| Background and objectiveMethylmalonic acidemia is an organic acidemia with autosomal recessive heredity. MMA can be classified into two types due to the defect of the methylmalonyl-CoA mutase (MCM) and the cofactor,5’-deoxyadenoxylcobalamin. According to the errors of cobalamin metabolism, conversion of vitamin B12 is divided into the cofactor forms methyl-Cbl (MeCb1) and adenosyl-Cbl (AdoCbl). Cb1C, Cb1D and Cb1F which belong to MeCbl type combined MMA and homocystinemia, whose coding genes have been identified as MMACHC, MMADHC and LMBRD1.In China patients cb1C type is claimed to be the most common disorder of MMA.MicroRNAs regulate many biological processes. As a important munber of microRNA, Let-7 has all important effect on variety of diseases, such as tumor,inflammation and so on. Let-7 is possible biomarker of MMA. The study focused on the a mumber of let-7 family, hsa-let-7g,for its role in the regulation of late onset MMA combined with homocystinemia. We also investigate the biological functions of hsa-let-7g, in order to obtain the necessary data for use in the evaluation of clinical diagnosis, treatment and prognosis of MMA. The study includes three parts as follows:Part 1 Expression of hsa-let-7g in MMAObjectiveTo find expression of hsa-let-7g of plasma circulation in MMA patients of late onset type.Materials and methodsTo select 21 patients of vitamin B12 valid of late onset type who were diagnosed in the First or Third Affiliated Hospital of Zhengzhou University from August,2011 to January,2016 as MMA group; All of them were tested methylmalonic acid analysis value of blood and urine by gas-chromatography mass spectrometry. To select 19 healthy patient relatives who haven’t any diseases as control group. We filed plasma and detected the expression level of hsa-let-7g by real-time quantitative PCR (Q-PCR) method.Results1.The results of GC/MS presents the urine methylmalonic acid zoom ratio is 2460.172±1380.529 of MMA group before treatment, while the urine methylmalonic acid zoom ratio decreased variantly is 579.572±440.642of all the patients,both groups are different significantly (P<0.01).The urine methylmalonic acid zoom ratio of control group is 7.784±2.912 (1.436-19.578), less than 100,compared with MMA group before and after treatment,the difference is remarkably (P<0.01).2.The results of Real-time PCR shows the mRNA lever of Hsa-let-7g in MMA group before treatment was different significantly higher than control group (P<0.01).Expression of hsa-let-7g of MMA group before and after treatment are different strongly (P<0.01)ConclusionHsa-let-7g in MMA patients with high Hcy was significantly higher than that in normal people.Compared with before treatment, expression of hsa-let-7g reductions in MMA group after treatment.Part 2 Prediction of hsa-let-7g targeting gene and verifying their relationshipObjectiveTo Predict the targeting gene of hsa-let-7g in MMA and verify the relationship between them.Methods1.We first identify the promoter region of hsa-let-7g in NCBI and Ensemble Genome Brower database. Then we predict the target gene of hsa-let-7g in MMA by bioinformatics.2.Based on our prediction, we cloned MMACHC-3’-UTR sequence which was predicted as target site of hsa-let-7g and mutMMACHC-3’-UTR onto psiCHECK2 plasmid. By co transfecting the hsa-let-7g mimics or mimics negative control into H293T cell line, the corresponding luciferase activity was measured.Results1.MMACHC in MMA is a target gene of hsa-let-7g.2. PsiCHECK reporter plasmids containing MMACHC-3’-UTR complementary site were constructed. Compared with blank control group, the luciferase activity changed significantly in mir group (P<0.05). Compared with NC control group, the luciferase activity changed strongly in mir group (P<0.05). The luciferase activity changed by hsa-let-7g combining with MMACHC 3’UTR.Compared with blank control group, the luciferase activity changed different significantly in hsa-let-7g inhibitor group (P<0.01).Compared with NC inhibitor group, the luciferase activity changed strongly in hsa-let-7g inhibitor group (P<0.05).with3. PsiCHECK reporter plasmids containing mutMMACHC-3’-UTR complementary site were constructed. There was no marked difference among mir group, blank control group orNC group(P>0.05).ConclusionMMACHC in methylmalonic academia clbC is a target gene of hsa-let-7gPart 3 Effects of hsa-let-7g on the expression of MMACHCObjectiveTo investigate hsa-let-7g effecting the expression of MMACHC based on the results of first two partsMethodsWe chose H293T cells to explore hsa-let-7g and hsa-let-7g inhibitor for further study..In this study Q- PCR were performed to reveal change of MMACHC mRNA and hsa-let-7g expression profiles of H293T samples. Expression levels of MMACHC protein was detected by Western blot.Results1.Compared with mimics negative control group, the expression level of MMACHCmRNA in hsa-let-7g mimics group was lower (p<0.05); While in hsa-let-7g inhibitor group is higher (p<0.05).2.Hsa-let-7g levels was found to be increased 48 hours post-transfection of hsa-let-7g mimics as compared with the mimics negative control group (p<0.05).3. Western Blotting revealed a significant down-regulation of protein expression levels of MMACHC in H293T (p<0.05).ConclusionHsa-let-7g downregulate the expression of MMACHC.
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