| BackgroundOur previous study proved that TLR4 played an important role in radioprotection.The activation of TLR4 increased the survival rate of irradiated mice evidently.Lipopolysaccharide（LPS）is the ligand of TLR4 and has fierce side-effect which limits its usage in clinic.As a result,it is urgent to find new target which plays important roles in the regulation of radiosensitivity in TLR4 signal pathway.Content of researchA.The role of miR-351 in radio-sensitivity and its target genes.1.Identification of differentially expressed miRNAs related to TLR42.Bioinformatic analysis of miR-3513.Up-and down-regulation of miR-351 in vitro4.Effects of miR-351 on radio-sensitivity in vitro5.Differentially expressed proteins related to miR-351 identified by iTRAQ6.Confirmation of target of miR-351 using Dual-Luciferase Reporter AssayB.The effects of miR-351 on hematopoiesis1.Establishment of miR-351 knock-out mouse2.Analysis of blood cells in peripheral blood in miR-351-/-mice3.Analysis of HSCs in miR-351-/-mice4.Analysis of myeloid progenitor in bone marrow in miR-351-/-mice5.Analysis of erythroid and megakaryocytic differentiation in miR-351-/-mice6.Analysis of lymphoid progenitor in miR-351-/-mice7.Effects of Bcl2l13 on repopulation and cell cycle of HSPCs in vitroC.The regulation of miR-351 in radio-sensitivity in CT26 cell1.The radio-protective effects of LPS in mice2.The regulation of LPS in IL-6,IL-11 and PGE2 in mice3.The radio-protective effects of LPS in CT26 cell4.The regulation of LPS in miR-351 level in vitro5.The regulation of miR-351 in radio-sensitivity in CT26 cell6.The regulation of miR-351 in the levels IL-6,IL-11 and PGE2 in CT26 cell7.The targeting effects of miR-351 on IL-6,IL-11 and PGE2.Methods1.Total RNA extractionCellular total RNA was extracted using a RNA extraction kit,quantified and stored at-80℃.2.Real time PCR assayMake RNA extracts,prepare the cDNA and run realtime PCR with Roche machine.3.Protein extractionCells were lysed using RIPA buffer.Then lysates were mixed with loading buffer forincubating at 100℃and stored at-20℃.4.Western blot assayLysates were separated by SDS-PAGE,followed by blotting onto polyvinyldifluoridemembranes and detection with different antibodies coupled with the ECL detection system.5.Apoptosis assayCells were labeled with AnnexinⅤand PI using a apoptosis detection kit and then analyzedby flow cytometry.6.Cell viability assayCells were treated with CCK-8 and incubated at 37℃.Then OD value was measured toevaluate the viability.7.TransfectionPlasmid or miRNA was transfected to cells using Lipofectamine 3000.At different time aftertransfection cells were objected to further experiment.8.Lentivirus packaging and infectionPlasmids for lentivirus packaging were transfected to 239T.After 48h,supernatants of cellcultures were collected and stored at-80℃for further experiment.For infection,lentivirusmixed with culture medium was added to cells.Then stable transgene cell line was selected.9.isobaric tags for relative and absolute quantitation（iTRAQ）Protein was extracted,digested and labeled with iTRAQ regent.Then mass spectrometryanalysis was conducted.10.miRNA micro arrayTotal RNA was extracted and labeled with Cy3.Then labeled RNA was purified,hybridizedand scanned for original images.Finally analysis of bioinformatics was conducted.11.Luciferase reporter assaysAll transfections were carried out with Lipofectamine 3000,according to the manufacturer’sinstructions.48 h after transfection,luciferase activities were measured using theDual-Luciferase Reporter Assay System（Promega）and normalized to Renilla luciferaseactivity.12.Establishment of miR-351-/-mouse strainThe miR-351-/-mouse strain was generated using CRISPR/Cas9 technology.13.DNA gel electrophoresisDNA in tissues was extracted and amplified by PCR.Then DNA gel electrophoresis wasconducted.14.Analysis of peripheral bloodMurine peripheral blood was collected and anti-coagulated.Then samples were analyzed usingan automatic blood cell analyzer.15.Bone marrow cells（BMCs）collectionMice were sacrificed and femurs were collected.BMCs suspensions were obtained bycrushing of the femurs and removal of the red blood cells.16.Spleenocytes and Thymocytes collectionMice were sacrificed and spleenocytes and thymocytes suspensions were obtained by gentlebreaking up of the tissues and removal of the red