Analysis Of Wnt1-targeting Mirnas And Its Interaction With Non-coding RNAs And Target Genes

Wnt signaling pathway is a signal pathway that is widely distributed in multicellular organisms and is highly evolutionarily conserved.The pathway is involved in cell proliferation,differentiation,apoptosis,cell polarity and cell migration and invasion.It plays an important role in the development and formation of multiple organs and in the pathophysiology of adult status.Wnt family members and mutations in Wnt related signaling pathways are closely related to many human diseases,such as fibrosis,metabolic syndrome,cancer,bone related diseases,and so on.Among them,Wnt 1 is one of the members of Wnt family,and its abnormal expression is related to human breast cancer and prostate cancer.Wnt 1 is essential for the development of the central nervous system and spinal cord,it will lead to abnormal frontotemporal dementia、 Alzheimer’s disease and other causes.Wnt 1 is also an autosomal recessive gene causing XV osteogenesis imperfecta（OI）.Over the past ten years with the understanding of the RNA scientific community increased rapidly,from simple information transfer molecules to the multiple functions of a class of gene expression regulation moleculars,many functional significance of non encoding RNA（ncRNA）has attracted more and more attention.There are a number of complementary binding sites of miRNA on circRNA and because of it lacks of poly（A）tail and 5 ’end let the deadenylation and degradation process unpredictable that play a strong role in the miRNA sponge,reduces the negative regulation of target gene miRNA.LncRNA and miRNA can be mutually regulated by competitive combination of corresponding miRNA response components（miRNA,response,element,MRE）,thus effectively controlling the subsequent transcriptional regulation of miRNA.The Wnt1-targeting miRNAs reported in the literature include let-7e、miR-21、 miR-34a、miR-122、miR-146a、miR-148a、miR-148 b and miR-152.Recent studies have shown that let-7e is involved in many physiological and pathological processes such as embryonic development、stem cell differentiation、inflammatory response and tumorigenesis.MiR-21 is highly expressed in HeLa cells and colon cancer cell lines（HCT-116）as well as in human glioblastoma multiforme.In vertebrates,miR-34 a mediates tumor cell proliferation and apoptosis through the accumulation and synergistic effects of a variety of target molecules.In addition,it exerts the function of tumor suppressor gene by regulating the invasion and metastasis of some tumor cells involved in hepatocyte growth factor receptor MET.MiR-122 is a liver specific high expression of miRNA,but it is decreased in liver cancer tissues.It plays an important role in regulating lipid metabolism and maintaining the normal function of liver.MiRNA-146 a plays an important role in the regulation of innate immune Toll like receptor signaling pathway.A variety of diseases,including autoimmune diseases、viral infections and cancers,may be related to changes in the expression of miRNA-146 a.MiR-148 a is down regulated in many cancers including gastric cancer、colorectal cancer、liver cancer and pancreatic cancer.MiR-148 b is down regulated in pancreatic cancer、gastric cancer and colorectal cancer.MiR-152 induces epigenetic silencing through DNA methylation in endometrial cancer and promotes endometrial cancer.It also targets DNA methyltransferase family 1（DNMT1）and is downregulated in liver cancer.Objective:8 Wnt1-targeting miRNAs have been reported in literature and 2 miRNAs highly predicted by bioinformatics both analysised by the interaction between circRNAs 、lncRNAs and target genes,and draw the relationship between the network.On the basis of this,a preliminary analysis of differential expression of circRNA and the target genes in patients’ bone tissue of osteogenesis imperfecta,which lays a foundation for the further discussion of the differences in the role of circRNA and osteogenesis imperfecta disease.Methods:Let-7e,miR-21,miR-34 a,miR-122,miR-148 a,miR-148b、miR-152、miR-130 and miR-130 b were predicted for their interacting circRNAs/lncRNAs and target genes by Starbase and miRWALK softwares,respectively,using the Cytoscape 3.2.1 to analyze the relationship between the 10 miRNAs and the predicted circRNAs/lncRNAs and target genes.The predicted target genes were analyzed by DAVID software,and RT-qPCR was used to analyze the expression of circRNA and target genes in the bone tissues of osteogenesis imperfect.Results: By bioinformatics analysis,the interaction between the Wnt1-targeting miRNAs and non encoding RNAs collects to six signaling pathway: Focal adhesion,Glioma,Melanogenesis,Notch,Prostate cancer and TGF-beta.We screened 10 circRNAs primers,including AKR1A1_hsa_circ₀00048 、BTBD7_hsa_circ₀00595 、 BTBD7_hsa_circ₀00847 、 C18orf1_hsa_circ₀01090 、PANK3_hsa_circ₀01308 、 PHIP_hsa_circ₀01319 、 EXOSC10_hsa_circ₀01352 、SLC25A3_hsa_circ₀01372 、 KIAA0922_hsa_circ₀01421and PSMD1_hsa_circ₀01736,and 13 target gene primers,including AKR1A1、BTBD7、C18orf1、EXOSC10、FZD5、KIAA0922、LEF、PANK3、HIP、PSMD1、SLC25A3、TCF and WNT1 in the experiment of primer optimization by RT-qPCR.Further studies show that in the bone tissues of osteogenesis imperfect patients,the expression of AKR1A1_hsa_circ₀00048、BTBD7_hsa_circ₀00847、FZD5 and SLC25A3 are down-regulated.Conclusions: AKR1A1_hsa_circ₀00048 and SLC25A3 in Focal adhesion,BTBD7_hsa_circ₀00847 in TGF-beta and FZD5 in Wnt signaling pathway,which from the results of 10 circRNA and 13 target genes with value of higher predicted by bioinformatics analysis from Wnt1-targeting 10 miRNAs,have different expression in the patients ’ bone tissue between osteogenesis imperfecta and DDH.The specific mechanism of of AKR1A1_hsa_circ₀00048,BTBD7_hsa_circ₀00847,FZD5 and SLC25A3 on osteogenesis imperfect is still unclear and needs further investigation.