MiR-9 Induces Expression Of PRDM1 In L428 Cells Through Inactivation Of TGF-β1/Smads Signal

BackgroundHodgkin’s lymphoma (HL) is one kind of highly malignant tumour of the lymphatic hematopoietic system, and can be classified as nodular lymphocyte predominant (NLPHL) and the typical Hodgkin’s lymphoma (CHL), the latter is the most common type of HL.The CHL, characterized by the rare and so-called Hodgkin/Reed-Stemberg(HR/S) tumour cells, with the background of predominant reactive inflammatory cells. Problems such as the origination and mechanism of H/RS cells is one of the concerns of the academia. A growing body of evidence now indicates that the occurrence of tumor is due to the result of the differentiation of normal cells disordered or blocked. Kuppers et al. found that the vast majority of H/RS cells was absent of B cell phenotype and early plasma cells differentiation potential was disordered compared with the characteristics of B cell differentiation. This research will focus on exploring the key targets which can reverse HR/S differentiation disorder.Current research suggests that reversing HR/S cell differentiation disorder need to open the expression of the key target PRDM1, which switching the cell to differentiate into plasma cells and inducing H/RS cells to turn back towards the end differentiation of B cells. Other research indicates that miR-9 can negatively regulate the expression of PRDM1 in L428. These prompt us to take miR-9 for granted as the key target that can reverse L428 differentiation disorder. Our preliminary research shows that PRDM1 expression is significantly increased after instantaneously silencing the expression of miR-9 in L428 cells, which further confirms that miR-9 can open the expression of the key target PRDM1 and put the way to differentiate into plasma cells, reversing L428 cells differentiation disorder. So, this study focuses on exploring the target genes of miR-9 in inducing L428 cells redifferentiation, as well as the target gene’s role in the differentiation and the related signaling pathways involved.Many related reports indicate that TGF-β1/Smads pathways involve the regulation of tumor differentiation and TGF-β receptors are more related to this topic. Early pre-experiment of our research group also predicted that the TGF-β1/Smads pathway played a role in L428 cell redifferentiation, and the expression mode of TGF-βR1 in seven B lymphoma cell lines was contrary to miR-9. Then, whether TGF-βR1 is the targeted regulation of genes of miR-9 in L428 cells and what the role of TGF-β1/Smads pathways is in L428 cells? These need to be further explored.Objective1. To observe and verify the effect of miR-9 on the expression of PRDM1, redifferentiation and biological behavior in L428 cells.2. To predict and identify the target mRNA of miR-9 induced expression of PRDM1 in L428 cells.3. To explore the signaling pathway related to miR-9 regulation of L428 cells redifferentiation and its signaling pathways involved in each member’s role.Methods1.Effects of miR-9 on expression of PRDM1、redifferentiation and biological behaviors of H/RS cells L428(1) We designed and constructed miR-9 lentiviral vector and transfected into L428 cells,the stable miR-9-silence expressing cell lines were obtained by FCM(flow cytometry) method; the stable miR-9-silence expressing cell lines were transfected with miR-9-mimics which might reverse the expression of miR-9; the expression of PRDM1 was detected by western blot and fluorescence confocal.(2) Fluorescence microscope was applied to detect cell adhesion; Phalloidin staing of cytoskeletal fractions was used to determine the expression of F-actin and western blot was used to measure the expression of Casp-9 and Cleaved-Casp-3 in miR-9 transfection group cell lines.(3) Flow cytometric analysis was applied to analyse the differentiation antigen expression(CHL diagnostic markers CD 15 and CD45、germinal center marker CD10、plasma cell markers CD38 and CD138) in L428 cells after miR-9 silencing expression.2. Predicting and characterizing the target mRNA of miR-9(1) Predicting the target mRNA with the index of miR-9 in the DIANA-microT, TargetScan, Pictar and miRanda prediction database, the four results would be intersected, which predicted a higher reliability and accuracy of target mRNA. We selected predicting target mRNA by prediction database and then tested and screened the best target mRNA and the relative expression was detected in7 B-cell lymphoma by qRT-PCR.(2) We designed and builted GV306-TGF-βR1-3’UTR and GV306-mutTGF-βR1-3’UTR vector and detected whether miR-9 could be combined with GV306-TGF-βR1-3’UTR area by luciferase reporter system.(3) Real-time PCR was used to detect the expression of miR-9 and TGF-βR1 in seven B lymphoid cell lines.Their correlation was detected by Pearman correlation analysis and western blot was applied to detect the expression of TGF-βR1 in miR-9 transfection group cell lines.3. The role of TGF-β1/Smads signal pathway and its related member in the role of miR-9 regulating L428 cells redifferentiation(1)Transduct the TGF-β1/Smads inhibitor SB431542 into L428-miR-9-inhibitor cell and TGF-β1/Smads activation TGF-β1 into L428 cell, respectively, and detect PRDM1 expression by western blot. Flow cytometric was applied to determine the effect of TGF-β1 stimulus in differentiation antigen expression of L428 cell after the transduction of TGF-β1/Smads activation TGF-β1 into L428 cell.