MiR-1273g-3p Was Purified By Targeting CNR1 To Regulate The Expression And Transport Of MiRNA-associated Protein Ku70 And Its Mutants In A549 Cells

MiRNA（microRNA）is a group of~22nt length non-coding small RNA molecules.Currently, miRNA have been found closely related to many pathological and physiologicalprocesses, such as growth and development, cell differentiation, apoptosis, tumor formationand viral infections. Currently, the processing and maturation of miRNAs has been clarified.First, the gene encoding miRNA is transcribed into a300~1000bp length nucleotides(primary miRNA,pri-miRNA) by RNA polymerase Ⅱ, then is cut by complexmicroprocessor which contains the Drosha and DGCR8, generating about60-70nt precursormiRNA (pre-miRNAs) with stem-loop structure. Exportin5recognizes the pre-miRNA andexport pre-miRNA into the cytoplasm depending on Ran-GTP mechanism. Then thepre-miRNA is cut into21~25nucleotides by Dicer (double-stranded RNA-specificendonuclease) with5’ phosphorylated and3’ end of2nt overhanged. The double-stranded RNAis not fully complementary but similar to the part paired of siRNA, The double strands RNAis a heterodimer constitutes of mature miRNA and miRNA*that complementary nucleotideschain. Finally, the dimer is unchained into single strand mature miRNA through theinteraction of helicase enzyme, and mature miRNA composing RISCs with some othermolecules binding to the3’ UTR of target mRNA by fully or partially complementary targetsequences.According to the sequence specificity of miRNAs，it is found that more than60%of themRNA could be targeted by miRNAs analyzed with bioinformatics. Moreover, miRNA has thefollowing three features:1. Time and space specificity，that is, different miRNAs express atspecific space and time. Different express profiling of miRNA is found in different cells andtissues, even in the different stages of the same cell.2. Multi-target effects. One miRNA maytarget to a variety of different mRNAs, and multiple miRNAs can target to same mRNA.3.Trimming. The effect of miRNA targeting to mRNA (degradation or translational repression)generally does not exceed50%. Lung cancer is the most common malignancy, one of the killers serious threat to humanlife, results in about one million people died in worldwide each year. Lung cancer is dividedinto small cell lung cancer (SLCC) and non-small cell lung cancer (NSCLC), NSCLCaccounts for approximately80~90%of lung cancer cases. Lung cancer is affected by manyfactors，performance of a complex multi-stage process. The pathogenesis of lung cancer is stillunclear. Currently, with the development of molecu-lar biology, the research about lung cancer is mainly focused on susceptibility genes intumor，cancer suppressor genes, apoptosis regulation and cell signaling. In recent years, Theemerging of miRNA provides a new platform and tool for people to better understand cancerat the molecular level.The endocannabinoid system is composed of the cannabinoid receptor type1(CNR1) andcannabinoid receptor type2(CNR2), their endogenous ligands (endocann-abinoids) and the enzymes that regulate endocannabinoid biosynthesis and degradeat-ion. CNR1is found in the central nervous system and the peripheral tissues, especially relatedto energy balance: adipose tissue, liver, skeletal muscle, etc. The growing literatures prove toendocannabinoid system plays an important role in the regulation of energy metabolism andcancer-related diseases, the main mechanism of action is affecting cell proliferation, cell cycle,cell migration, angiogenesis and promote tumor cells programmed death in cancer cells.The purpose of our study is to investigate the target gene of miR-1273g-3p and itsbiological function in A549cells.The main results are as follows:1. Detecting miR-1273g-3p expression in non-small cell lung cancer cells (H1299, H460and A549)We found miR-1273g-3p was highly expressed in H1299and H460cells by miRNA chipand was confirmed by experimental results also showed that the expression level ofmiR-1273g-3p was higher than U6in H1299, H460and A549cells, indicating that theunnormal high expression pattern of miR-1273g-3p.2. Identification and validation of miR-1273g-3p’s target gene Through bioinformatics prediction, it is found CNR1, MGA, ZFX, SYNCRIP andAPBB2were the potential targets of miR-1273g-3p. After transfection the miR-1273g-3p mimics into A549cells, we found that the mNRA level of CNR1was significantlydownregulated, but the mRNA levels of MGA, ZFX, SYNCRIP and APBB2had nosignificant changes, indicating that CNR1may be miR-1273g-3p target. With luciferaseexperiments, it was confirmed that miR-1273g-3p targeted to the two target sites of CNR13’UTR. Western blot assay further onfirmed that CNR1expression levels can be reduced bymiR-1273g-3p in A549cells.3. The biological function of miR-1273g-3p targeting to CNR1We transfected miR-1273g-3p mimics, miR-1273g-3p inhibitor and CNR1siRNA intoA549cells, and then