Studies On The Role And Related Mechanisms Of MiRN-422a In Osteosarcoma

With the development of modern medicine, people know more about the tumor，andrealize that the tumor is various environmental and genetic factors cause DNA damage inthe form of synergy or sequential in the body, activate the proto-oncogene and mutations intumor suppressor genes, and apoptosis regulation genes change, etc., cause abnormalexpression level, makes the target cell transformation and cloned abnormally growth，eventually form malignant tumor. Local invasion and distant metastasis are the two mostimportant features of malignant tumor, which are also the main causes of the high fatalityrate of malignancy, but the treatment on the two characteristics of tumor hasn’t been foundyet.During the treatment of the tumor, accounting for about99%of the non-codingsequences of the human genomes have caused the attention of the scientists, of which themost striking one is the finding of micrornas (miRNA). It combined with target genemRNA3’UTR to regulate gene expression, play an important role in the development oftumor. Through screening high-throughput micrornas chip we found that miR-133a,miR-143and miR-422a significantly decrease in the osteosarcoma. There are manyresearches are conducted on miR-133a and miR-143, while people’ understanding ofmiR-422a is not that much, whose mechanism in the osteosarcoma is still unknown yet.Therefore in this paper, we have studied MiR-422a’ biological function and its mechanismin the osteosarcoma. We found that miR-422a significantly reduced in osteosarcoma celllines and osteosarcoma clinical samples. Functional studies show that miR-422a has theinhibitory effect of antiapoptotic and the function of blocking cells in the period of G1/G0,then inhibiting tumor growth. Through further research, bioinformatics prediction andexperiment confirmed that applied to the target genes of miR-422a, the result shows thatmiR–422a is a inhibitor in the growth of tumor by targeting and inhibit the expression ofBcl2l2and Kras. In conclusion, our data explains the role of miR-422a in the pathogenesisof osteosarcoma, and implicates its potential in cancer therapy.Part I: because of the methylation of histone H3K9me3, H3K27me3and thedemethylation of H3K4me3, miR-422a shows a lower expression in osteosarcoma.First, through the qRT-PCR experiments we confirmed that miR-422a reduced inosteosarcoma cell lines and osteosarcoma clinical samples, and studied the reducingmechanism of miR-422a. It was found that the expression of miR-422a in osteosarcoma cell lines MG63and U2OS expressed lower. Analysis of miR-422a expression in theclinical sample of osteosarcoma patients shows that miR-422a has also expressed lower.Through chromatin immune co-precipitation technique (ChIP) combined with qRT-PCR,we study the molecular mechanism of miR-422a expression change, and find in relatedcell lines of miR-422a upstream promoter regions inhibiting histone modificationH3K9me3, H3K27me3and H4K20me1and activation modification of histone H3K4me3,H3K36me3and H3K79me2methylation. Results showed that inhibiting histonemodifications in osteosarcoma cell lines H3K9me3and H3K27me3caused significantlyincreasing, while activation of histone modification H3K4me3significantly reducing. Wehave verified this point with osteosarcoma tissue clinical samples validation, and theconclusion is in keeping with the result from osteosarcoma cell lines.Part II: miR-422a inhibits the growth of tumor growth by inhibitingantiapoptotic effect and blocking cells in the G1/G0In order to explore the physiological function of miR-422a in the osteosarcoma,we analyzed the influence that either higher or lower expression of miR-422a effect onosteosarcoma cell line proliferation. It is found that higher expression of miR-422a canobviously inhibit cell proliferation, while transfection of cell line in miR-422a inhibitorlead to promote cell proliferation. These results suggest that miR-422a is an importanttumor suppressor. In addition, we also confirmed that the growth of tumor could be inhibitby subcutaneously injection into nude mice tumor with miR-422a inhibitor. After that, westudied the molecular mechanism of miR-422a with lower expression promoting growth ofosteosarcoma cells, which maybe come from promoting tumor cell apoptosis, or inhibitingcell proliferation. We detect the number of osteosarcoma cells apoptosis and the cell cycleby transfecting miR-422a mimics and its control RNA by the Annexin V/PI double dyeexperiment. The results showes that miR-422a itself does not induce the apoptosis ofosteosarcoma cells, but it can inhibit apoptosis related gene expression and promoteapoptosis of osteosarcoma cells in the bad environment; on the other hand, a high miR-422a expression can inhibit the osteosarcoma proliferation by blocking osteosarcoma cellsin G0/G1phase. Part III: miR-422a target Bcl2l2and Kras leads to inhibit the growth ofosteosarcomaIn this part, we discussed which section the miR-422a target molecules play the rolein could inhibit the growth of osteosarcoma. Through miRecords mirecords.biolead.org/(http://) online software, we found that miR-422a owns conservative gummings in loci3-9and1557-1564of Bcl2l23’UTR region, loci286-309of Kras3’ UTR region and loci1904-1926of Nras3’UTR region. So we use double fluorescent to report the activity ofkits luciferase, and the results shows that miR-422a can play a role of negative regulationby effecting on Bcl2l23’UTR (loci1557-1557) and Kras3’ UTR (loci286-309). Whileloci3-9of Bcl2l23’UTR and loci1904-1926of Nras3’ UTR appear false positivereaction, which are not potential action sites of miR-422a. In addition, we detected theexpression level of mRNA and protein of Bcl2l2and Kras in cells after transfectingmiR-422a quasi content. According to the change of expression level, we preliminaryconfirmed that miR-422a targeted Bcl2l2and Kras could inhibit tumor growth. In order tofurther test that, we synthesized interference RNA of two genes, Bcl2l2and Kras, to rescuethe target genes by experiment. Results proved that miR-422a expressed down-regulatedduring osteosarcoma expression, and inhibit the growth of osteosarcoma by targetingBcl2l2and Kras.