Study On The Function Of MiR-146a And The Mechanism In The Development Of Inflammatory Bowel Disease

Background:Inflammatory bowel disease（IBD）is a chronic,recurrent inflammatory disorder that includes Crohn’s disease and ulcerative colitis.Typically,the main clinical features of inflammatory bowel disease are diarrhea,abdominal pain,and,in the case of ulcerative colitis,perianal bleeding.These disorders are caused by immune disorders caused by a combination of genetic and environmental factors.Most IBD were diagnosed among adults with aged 30 to 40 and leading to patients’disability.There are about 6-8 million IBD patients worldwide,and IBD drugs have limited efficacy.The most clinically used drug for IBD is anti-tumor necrosis factor（TNF-α）antibody,but its efficacy rate is only about 50%,and the risk of systemic toxicity increases over time.This makes it necessary for scientists to discover a more effective and safer target and drug for IBD.miRNAs are a class of short,endogenous single-stranded noncoding RNAs（approximately 22 nts）that regulate the gene expression.They inhibit protein production by selectively binding complementary m RNAs.The miR-146 family consists of two member genes,miR-146a and miR-146b.Both miR-146a and miR-146b are regulated by the nuclear factor kappa-B（NF-κB）,a transcription factor that activates B cells,thereby regulating the inflammatory response.Both miR-146a and miR-146b are involved in the regulation of inflammation which is the most focused areas recently within miR-146 studies.Compared with traditional biopharmaceuticals and small molecule compounds,oligonucleotide drugs showed the characteristics of strong specificity,multiple drug targets,long half-life（most commonly maintained for several months）,and low immunogenicity.Objective:To elucidate the functional and molecular mechanism of miR-146a in the development of IBD,and to study the effect of similar oligonucleotide drugs PL18/PL19 on the effect of miR-146a on mouse models of IBD and the inflammatory factors,and to explore the mechanism of action of PL18/PL19 in slowing IBD,so as to provide a scientific basis for the development of new drugs for IBD in the role of miR-146a in the inflammatory response of the immune system.Methods:1.10 2-6 months female C57BL/6N mice and 11 2-6 months female miR-146a knockout mice.11 2-6 mice model and enteritis modeling for 7 days),normal control group（free drinking sterilized water for 7 days）.Disease progression at a single time point was observed by disease activity index,HE staining was used to observe the colonic injury of mice in each group.Real Time Quantitative PCR（RT-q PCR）and Western Blot（WB）were used to measure the expression of genes and proteins ofmiR-146arelated inflammatory.At the same time,RNA sequencing was performed to see the gene network of miR-146a regulating inflammation production.2.Mi R-146a-like oligonucleotide drugs were selected using a dual-luciferase reporter assay.Male and female miR-146a^-/-mice aged 2-4 months were randomly divided into three groups:untreated control group（n=13）,saline-treated DSS model group（n=13）and DSS model group（n=13）treated with small nucleic acid PL-18+PL-19 of miR-146a.Three groups of mice were fasted for 24 hours after measuring body weight,and then 2%DSS solution was added to the drinking water,the time point was recorded as the starting time of the experiment.Mice were weighed one day before and daily after DSS administration,and feces and hematochezia were scored.To investigate whether miR-146a-like oligonucleotide drugs could effectively alleviate the severity of enteritis in miR-146a^-/-mice;RT-q PCR and WB were used to determine the gene and protein expression related to inflammation in order to further investigate whether miR-146a treatment inhibited most inflammatory factors in IBD disease.3.7weeks wild-type C57BL/6 male mice were randomly divided into four groups:untreated control group（n=5）,DSS group（n=9）,saline-treated DSS group（n=8）,and DSS model group（n=9）（treated with miR-146a-like oligonucleotide drugs PL-18+miR-146a-like oligonucleotide drugs PL-19）.Four groups of mice were fasted for 24hours after measuring body weight,after which 2.5%DSS was added to the drinking water,which was recorded as the starting time of the experiment.Mice were weighed one day before and daily after DSS administration,and feces and hematochezia were scored.miR-146a-like oligonucleotide drugs PL-18（0.0065 ng/kg）and miR-146a-like oligonucleotide drugs PL-19（0.00325 ng/kg）were injected intraperitoneally once 24hours after DSS administration,and then every other day for a total of three doses.DSS concentrations decreased to 2%on day 2 and stopped giving DSS on day 6.Mice were scored on day 7,after which they were sacrificed by cervical dislocation,colons were dissected,and their length was measured to investigate whether miR-146a-like oligonucleotide drugs could further regulate inflammatory levels in wild-type mice.The expression of inflammatory factors at the gene and protein levels was determined by RT-q PCR and WB methods to further investigate whether miR-146a treatment could inhibit the expression of most inflammatory factors in wild-type mouse IBD disease.Results:1.miR-146a^-/-mice are more susceptible to inflammatory bowel disease.2.RNA sequencing revealed that miR-146a inhibited the number of DSS-induced IBD genes up to 90%,The most relevant up-regulated inflammatory cytokines were Mmp3,Mmp8,Mmp10,Il6,Il1α,Cxcl2,Cxcl3 and S100a9,respectively.The down-regulated inflammatory factors were Lcn2,Serpine2,Saa3,Ccl3 and Csf3.At the meanwhile,in the colon of DSS-induced wild-type mice,11 of the genes with a 2-fold up-regulation of expression were associated with pain promotion.These results indicated that,compared to wild mice,miR-146a^-/-mice had more significant pain manifestations after being induced into IBD by DSS.3.12 miR-146a-like oligonucleotide drugs were screened to significantly inhibit luciferase expression in the PGL4.17-Luc2-miR-146a-Response plasmid.4.miR-146a-like oligonucleotide drugs inhibited the expression of inflammatory factors,which were expressed in miR-146a^-/-mice in IBD.5.miR-146a-like oligonucleotide drugs could alleviate IBD in WT mice.Conclusions:miR-146a prevented the initiation and progression of IBD by regulating the expression of most genes in the IBD gene network,and miR-146a functionally similar oligonucleotides did significantly alleviate DSS-induced IBD in mice.Inflammatory factors play a certain therapeutic protective role in enteritis models,and these results provide experimental evidence and new theoretical basis for the further development of miR-146a oligonucleotide drugs as a potential preventive drug for the treatment of IBD and even in people at risk of immune-related inflammation.