blood cells.17.Flow cytometry analysisStain the cells with different antibodies,and then analyze with flow cytometry.Analyze thedata with special software.18.Mouse hematopoietic stem and progenitor cell isolation and cultureBMCs were obtained and red blood cells were removed.Mouse HSPCs were purified using akit（stem cell）of immunomagnetic beads method.HSPCs were cultured in the medium ofStemSpan SFEM with IL-3（20ug/ml）,SCF（100ug/ml）and Flt-3（100ug/ml）.19.Transplantation experimentsFor transplantation of total bone marrow cells,mice were irradiated at a dose of7.5Gy（1Gy/min）.These mice were then reconstituted with 5×106 freshly isolated bonemarrow cells in 0.2 ml PBS within 24 h of irradiation.20.Cell cycle analysisThe cell cycle status of HSCs were detected using PI staining method and analyzed by flowcytometry.21.Statistical analysisSPSS18.0 and Graphpad Prism5 were used for statistical analysis.For two independentsamples,unpaired Student’s t test or Wilcoxon rank sum test was performed.To comparethree or more samples,one-way ANOVA,two-way ANOVA or Kruskal-Wallis H testwas performed.Theαlevel was set at 0.05.ResultsA.The role of miR-351 in radio-sensitivity and its target genes.1.miRNA micro array showed that miR-351 was most markedly up-regulated.miRNA micro array was used to identify differentially expressed genes between wild type mice and TLR4-/-mice treated with irradiation.Results showed that miR-351 was most markedly up-regulated in TLR4-/-mice.2.Bioinformatics analysis showed that miR-351 could be regulated by several potential TFs.Bioinformatics analysis of AliBaba2 and Gene-Regulation were conducted and found that the promoter region of miR-351 could be potentially targeted by 8 TFs.3.Up-and down-regulation of miR-351 in cellsPCR showed that the fold changes of up-regulation of miR-351 were 250 in NIH/3T3,550 in BALB/3T3 and 123 in 3T3-L1.miR-351 inhibitor down-regulated the level of mir-351 by 40%in cells.4.miR-351 up-regulation increased the radio-sensitivity in vitroThe up-regulation of miR-351 increased the apoptosis and decreased the viability of cells treated with irradiation.In addition,down-regulation of miR-351 alleviated radiation-induced injuries in vitro.5.Identification of targeted genes of miR-351 by iTRAQiTRAQ was used to identify genes targeted by miR-351.Results showed that 249genes,86 genes and 214 genes were identified in BALB/3T3,3T3-L1 and NIH/3T3respectively.6.miR-351 down-regulated Bcl2l13 in 3’UTR dependent mannerLuciferase reporter assays showed that 3’UTR activity of Bcl2l13 dicreased by28%（P<0.01）after miR-351 treatment.However,the mutation of 3’UTR of Bcl2l13reversed this effect（P<0.01）.B.The effects of miR-351 on hematopoiesis1.Generation of miR-351-/-mouseThe miR-351-/-mouse strain was generated using CRISPR/Cas9 technology.DNA sequencing confirmed the knock-out of miR-351.2.Deletion of miR-351 promoted hematopoiesisAnalysis showed that the numbers of BMCs of miR-351-/-mouse was larger than that of wild type mouse（P<0.05）.Bone marrow transplantation showed that numbers of BMCs,WBC,RBC and platelate in recipients of miR-351-/-increased（P<0.05）.3.miR-351-/-mouse showed a larger pool of HSCsThe number of HSCs（LSK cells）was larger in miR-351-/-mouse（P<0.05）.Furthermore,the proportion of Flt3+LSK cells is increased.4.Deletion of miR-351 promoted the self-renew of HSCsDeletion of miR-351-/-had a higher fraction of LSK cells in the S-G2-M phase of the cell cycle.5.Deletion of miR-351 promoted the differentiation of myeloid progenitor.The numbers of CMP,GMP and MEP were larger in miR-351-/-mouse（P<0.05）.The number of myeloid cells（CD11b+Gr-1+）also increased in miR-351-/-mouse（P<0.05）.6.Deletion of miR-351 promoted erythroid and megakaryocytic differentiation.The numbers of erythroid and megakaryocytic progenitor in miR-351-/-mouse were larger（P<0.05）.7.miR-351 deletion showed no effect on lymphoid differentiationResults showed that there was no difference in the number of CLP,T cells and B cells between miR-351-/-mouse and wild type.8.Bcl2l13 promoted the repopulation of HSPCs in vitroOverexpression of Bcl2l13 increased the proportion of HSPCs in the S-G2-M phase of cell cycle.PCR showed that Bcl2l13 down-regulated the levels of Cdkn1a and Bmi-1,while up-regulated the levels of HoxB4 andβ-catenin（P<0.05）.C.The regulation of miR-351 in radio-sensitivity