cell proliferation was detected by MTS method. Results showed thatthere was no significant change of cell proliferation among transfection groups and in controlgroup. Cell migration assay showed that the A549cells transfected miR-1273g-3p mimics andCNR1siRNA could induce migration of A549cells, and anti-mir-1273g-3p could reverse thisphenomenon.In summary, we examined the miR-1273g-3p expression in non-small cell lung cancercells, and discovered and proved that miR-1273g-3p targeted CNR1and promoted cellmigration. Ku protein is a evolutionarily conserved DNA-binding protein. The homologous Kuproteins are found in different species, Ku protein is widely present in different tissues, themRNA is abundant in the thymus and lymphocytes, Ku mainly presents in the nucleus, is alsoreported presence in the cell membrane and intracellular.Human protein Ku is a heterodimer structure formed two polypeptide chains bynon-covalent bond tightly binding, called Ku80(XRCC5) and Ku70(XRCC6), respectively.Ku80gene is located on2q33-34, encoding732amino acids, molecular weight is about83kDa.Ku70gene is located on human chromosome22q13, encoding609amino acids (AA), therelative molecular mass is69kDa. Ku80interacts with Ku70by a certain area. The20kDa ofKu70C-terminal and32kDa of Ku80C-terminal could form heterodimer. It is reported thatKu80449-477AA is an important region to the interaction between the two subunits. Wangreported the Ku80371-510AA is interaction site with Ku70, the179-732AA of Ku80Cterminal is required to DNA binding, the536-609AA of Ku70is the DNA-binding coredomain. Ku protein stability may depend on the interdependence of the two subunits.Ku70/Ku80subunits formed the heterodimer structure in close connection with theDNA dependent protein kinase catalytic subunit (DNA-PKcs), formed DNA-dep-endent protein kinase (DNA-PK), plays an important role in the non-homologous end binding(NHEJ) repair pathway of the double-stranded DNA breaks (DSBs). In addition, Ku proteinalso has an important role in maintain telomere structure. Ku depl-eted cause mouse fibroblasts chromosomal aberrations and occur of lymphoma. Ku activitymay be associated with tumor development. Ku DNA terminal binding protein (DEB) activityassociated with tumor resistance. Thus, Ku as a potential target for cancer therapy wasconcerned by researcher.We focused on the DNA binding property and DNA helicase activity of Ku70, Whetherthese properties could be applied to the RNA sequences? It is reported that Ku protein caninteract with an internal ribosome entry site sequence (IRES). We found that IRES have a typical hairpin structure similar to microRNA primary transcripts (pri-miRNA) and theprecursor microRNA (pre-miRNA).The purpose of our study is to expression the Ku70protein and its mutants in E.colisystem. The main results were as follows:1. Comparing Ku70protein expression in H1299and H460cells.Total protein and RNA were extracted seperately from H1299and H460cells, and theKu70level was detected with WesternBlot and Results showed that the express level of Ku70was higher in H1299cells than that in H460cells.2. Construction of pcDNA6-Ku70, pcDNA6-Ku70△α/β, pcDNA6-Ku70△DNA andexpression in eukaryotic cells.a. Obtaining the DNA sequence. synthesis primers of Ku70, Ku70deletion α/β domain andKu70deletion of DNA domain.b. Obtaining recombinant strains and identification. The vectors were digested by restrictionenzymes, then ligated by T4ligase, and then transformed into DH5α competent cells, PCRidentification and sending samples sequencing to obtain positive clones.c. Detecting the expression of recombinant plasmids. The eukaryotic expression plasmids ofpcDNA6-Ku70, pcDNA6-Ku70△α/β, pcDNA6-Ku70△DNA were transfected into HeLacells individually and identified by using Myc-tagged antibody to test the expression of theproteins.3. Construction of pET28a-Ku70, pET28a-Ku70△α/β and pET28a-Ku70△DNA vectors andexpression in E.coli.a. Obtaining the target sequences. Synthezing primers and hook Ku70, Ku70deletion α/βdomain, Ku70deletion of DNA domain sequence.b. Obtaining recombinant strains. The vectors were digested by the same restriction enzyme,and then connected by T4ligase, and then transfected into DH5α competent cells, PCRidentification and sequencing of samples to obtain positive clones.c. Obtaining a recombinant plasmids and detection of the expression of recombinant proteins.The pET28a-Ku70, pET28a-Ku70△α/β and pET28a-Ku70△DNA plasmids were transformed into BL21competent cells, using IPTG induce. SDS-PAGE results showed that the threerecombinant plasmids are able to express, and the expression level reached nearly40%.4. Purifing Ku70, Ku70△α/β and Ku70△DNA recombinant proteinsAfter two hours culture of bacteria, adding IPTG to the final concentration of1mM, thenthe expression of recombinant proteins were detected by SDS-PAGE. The proteins werepurifiedthrough1mL Ni columns then identified by western blot.