| ObjectivesTo explore the effect of Lnc78583,a kind of new long non-coding RNA(lncRNA)associated with inflammatory bowel disease(IBD),in the pathogenesis and development of IBD by high-throughput sequencing technology.To explore the effects of Lnc78583 on the onset and development of IBD through human umbilical cord mesenchymal stem cell derived exosome(huc MSC-Ex).To explore the clinical significance of huc MSC-Ex in IBD patients using intestinal mucosal epithelial tissue.Methods1.The detection of the expression of lncRNAs in IBD patients through high-throughput sequencing technologyTwo cases each of colon tissue from IBD patients and human normal colorectal mucosal epithelial cells(FHC)were collected to construct a lncRNA libraries,Illumina sequencing was performed and sresults analyze,followed by screening new lncRNAs related to IBD,which required high tissue specificity and significant expression differences.QRT-PCR was performed on FHC and the new lncRNAs-lnc78583 was screened by comparing the detection results and the high-throughput sequencing results.The targeted mRNA-HOXB13 was found by high-throughput sequencing and the contents of lnc78583 and HOXB13 in colon tissues of IBD patients were detected by QRT-PCR and IHC.2.The effects of lnc78583 in the cell inflammation model of FHCLipopolysaccharide(LPS)was used to stimulate FHC to construct an IBD inflammatory model,followed by the determination of the levels of lnc78583,HOXB13,and inflammatory factors by QRT-PCR.Plasmids containing lnc78583 was transfected into FHC and the levels of lnc78583,HOXB13,and inflammatory factors were determined by QRT-PCR.Cell proliferation was assessed by CCK8 assay,and the expression of p NF-κB and NF-κB was detected by western blot.3.The effects of lnc78583 on miR3202 in the cell inflammation model of FHCThe targeted miRNA-miR3202 related of lnc78583,HOXB13,was predicted on the miRDB database.FHC was treated with LPS and the content of miR3202 was measured by QRT-PCR.Plasmids containing lnc78583 was transfected into FHC and the levels of lnc78583 and miR3202 was detected by QRT-PCR.The miR3202 mimics and inhibitors were transfected into FHC,the levels of miR3202 were detected by QRT-PCR,and the expression of p NF-κB and NF-κB was detected by western blot.4.The effects of huc MSC-Ex on the lnc78583-HOXB13/miR3202 pathway in the cell inflammation model of FHCHuman umbilical cord mesenchymal stem cells were cultured,huc MSC-Ex were extracted,and verified by NTA,electron microscopy,and western blot.Plasmids containing lnc78583 were transfected into FHC,followed by treating the FHC with huc MSC-Ex and LPS.CCK8 assay was used to assess the proliferation of FHC cells,the levels of lnc78583,HOXB13,and miR3202 were measured by QRT-PCR,and the expression of p NF-κB and NF-κB was detected by western blot.Results1.The detection of the expression of lncRNAs in IBD patients through high-throughput sequencing technologyColon tissue from IBD patients and cell lines of FHC,were sequenced with high-throughput sequencing technology,identifying 397 genes with significant differential expression,including 72 lncRNAs and 325 mRNA.The 72 lncRNAs contained 31 known and 41 new lncRNAs named after MSTRG.QRT-PCR was performed on FHC cells and the results were compared with the above sequencing results,where 8 lncRNAs were found to be consistent,including five up-regulated MSTRGS and three down-regulated MSTRGS.Colon epithelial cells from 11 IBD patients were collected as the case group and FHC cells were used as the control group.Eight(8)lncRNAs were detected by QRT-PCR.The results showed that MSTRG 78583 was significantly reduced in colon tissues of IBD patients.The results of high-throughput sequencing showed that the targeted gene of MSTRG 78583 was HOXB13.QRT-PCR and IHC were used to assess the level of HOXB13 in colon tissues of IBD patients.The results showed that HOXB13 gene and protein levels were significantly decreased in IBD patients.Therefore,MSTRG 78583 were selected as the further target and renamed as lnc78583.2.The effects of lnc78583 in the cell inflammation model of FHCPlasmids containing lnc78583 was transfected into FHC,and FHC were treated with LPS of different concentrations.It was found that the level of lnc78583 decreased with increasing LPS concentration,thus 100ng/m L was selected as the key concentration of LPS treatment.FHC treated with 100ng/m L LPS was constructed as the experimental group with FHC(no LPS treatment)as the control group.QRT-PCR was used to detect the level of lnc78583,HOXB13,tumor necrosis factor(TNF-α),interleukin 6(IL-6),and IL-10 in the two groups.The results showed that lnc78583,HOXB13 and IL-10 were significantly decreased while TNF-α and IL-6 were highly expressed in the experimental group.Plasmid containing lnc78583 was transfected into FHC and divided into the following groups: FHC with lnc78583 plasmids and LPS treatment as the experimental group,FHC with pcDNA3.1-EGFP plasmidsbut without lnc78583 gene,LPS treated FHC as the control group,and FHC without plasmid transfection as the blank group.QRT-PCR was used to measure the contents of lnc78583,HOXB13,TNF-α,IL-6,and IL-10.The results showed that the lnc78583、HOXB13、and IL-10 were significantly increased in the experimental group.The results of CCK8 assay and the determination of Lactate dehydrogenase(LDH)showed that overexpression of lnc78583 could improve the cell proliferation and decrease the release of LDH.The results of western blot showed that overexpression of lnc78583 could increase the expression of HOXB13 and decrease p NF-κB.3.The effects of lnc78583 on miR3202 in the cell inflammation model of FHCThe targeted miRNA related of lnc78583 and HOXB13 was predicted on the miRDB database,miR3202 was found to contain binding sites to lnc78583 and HOXB13.FHC treated with 100ng/m L LPS as the experimental group and FHC with no LPS treatment as the control group were constructed,and QRT-PCR was used to measure the level of miR3202.The results showed that the level of miR3202 in FHC increased after LPS stimulation.Plasmid containing lnc78583 was transfected into FHC and the following groups set up: FHC with lnc78583 plasmids and LPS treatment as the experimental group,FHC with pcDNA3.1-EGFP plasmids without lnc78583 gene,LPS treated FHC as the control group,and FHC without plasmid transfection as the blank group.The levels of lnc78583 and miR3202 were determined by QRT-PCR.The results showed that the level of lnc78583 was significantly increased in the experimental group but miR3202 decreasedcompared to the control group.Mi R3202 mimics and inhibitors were transfected into FHC and the levels of HOXB13 and miR3202 determined by QRT-PCR.The results showed that miR3202 was overexpressed in mimics group while HOXB13 was lower than the other groups.The expression of p NF-κB and NF-κB was detected by western blot,where the results showed increased p NF-κB in mimics group.4.The effects of huc MSC-Ex on the lnc78583-HOXB13/miR3202 pathway in the cell inflammation model of FHCHuman umbilical cord mesenchymal stem cells were cultured,huc MSC-Ex were extracted,and verified by NTA,electron microscopy,and western blot.Plasmid containing lnc78583 was transfected into FHC and different groups set up: FHC treated with huc MSC-Ex + LPS as the experimental group,FHC treated with LPS as the control group,and FHC with no treatment as the blank group.CCK8 assay was used to detect the proliferative ability of the 3 groups of cells.The results showed that the proliferation of FHC in the LPS group was lower than that in the blank group,but higher in the huc MSC-Ex + LPS group compared to the LPS group.The LDH content of the huc MSC-Ex + LPS group was lower than the LPS group.Moreover,the levels of lnc78583、HOXB13、IL-10,and miR3202 were detected by QRT-PCR,where results showed that lnc78583,HOXB13,and IL-10 in the huc MSC-Ex + LPS group were higher than in the LPS group,while the level of miR3202 in huc MSC-Ex + LPS group was lower than that in the LPS group.The expression levels of HOXB13,p NF-κB,and NF-κB in the three groups were assessed by western blot.The results further confirmed that the content of HOXB13 in the huc MSC-Ex + LPS group was higher than that in the other two groups,while the expression of p NF-κB was decreased.ConclusionsIn this study,the new lncRNA-lnc78583 was successfully found in FHC.High-throughput sequencing results showed that its target gene is HOXB13.The levels of lnc78583 and HOXB13 in intestinal mucosal epithelial tissues of IBD patients were significantly reduced and the same results were found in FHC inflammation model with LPS treatment,suggesting that lnc78583 can inhibit FHC inflammation by targeting HOXB13.Due to the damage repair effect of huc MSC-Ex in many diseases including IBD,we also investigated whether huc MSC-Ex regulates lnc78583 during IBD remission.It was found that the levels of lnc78583 and HOXB13 were significantly increased in FHC with huc MSC-Ex + LPS treatment,suggesting that huc MSC-Ex alleviated LPS-induced FHC inflammation by regulating the lnc78583-HOXB13 pathway.We explored the binding ability of miRNA-mi3202 to lnc78583 and HOXB13,and found that the overexpression of lnc78583 significantly inhibited the expression of miR3202,suggesting that lnc78583 alleviates FHC cell inflammation by mediating the HOXB13/miR3202 axis.In conclusion,the level of lnc78583 in intestinal mucosal epithelial tissues of IBD patients is significantly increased and huc MSC-Ex can alleviate FHC cell inflammation through modulating the huc MSC-Ex-HOXB13/miR3202 pathway,which has potential clinical application value in the diagnosis and treatment of IBD. |
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