An Investigation of the Effects of B7-H4 Gene rs10754339 and miR-125a Gene rs12976445 on Cancer Susceptibility

Objective To investigate the effects of the B7-H4 gene rs10754339 and miR-125a gene rs12976445 on cancer susceptibility through a case-control study and meta-analysis.Methods A total of 1,490 cancer patients(lung/gastric/liver/:550/460/480)and 800 controls were recruited in this case-control study.The meta-analysis was performed by pooling the data from previous related studies and the present study.Results The results of this study showed that in the Hubei Han Chinese population,the rs10754339gene was significantly associated with the risk of lung and gastric cancer but not liver cancer,and the rs12976445 gene was significantly associated with the risk of lung cancer but not liver or gastric cancer.The meta-analysis results indicated that rs10754339 and rs12976445 contributed to cancer susceptibility in the Chinese population and also revealed a significant association between rs10754339and breast cancer risk,as well as between rs12976445 and lung cancer risk.Conclusion The B7-H4 gene rs10754339 and miR-125a gene rs12976445 may be the potential genetic markers for cancer susceptibility in the Chinese population,which should be validated in future studies with larger sample sizes in other ethnic populations.

AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Experimental Study on the Antitumor Effect of Mouse B7-1 Gene

Summary: Mouse B7 1 cDNA was cloned by RT PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named pCD mB7 1, in which the B7 1 cDNA was identified to be consistent with the data from other researchers. pCD mB7 1 plasmid was transfected into B16(F0) cells, and effective expression of mB7 1 in these tumor cells could be detected till the 6th month by RT PCR and RNA hybridization. Specific cytotoxity assay of lymphocytes was conducted after culturing with tumor cells and the results demonstrated that B16 cells transfected with B7 1 gene were more effective than B16 wt and B16 neo in inducing specific cytotoxity of lymphocytes against B16 wt cells. It is suggested that expression of B7 1 gene in tumor cells could enhance the immunogenicity and induce the effective antitumor immunity.

Construction of Eukaryotic Expression Vector Containing B7-1/GFP Gene and Its Expression in Osteosarcoma Cell Line

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.

High-resolution genetic mapping and identification of candidate genes for the wheat stem rust resistance gene Sr8155B1

Stem rust,caused by Puccinia graminis f.sp.tritici(Pgt),threatens global wheat production.Development of cultivars with increased resistance to stem rust by identification,mapping,and deployment of resistance genes is the best strategy for controlling the disease.In this study,we performed fine mapping and characterization of the all-stage stem rust resistance(Sr)gene Sr8155B1 from the durum wheat line 8155-B1.In seedling tests of biparental populations,Sr8155B1 was effective against six Chinese Pgt races tested.In a segregating population of 5060 gametes,Sr8155B1 was mapped to a 0.06-cM region flanked by markers Pku2772 and Pku43365,corresponding to 1.5-and 2.7-Mb regions in the Svevo and Chinese Spring reference genomes.Both regions include several typical nucleotide-binding leucine-rich repeat(NLR)and protein kinase genes that represent candidate genes.Among them,three NLR genes and three receptor-like protein kinases were highly polymorphic between the parental lines and their transcripts were upregulated in the homozygous resistant line TdR2 relative to its susceptible sister line TdS4.Four markers(Pku2772,Pku43365,Pku2950,and Pku3721)developed in this study,together with seedling resistance responses,correctly predicted Sr8155B1 absence or presence in 78 tetraploid wheat genotypes tested.The presence of Sr8155B1 in tetraploid wheat accessions CItr 14916,PI 197492,and PI 197493 was confirmed by mapping in three F_(2)populations.The genetic map and linked markers developed in this study may accelerate the deployment of Sr8155B1-mediated resistance in wheat breeding programs.

GM-CSF GENE OR B7-1 GENE MODIFIED MURINE EL-4 CELLS VACCINE

Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced BL-4 cells can be used as a good tumor vaccine. The combination of the two kinds of vaccine may have potential application value in human cancer treatment.

