RNAi Screening Identifies KAT8as A Key Molecule For Lung Cancer Cell Survival

ObjectiveHistone acetylation, one of the most extensively characterized epigeneticmodifications, is controlled by histone acetyltransferases (HATs) and histonedeacetylases (HDACs). HATs catalyzetransfer of acetyl groups to NH2-terminallysine residues in histones, which results in more open conformation of nucleosomesand increased accessibility of regulatory proteins to DNA, whereas HDAC activitycauses chromatin condensation and transcriptional repression. HDACs have beenextensively studied and several HDAC inhibitors are currently in clinical trials aspotential anticancer treatment. Recently, growing evidences suggest that in addition toHDAC inhibitors, HAT inhibitors may also have therapeutic potential. It has beenfound that abnormal activities of HATs are linked to a number of human diseases,especially cancer. HATs are involved in many key cancerrelated processes, includingtranscription regulation of a number of cancerrelated genes, DNA damage responseand repair, as well as DNA replication. Moreover, HATs gene expression profiles havebeen reported to be associated with pathological and clinical outcomes in breastcancer. HATs are therefore promising cancer therapeutic targets. Nonetheless, cellularfunctions and molecular mechanisms of HATs in cancer remain largely unclear.although alteration of HAT activity has been found to be common in cancer.Elucidation of function of different HATs in different cancer types will be helpful tobetter understand the consequences of histone and general protein acetylation and potentially provide novel therapeutic approaches for the treatment of cancer, RNAinterference (RNAi) genetic screens have been successfully used to identify genesinvolved in a phenotype. To systematically address the function of HATs in lungcancer cell growth and viability, we carried out an RNAi screen for genes involved inthe regulation of A549lung cancer cell viability. We performed the screen using alentiviral short-hairpin RNA (shRNA) library targeting the entire human HATs.Evaluation of histone acetylation enzyme in the role of lung cancer cell growth andsurvival, to explore the possible mechanism and treatment strategy of thedevelopment of lung cancer.Methods:According to known acetyltransferase mRNA sequence, design shRNA primer,with lentiviral as the carrier, to build one acetyltransferase shRNA lentiviral viruslibrary, RNAi in A549lung cancer cell line selected, determined the positive gene,observe the influence of cell survival condition and to explore its mechanism aftersilencing positive gene in lung cancer line A549cell:(1) According to person acetyltransferase mRNA sequence, through software,everyone acetyltransferase design8pairs of primers with pUCTP as the carrier, eachof acetyltransferases builds eight shRNA lentiviral recombinant plasmid, transformatsfeeling state of GeneHogs bacteria, to identify the positive colony by PCR andenzyme digestion.(2) Each of acetyltransferases has eight shRNA lentiviral recombinant plasmid,every4shRNA lentiviral recombinant plasmids mixed packing with293T cells,eachof acetyltransferases get two lentiviral thick liquids.(3) Acetyltransferases shRNA lentiviral library infected lung cancer cell lineA549, after72hours, with fluorescent microscope to observant the efficiency ofinfection, after120hours, to detect the efficiency of infection by with MTS method.(4) People acetyltransferase shRNA lentiviral library infected lung cancer cellline, to observed the efficiency by MTS method and to chosen the significantlyaffection on proliferation.(5) Four shRNA lentiviral recombinant plasmids of target genes selected, respectively packaged with293T cells, infected with A549cells, after120h, with theMTS method to detect the infection.(6) The positive shRNA lentiviral selected infected A549cells, after96hours,to extract the total RNA and protein respectively, with qPCR and Western blotto test silence efficiency of target genes.(7) Target genes shRNA lentiviral infected A549, after96hours, to detect thecell cycle phase (G1, G2, S and M) with flow cytometry.(8) Target genes shRNA lentiviral infected A549, after96hours,to extract thetotal protein, with Western blot to detect protein involved in cell proliferation cycle.Results:(1) Successfully to construct the acetyltransferase (HAT) RNAi lentiviral library:for16acetyltransferases, to build the acetyltransferase shRNA recombinant plasmids,the acetyltransferase shRNA recombinant plasmids transformated GeneHogs bacteria,to determine the positive clones by PCR and enzyme digestion, to package with293Tcells, get acetyltransferases shRNA lentiviral library.(2) Acetyltransferase shRNA lentiviral library infected A549cells, after96hours,to test the proliferation of A549cell by MTS.There are eight gene that significantlyinfluenced the proliferation of A549cells, respectively: CREBBP-2,2, EPC1EP300-2, ESCO1-1,2,1, HAT1-KAT2A, KAT8-2-2, KAT5-1.EP300and KAT5related totumor cells,which had been reported previously. By the analysis of literature,we foundthat KAT8was’t to study in lung cancer.(3) HAT shRNA lentiviral library infected A549cells, found that KAT8-2: KAT8-e, KAT8-f, KAT8-g, KAT8-h mixed packing lentiviral, had significantly effect on theproliferation of A549cells. KAT8-e, KAT8-f, KAT8-g, KAT8-h shRNA recombinantplasmids packaging by239T respectively, to infectA549cells, to test silenceefficiency by MTS. KAT8was effectively silenced by shKAT8-f shRNA lentiviral.(4) The shKAT8–f lentiviral infected A549cells, after96h, to extract total RNAand protein, to detect silence efficiency of KAT8b y qPCR and Western blot onmRNA and protein levels in A549cells(5) The shKAT8-f lentiviral infected A549cells,after96h,KAT8was silenced,find to arrest the cell cycle on G2/M phase in A549cells by flow cytometry.(6) The shKAT8-f lentiviral infected A549cells, after96h, to extracted the totalprotein, by Western blot test, finding the expression of cyclin D1, cyclin B1, p-AKT,p–ERK declined,but the expression of p53rised.Conclusion(1) HAT shRNA library was successful builded and selected which is one of theimportant gene to affect A549lung cancer cell survival(2) By using RNAi technology, KAT8was silenced,it is proved that KAT8performs the function of oncogenes in lung cancer in A549cell line.