in CT26 cell1.Low dose of LPS protected mice from radiation-induced injuries via TLR4High dose of LPS（>0.5mg/mouse）was highly toxic,while 100%of mice treated with low dose of LPS（0.05/mouse and 0.1/mouse）survived.LPS increased the survival rate of irradiated mice with different doses of radiation（P<0.05）.However,LPS showed no radio-protective effects in TLR4-/-mice.2.LPS up-regulated the levels of IL-6,IL-11 and PGE2 via TLR4LPS increased the levels of IL-6,IL-11 and PGE2 in mice treated with or without irradiation（P<0.05）.However,the effects of LPS were decreased in TLR4-/-mice.3.Low dose of LPS alleviated radiation-induced injuries in CT26High dose of LPS（>10ug/ml）decreased the cell viability and low dose of LPS（<1ug/ml）increased cell viability.LPS（1ug/ml and 0.1ug/ml）treatment increased the viability of CT26 cells at different doses of radiation（P<0.05）.ELISA showed that LPS treatment increased the levels of IL-6,G-CSF,TNF,IL-11 and PGE2 in CT26（P<0.05）.Moreover,treatment with IL-6,IL-11 and PGE2 increased the viability of CT26 cells after radiation（P<0.05）.4.LPS down-regulated miR-351 in CT26Treatment with LPS down-regulated the level of miR-351 in CT26 cells in dose-dependent manner（P<0.05）.5.Overexpression of miR-351 increased the radio-sensitivity of CT26 cellsUp-regulation of miR-351 decreased the viability of CT26 cells after irradiation（P<0.05）,while down-regulation of miR-351 increased the viability of irradiated cells（P<0.05）.Furthermore,miR-351 reversed the radio-protective effects of LPS in vitro.6.Up-regulation of miR-351 decreased the levels of IL-6,IL-11 and PGE2Overexpression of miR-351 decreased the levels of IL-6,IL-11 and PGE2 in CT26cells（P<0.05）.Moreover,up-regulation of miR-351 reversed the up-regulating effects of LPS on IL-6,IL-11 and PGE2.miR-351 decreased the radio-protective effects of LPS in vitro.However,IL-6,IL-11 and PGE2 increased the viability of irradiated cells after treatment of LPS and miR-351.7.miR-351 down-regulated IL-11 in 3’UTR dependent mannerLuciferase reporter assays showed that miR-351 treatment decreased the activity of3’UTR of IL-11（P<0.05）.However,mutation of 3’UTR of IL-11 reversed the effect of miR-351.DiscussionIn this study,miRNA micro array was used to identify the miRNAs related to TLR4signal pathway.Our data showed that miR-351 increased most evidently in TLR4-/-mice treated with radiation.Then we found that up-regulation of miR-351 increased radio-sensitivity in vitro.iTRAQ was performed to identify targeted genes of miR-351.Our results showed that miR-351 down-regulated Bcl2l13 through targeting 3’UTR.Bcl2l13 is a member of Bcl2 family and is related to cell apoptosis.Researches confirmed that Bcl2l13 inhibited apoptosis and is up-regulated in tumor tissues.Therefore,our results indicated that miR-351 increased radio-sensitivity by targeting Bcl2l13.Bone marrow is highly sensitive to irradiation.It is of great importance to investigate the hematopoiesis.Based on the study of miR-351-/-mouse,we found that deletion of miR-351 promoted hematopoiesis.Also,we investigated the HSCs in miR-351-/-mouse and found that miR-351-/-mouse exhibited a larger pool of HSCs and myeloid cells.Furthermore,our results showed that deletion of miR-351 promoted the entry of HSCs into the cell cycle.Overexpression of Bcl2l13 also increased the proportion of HSPCs in the S-G2-M phase of cell cycle in vitro.In conclusion,deletion of miR-351 up-regulated the level of Bcl2l13,which promoted the repopulation of HSCs resulting the increase of hematopoiesis.Next we investigated the regulation of miR-351 in the radio-sensitivity in CT26 cells.Our results showed that LPS alleviated the radiation-induced injuries in CT26 cells which was reversed with the treatment of miR-351.Furthermore,we found that miR-351decreased the levels of IL-6,IL-11 and PGE2 which were reported to be radio-protective experimentally.At last,luciferase reporter assays confirmed that miR-351 down-regulated IL-11 through target 3’UTR.Our results provided new targets for radio-sensitizing on tumor radiotherapy.In a word,this study identified miR-351 related to TLR4 signal pathway,which played an important role in the regulation of radio-sensitivity,indicating its promising potential in radio-protection and tumor radiotherapy. |
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