Synergetic anticancer effect of combined quercetin and recombinant adenoviral vector expressing human wild-type p53,GM-CSF and B7-1 genes on hepatocellular carcinoma cells in vitro

AIM: This study investigated the anti-cancer effect ofcombined quercetin and a recombinant adenovirus vectorexpressing the human p53, GM-CSF and B7-1 genes(designated BB-102) on human hepatocellular carcinoma(HCC) cell lines in vitro.METHODS: The sensitivity of HCC cells to anticancer agentswas evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of cells infectedwith BB-102 was determined by trypan blue exclusion. Theexpression levels of human wild-type p53, GM-CSF and B7-1genes were determined by Western blot, enzyme-linkedimmunosorbent assay (ELISA) and flow cytometric analysis,respectively. The apoptosis of BB-102-infected or quercetin-treated HCC cells was detected by terminal deoxynucleotidyltransferase (TdT) assay or DNA ladder electrophoresis.RESULTS: Quercetin was found to suppress proliferation ofhuman HCC cell lines BEL-7402, HUH-7 and HLE, with peaksuppression at 50 μmol/L quercetin. BB-102 infection wasalso found to significantly suppress proliferation of HCC celllines. The apoptosis of BB-102-infected HCC cells was greaterin HLE and HUH-7 cells than in BEL-7402 cells. Quercetin didnot affect the expression of the three exogenous genes inBB-102-infected HCC cells (P＞0.05), but it was found to furtherdecrease proliferation and promote apoptosis of BB-102-infected HCC cells.CONCLUSION: BB-102 and quercetin synergeticallysuppress HCC cell proliferation and induce HCC cell apoptosis,suggesting a possible use as a combined anti-cancer agent.

Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1 genes co-transfected liver cancer cell vaccines

AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice.METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards.The immunoprotection was investigated and the reactive T cell line was assayed.RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes cotransfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7vaccine) on the onset of tumor formation was the strongest.When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, χ2=7.70-11.69, P＜0.05) and the implanted tumor was the smallest (z =3.20-44.10, P＜0.05).The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively.The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P＜0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5±2.5%,65.0±2.9%, 83.1±1.5% respectively, compared with other groups, P＜0.05).CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.

ANTITUMOR IMMUNITY AND VACCINE EFFECT INDUCED BY IL-12 SYNERGIZES B7-1 GENE TRANSFECTED CELLS

Objective: To study the synergic effects of IL-12 and B7-1 transfectant on antitumor immunity in vivo. Methods: The retrovirus vector encoding mIL-12 and mB7-1 gene was tranfected into EL-4 thymic lymphoma cells respectively.The cells were used as tumor vaccine and the therapeutic effect was observed. Results: In contrast to the miceimmunized with EL-4/Wt or EL-4/Neo groups, thetumorigenicity of EL-4/IL-12 transfectant was decreased(P<0.001). The EL-4/IL-12 and EL-4/B7-1 cells irradiatedwith 60Co showed significant systematic protective effectsagainst the rechallenge of EL-4/Wt. 60Co irradiatedEL-4/IL-12 cells delayed the occurrence of tumor andprolonged the survival period of tumor bearing mice.Combination of the vaccines of EL-4/IL-12 and EL-4/B7-1 resulted in the enhanced therapeutic effect compared witheach single transfectant group (P<0.001). Conclusion: The results showed that IL-12 transduced cells could enhancethe antitumor immunity of host as cancer vaccine.Combination of the EL-4/IL-12 and EL-4/B7-1 transfectant could improve immunity of host and is a prospect cancervaccine.

Nonsense variant of ATP8B1 gene in heterozygosis and benign recurrent intrahepatic cholestasis: A case report and review of literature