(2) Western blot was conducted to detect the expression changes of Smad2、 Smad3、P-Smad2、P-Smad3、Smad4 gene after miR-9 silencing expression and TGF-β1/Smads activation TGF-β1 into L428 cell.The expression of Smad2、Smad3、 TGF-βR1 in L428-miR-9-inhibitor and Smad4 in L428 was knocked down by siRNA. The efficacy of transfection and expression of PRDM1 after transfection was confirmed by western blot.Statistical analysisAll images are representative of at least three independent experiments. QRT-PCR assays are performed in triplicate for each experiment. The data shown are presented as the mean±standard derivation (SD) for three independent experiments. The SPSS software 13.0 are used for the statistical analysis. Differences are considered statistically significant at p< 0.05, assessed using the one-way ANOVA for assays with qRT-PCR.Results1.Effects of miR-9 on expression of PRDM1、redifferentiation and biological behaviors of L428 cells(1)Fluorescence confocal and western blot results showed that the expression of PRDM1 was significantly increased after transfected with miR-9 silence expression vector, while the expression of PRDM1was reversed after transferring miR-9 mimics into the stable miR-9-silence expressing cell lines.(2) Fluorescence microscope、Phalloidin staing of cytoskeletal fractions and western blot results showed separately that cell adhesion and the expression of F-actin、Casp-9 and Cleaved-Casp-3 were significantly increased after transfecting with miR-9 silence expression vector, while the expression of F-actin、Casp-9 and Cleaved-Casp-3 were reversed after transferring miR-9 mimics into the stable miR-9-silence expressing cell lines.(3) Flow cytometric results showed that germinal center marker CD10、Plasma cell markers CD38 and CD138 were significantly increased after transfecting with miR-9 silence expression vector in L428 cells,while CHL diagnostic markers CD 15 decreased and CD45 increased.2. Prediction and validation the target mRNA of miR-9(1) Results showed that TGF-βR1 (predicted by four common databases) had the high predictive values and was most closely related to B cells differention in7 B-cell lymphoma detecting the relative expression of TGF-β1/Smads by qRT-PCR.So TGF-βR1 was chosen as the miR-9 target.(2)Luciferase reporter system showed that the luciferase activity was increased significantly especially in miR-9 group compared to blank group, NC group or mut TGF-βR1-3’UTR after transfection of wild-type TGF-βR1-3’UTR plasmid. The above results indicate that miR-9 activates the activity of luciferase through the binding sites of TGF-βR1-3’UTR region.(3)Real-time PCR results showed that the relative expression of TGF-βR1 was higher in KM3 and RPMI8226 cell lines than that in L428 cell lines, while miR-9 was lower in KM3 and RPMI8226 cell lines than that in L428 cell lines.Western blot results showed that the expression of TGF-βR1 was significantly increased after transfected with miR-9 silence expression vector, while the expression of TGF-βR1 was reversed after reversed the expression of miR-9.3. TGF-β1/Smads signal pathway and its related members in the role of miR-9 regulated L428 cells redifferentiation(1)Western blot results showed that the expression of PRDM1 was significantly increased after tranducing TGF-β1/Smads activation TGF-β1 into L428 cells, while the expression of PRDM1 was reversed after transduction of the TGF-β1/Smads inhibitor SB431542 into L428-miR-9-inhibitor cells.Flow cytometric results showed that germinal center marker CD10、plasma cell markers CD38 and CD138 were significantly increased after tranducing TGF-β1/Smads activation TGF-β1 into L428 cells,while CHL diagnostic markers CD 15 decreased and CD45 increased.(2)Western blot results showed that the expression of Smad3、P-Smad2 and P-Smad3 was significantly increased and Smad4 decreased while Smad2 had no change after miR-9 silencing expression and TGF-β1/Smads activation TGF-β1 into L428 cells.The expression of Smad2、Smad3、TGF-βR1 in L428-miR-9-inhibitor and Smad4 in L428 cells was knocked down by siRNA. The expression of PRDM1 was significantly increased after the expression of Smad2、Smad3、TGF-βR1 in L428-miR-9-inhibitor cells were knocked down by siRNA while the expression of PRDM1 was significantly decreased after the expression of Smad4 in L428 knocked down by siRNA.Conclusion1.miR-9 negatively regulates the expression of PRDM1 which induced redifferentiation in L428 cells, and miR-9 can also inhibit cell adhesion conglobation、apoptosis and cytoskeleton remodeling.2.miR-9 can combine with TGF-βR1 in the 3’UTR region directly,and targeted-regulate the expression of TGF-PR1.3.miR-9 regulates redifferentiation in L428 cells through the TGF-β1/Smad2/ Smad3/Smad4 signaling pathways, and TGF-β1/Smad2/Smad3/Smad4 signal path each member can regulate the expression of PRDM1 which was the switch of plasma cells differentiation.