Association of MicroRNA Polymorphisms With Hepatocellular Carcinoma in an Iranian Population

BACKGROUND: Single nucleotide polymorphisms (SNPs) can modulate various biological processes by influencing microRNA (miRNA) biogenesis and altering target selection. Common SNPs may alter the processing of miRNA and may be associated with hepatocellular carcinoma (HCC). We investigated the relationship between miR-499A>G, miR-149C>T, miR-196a2T>C, and miR-146aG>C and HCC susceptibility, examining the interaction of the miRNAs with hepatitis B virus (HBV). METHODS: We evaluated the associations of miR-499A>G (rs3746444), miR-149C>T (rs2292832), miR-196a2T>C (rs11614913), and miR-146aG>C (rs2910164) with HCC susceptibility in 100 HCC patients (70 males and 30 females) and 120 healthy controls (70 males and 50 females), using the PCR-restriction fragment length polymorphism method. RESULTS: For miR-499A>G, the frequencies of the AG genotype and G allele were higher in female HCC patients than in female controls (P=0.02 and 0.045, respectively). The frequency of the A allele was higher in HBV-positive HCC patients than in controls (P=0.019). For miR-149C>T, the frequency of the CC genotype was higher in female HCC patients than in female controls (P=0.009). For miR-196a2T>C, the frequencies of the CT and CC genotypes and the C allele were higher in HBV-positive HCC patients than in controls (P C polymorphisms did not differ between HCC patients and controls. CONCLUSIONS: miR-499A>G, miR-149C>T, and miR-196a2T>C were associated with the development of HCC in women and/or that of HBV-related HCC. They can be considered genetic risk factors for the development of HCC among Iranians.

Differentiation of definitive endoderm from human induced pluripotent stem cells on hMSCs feeder in a defined medium

Background: The Definitive Endoderm [DE] differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells [hiPSCs] on an appropriate feeder in a more defined medium

Methods: Human Induced Pluripotent Stem Cells [hiPSCs] were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3- ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts [MEFs] and in the fifth method were human adult bone marrow Mesenchymal Stem Cells [hMSCs]. DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry

Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences

Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine

Interleukin-17 gene expression and serum levels in acute rejected and non-rejected liver transplant patients

Interleukin-17 [IL-17], as a potent proinflammatory cytokine, has a critical role in post liver transplant outcomes. However, there is not much information about the effects of IL-17 cytokine on acute liver rejection. To evaluate the role of IL-17 in post-liver transplant acute rejection. Ninety seven adult liver transplant patients who enrolled in this cross sectional study were divided into Non- Acute Rejected [Non-AR] and Acute Rejected [AR] patient groups. Three blood samples were collected from each patient in days 1, 4 and 7 post liver transplantation. The IL-17 mRNA levels were evaluated using an in-house real time PCR protocol. IL- 17 protein levels were also analyzed in Non-AR, AR and also control groups using ELISA method. The IL-17 mRNA expression level continuously increased in AR patients in all days of follow-up post liver transplantation. IL-17 expression was, however, down regulated after day 4 post-transplant follow-up in Non-AR patients. Both IL-17 mRNA expression and protein levels were also significantly increased in AR patients compared with Non-AR ones. Based on these findings, significant increase of IL-17 mRNA and protein levels in AR patients highlights the important role of IL-17 in acute liver rejection

Anterior shoulder dislocation and ipsilateral humeral shaft fracture

Simultaneous dislocation of shoulder and humeral shaft fracture is a rare injury, and there is no clear protocol for its treatment. Herein we present a case of a 15-year-old boy, who suffered from a job-related accident and sustained fracture of humeral shaft associated with ipsilateral anterior shoulder dislocation and fracture of greater tuberosity 15 years ago. He received closed reduction of both injuries and coaptation plaster splint for four weeks, followed by Sarmiento splint at that time. Fifteen years after the injury, he has no problem related to the previous injury, and does not experience any episode of shoulder instability

Intrascrotal solitary neurofibroma: a case report and review of the literature

Solitary Neurofibroma of the scrotum is an extremely rare benign tumor, particularly when it is not associated with neurofibromatosis type I. To the best of our knowledge, less than 10 cases have been reported in the English literature. Herein, we report a 52-year-old man with the diagnosis of scrotal solitary neurofibroma

Evaluation of the immunomodulatory effect of curdlan on maturation and function of mouse spleen-derived dendritic cells

T helper 1 and T helper 17 cells play important roles in immunity against foreign invaders. Differentiation of these Th subsets is affected by state of maturation and cytokines that are produced by dendritic cells [DCs]. Curdlan is a linear [1-3]-beta- glucan and has shown activity against tumors and infectious agents. This study aims to investigate whether curdlan plays its role through affecting the maturation and cytokine production by DCs. DCs were isolated from the spleen of BALB/c mice by MACS method. After an overnight culture of DCs in the presence of curdlan, the expression levels of CD40, CD86, and MHC-II molecules were determined by flow cytometry. The production of cytokines involved in Th1 and Th17 cell differentiation [IL-12 and IL-6, respectively] was also evaluated by ELISA. Lipopolysaccharide [LPS] treated and untreated cells were considered as positive and negative controls, respectively. The results of this study did not show a significant difference in the levels of surface expression of CD40 [p=0.82], CD86 [p=0.79], and MHC class II [p=0.84] molecules upon exposure to curdlan. However, LPS increased the intensity of CD40 expression on dendritic cells [p=0.04]. In addition, it was revealed that curdlan-exposed DCs are not able to produce a significant amount of IL-6 and IL-12 cytokines. Conversely, LPS-treated DCs were able to make a significant amount of IL-12 [p=0.005]. The results of the present study suggest that curdlan has no effect on Th1 or Th17 differentiation while LPS may induce Th1 deviation by induction of CD40 expression and IL-12 production.

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64