human serum (NHS). The release of (51)Cr was measured by gamma-ray counter. Heat inactivated NHS (HINHS) was used as control. RESULTS: Two isoforms (isoform 1 and isoform 2) were amplified from the PED cells. The expression of alpha1, 3-GT in the PED cells transfected with SiRNA-1 was lower by 70% in comparison with the mock group (69% for the isoform 1 and 72% for the isoform 2, both P < 0.05). However, the expression of alpha1, 3-GT in the PED cells transfected with SiRNA21 was not different from those in the mock group and mismatch group (both P > 0.05). Flow cytometry showed that the average fluorescence intensity of the PED cells transfected with SiRNA-1 was 52, 9, significantly lower than that of the mismatch group and mock group (493.9 and 5-5.7 respectively, both P < 0.0). Fluorescence microscopy observed the "silence effect" of alphaGal after SiRNA-1 transfection. Added with 20% and 40% NHS, the cell dissolution rate of the SiRNA transfection group was lower than that of the mock group by 70% and 60% respectively. CONCLUSIONS: alpha1, 3GT gene silencing actually occurs following transfection of SiRNA-1. Porcine endothelial cells can be the targets of RNAi.

[Study on human leukocyte antigen G1 reducing xeno-cell-rejection by transfecting porcine endothelial cells].

OBJECTIVE: To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92 (NK-92). METHODS: By use of liposomes pack, the pcDNA3. 0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by 51Cr-release assays. RESULTS: HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein and RNA. It also could be found that the survival rate of transfected PECs was much higher than that of non-transfected PECs, when both of them faced the lysis mediated by human PBMC and NK-92. After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (P<0.05). CONCLUSION: The PECs transfected by HLA-G1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.