Establishment Of Screening Method For Specific Gene-Targeting MiRNAs With Identification And Function Study Of Non-Coding RNA Gene Z38

Part I Establishment of Screening Method for Specific Gene-Targeting miRNAsIn mammals and other eukaryotes,98%of the genome sequences transcript a large number of non-coding RNAs (ncRNAs). MicroRNAs (miRNAs),one kind of ncRNAs, are paid much attention after being found. Especially after the Nobel Prize award the work about miRNAs in2006,miRNAs research become one of the hottest research fields. Now the miRNAs research emphasis is transferred from finding new miRNAs to miRNAs function research. So far,we have known that miRNAs play very improtant roles in cell proliferation、apoptosis、 organogenesis、hematopoiesis、tumorgenesis and so on.The research of miRNAs function must resolve two problems:finding miRNAs downstream target genes and finding specific gene-targeting miRNAs. Generally, method of resolving the two problems includes two steps: prediction of target genes or miRNAs by softwares,then checking them by experiments. But prediction by softwares may not be the best choose, so recent years many methods based on experiments for finding target genes have been found.Thus we want to discover an experimental method to find specific gene-targeting miRNAs directly.So far, that miR-21can regulate programmed cell death protein 4(PDCD4) by binding3’UTR of PDCD4is well confirmed. Therefore,we take miR-21and PDCD4as a model to make our method.Firstly, we cloned3’UTR of PDCD4into pEGFP-C1plasmid with a stop codon between EGFP and3’UTR; Then EGFP-PDCD43’UTR plasmid and Ago plasmid co-transfected HeLa cells;After that we added biotin labeled antisense mix for EGFP mRNA into transfected HeLa cells lysate,and then we used streptavidin beads to pull-down EGFP-PDCD43’UTR mRNA and miRNAs by binding biotin moleculars. We used Trizol to collect the RNA and reverse transcripted the miRNAs by miRNAs RT kit. At last,we used primers for miR-21to PCR and abtained the band of miR-21. So,we successfully make a new method to isolate and identify specific gene-targeting miRNAs. We can use it to research miRNAs function further. Part II Identification and Function Study of Non-Coding RNA Gene Z38Except small RNAs like miRNAs and piRNAs, long non-coding RNAs about300bp-10kb also play key roles in gene transcription and translation,cell differentiation and ontogeny, heredity and epigenetics. Taken together, they form a highly complicated RNA network in cells.Z38gene is first isolated from mouse breast cancer tissue by suppression subtractive hybridization (SSH) method by our laboratory. Its homologous gene of human is about2.7kb and overexpresses in human breast cancer with the ability of promoting cell proliferation. Inhibiting Z38expression will reduce cell activities and result in apoptosis.For further research,we used in vitro transcription system、antibody synthesis、immunofluorescence technique and cell localization to identify that Z38gene is a long non-coding RNA. Then we detected the Z38gene expression level in human cancer tissues and cancer cell lines. After that we designed siRNAs for Z38gene and proved that they can interfere with Z38gene transcripts. At last we used Z38siRNAs to treat human breast cancer cell line MDA-MB-231formed tumors on BALB/c nude mice,and we found that Z38siRNAs can inhibit tumor proliferation compared with controls.Experiments above suggest that Z38gene has important function in breast cancer. It gives us a new idea that we can cure breast cancer by inhibiting Z38gene expression.