BACKGROUND Benign recurrent intrahepatic cholestasis is a genetic disorder with recurrent cholestatic jaundice due to ATP8B1 and ABCB11 gene mutations encoding for hepato-canalicular transporters.Herein,we firstly provide the evidence that a nonsense variant of ATP8B1 gene(c.1558A>T)in heterozygous form is involved in BRIC pathogenesis.CASE SUMMARY A 29-year-old male showed severe jaundice and laboratory tests consistent with intrahepatic cholestasis despite normal gamma-glutamyltranspeptidase.Acute and chronic liver diseases with viral,metabolic and autoimmune etiology were excluded.Normal intra/extra-hepatic bile ducts were demonstrated by magnetic resonance.Liver biopsy showed:Cholestasis in the centrilobular and intermediate zones with bile plugs and intra-hepatocyte pigment,Kupffer’s cell activation/hyperplasia and preserved biliary ducts.Being satisfied benign recurrent intrahepatic cholestasis diagnostic criteria,ATP8B1 and ABCB11 gene analysis was performed.Surprisingly,we found a novel nonsense variant of ATP8B1 gene(c.1558A>T)in heterozygosis.The variant was confirmed by Sanger sequencing following a standard protocol and tested for familial segregation,showing a maternal inheritance.Immunohistochemistry confirmed a significant reduction of mutated gene related protein(familial intrahepatic cholestasis 1).The patient was treated with ursodeoxycholic acid 15 mg/kg per day and colestyramine 8 g daily with total bilirubin decrease and normalization at the 6th and 12th mo.CONCLUSION A genetic abnormality,different from those already known,could be involved in familial intrahepatic cholestatic disorders and/or pro-cholestatic genetic predisposition,thus encouraging further mutation detection in this field.

Expression of IGF-Ⅱ,p53,p21 and HBxAg in precancerous events of hepatocarcinogenesis induced by AFBI and/or HBV in tree shrews

INTRODUCTIONIn order to study the relationship between oncogeneexpression and HCC generation,we observed theprecancerous hepatic GGT loci,IGF-Ⅱ,p53 andp21 expression during hepatocarcinogenesis of treeshrew induced by hepatitis B virus (HBV) and/oraflatoxin B1 (AFB1).

The Effects of Dwarfing Genes (Rht-B1b, Rht-D1b, and Rht8) with Different Sensitivity to GA_3 on the Coleoptile Length and Plant Height of Wheat

Understanding the effects of wheat dwarfing genes on the coleoptile length and plant height is crucial for the proper utilization of dwarfing genes in the improvement of wheat yield. Molecular marker analysis combined with pedigree information were used to classify wheat cultivars widely planted in major wheat growing regions in China into different categories based on the dwarfing genes they carried. The effects of the dwarfing genes with different sensitivity to gibberellins （GA3） on the coleoptile length and plant height were analyzed. Screening of 129 cultivars by molecular marker analysis revealed that 58 genotypes of wheat contained the dwarfing gene Rht-B1b, 24 genotypes of wheat contained Rht-D1b gene and 73 genotypes of wheat possessed Rht8 gene. In addition, among these 129 cultivars, 35 genotypes of wheat cultivars contained both Rht-B1b and Rht8 genes and 16 genotypes of wheat cultivars contained both Rht-D1b and Rht8 genes. Wheat cultivars with the dwarfing genes Rht-B1b or Rht-D1b were insensitive to GA3, while the cultivars with the dwarfing gene Rht8 were sensitive to GA3. Most of the wheat genotypes containing combination of Rht8 gene with either Rht-B1b or Rht-D1b gene were insensitive to GA3. The plant height was reduced by 24.6, 30.4, 28.2, and 32.2%, respectively, for the wheat cultivars containing Rht-B1b, Rht-D1b, Rht-B1b ＋ Rht8, and Rht-D1b ＋ Rht8 genes. The plant height was reduced by 14.3% for the wheat cultivar containing GA3-sensitive gene Rht8. The coleoptile length was shortened by 25.4, 31.3, 28.4 and 31.3%, respectively, in the wheat cultivars containing Rht-B1b, Rht-D1b, Rht-B1b ＋Rht8 and Rht-D1b ＋ Rht8 genes, while the coleoptile length was shortened only by 6.2% for the wheat cultivar containing Rht8 gene. We conclude that GA3-insensitive dwarfing genes （Rht-B1b and Rht-D1b） are not suitable for the wheat improvement in dryland because these two genes have effect on reducing both plant height and coleoptile length. In contrast, GA3- sensitive dwarfing gene （Rht8） is a relatively ideal candidate for the wheat improvement since it significantly reduces the plant height of wheat, but has less effect on the coleoptile length.

Interleukin-1β gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection

AIM： To examine the effect of interleukin-l-beta （IL-113） promoter region C-511T and IL-1 receptor antagonist （IL-1RN） polymorphism among the patients with chronic hepatitis B virus （HBV） infection （HCC and non-HCC）. METHODS： Genomic DNA from 136 Thai patients with chronic HBV infection （HCC =46 and non-HCC= 90） and 152 healthy individuals was genotyped for IL-113 gene polymorphism （-511） using polymerase chain reaction with sequence specific primers （PCR-SSP）. The variable number of tandem repeats （VNTR） of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by X^2 test. RESULTS： IL-1B-511 genotype c/c was found to be significantly different in patients with HCC when compared with healthy individuals （P = 0.036, OR = 2.29, 95%CI = 1.05-4.97） and patients without HCC （P=0.036, OR= 2.52, 95%CI=1.05-6.04）. Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control （P=0.033, OR= 1.72, 95%CI=1.04-2.84）. However, no significant association in IL-1RN gene was found between the two groups. CONCLUSION： IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin （IL）-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

Cloning and expression of HLA-B7 gene

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miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene

MicroRNAs(miRNAs) have been widely identified in porcine testicular tissues and implicated as crucial regulators of proliferation, apoptosis, and differentiation in porcine spermatogenesis related cells. However, the function roles of most of the miRNAs that have been identified in Sertoli cells are poorly understood. In the present study, six experiments were conducted to study the regulatory role of miR-10b in porcine immature Sertoli cells. In experiment 1, the results showed that the relative mRNA expression level of miR-10b in porcine testicular tissues decreased quadratically(P<0.001) with increasing age, while the relative mRNA expression level of DAZAP1 gene increased(P<0.001). In addition, the mRNA expression of miR-10b was negatively(P<0.01) correlated with DAZAP1 mRNA expression(r=–0.550). In experiment 2, the results from the bioinformatic analysis and a luciferase reporter assay demonstrated that miR-10b directly targeted the DAZAP1 gene in porcine immature Sertoli cells. DAZAP1 mRNA and protein expressions were both regulated(P<0.05) by miR-10b. In experiments 3 to 5, the over-expression of miR-10b or the siRNA-mediated knockdown of the DAZAP1 gene promoted(P<0.05) porcine immature Sertoli cell proliferation, as determined by the Cell Counting Kit-8(CCK-8) assay and the 5-Ethynyl-2′-deoxyuridine(EdU) assay. However, an annexin V-FITC/PI staining assay and the expression of cell survival-related genes indicated that over-expression of miR-10b or knockdown of DAZAP1 had no effect(P>0.05) on porcine immature Sertoli cell apoptosis. In experiment 6, the co-transfection treatment results showed that miR-10b promoted(P<0.05) porcine immature Sertoli cell proliferation by targeting DAZAP1 gene. Overall, these experiments demonstrated that miR-10b promotes porcine immature Sertoli cell proliferation by targeting the DAZAP1 gene.

Correlation between single nucleotide polymorphism of rs3811047 in IL-1 F7 gene and rheumatoid arthritis susceptibility among Han population in central plains of China

Objective:To discuss the association between single nucleotide polymorphism(SNP) of rs3811047 in IL-1 F7 gene and rheumatoid arthritis(RA) susceptibility among the Han population in central plains of China.Methods:A total of 276 RA patients admitted to our hospital from December 2009 to December 2011 together with 276 healthy physical examinees in the same period were chosen as the subjects.The typing for rs3811047 SNP in IL-1 F7 gene was carried out by using ligase detection reaction and polymerase chain reaction technique.And the frequency of each allele and genotypes distribution was calculated so as to evaluate the association between genotype distribution and RA susceptibility.Results:The frequency of A allele of rs3811047 in IL-1 F7 gene in RA group and control group was 16.27%and 17.68%,respectively,and that of G allele in two groups was 83.73%and 82.32%,respectively.The difference between two groups wasn’t statistical significant(P 】0.05).The frequency of genotype AA,AG and GG in RA group was 2.19%,27.84%and 69.97%,respectively,while that in control group was 2.94%,29.78%and 67.28%, respectively.The difference of distribution of three genotypes was not statistically significant (P 】0.05).RA patients with A allele were better than those without A allele in joint swelling index, rest pain,HAQ scoring and blood sedimentation.There was significant difference between two groups in above indexes(P【0.05/P【0.01).Conclusions:No significant correlation between RA susceptibility among the Han population in central plains of China and rs3811047 SNP inIL-1 F7 gene is observed.However,A allele of rs3811047 has certain influence on the condition of RA patients.