Structural And Functional Studies Of C1orf109Gene Encoding The Substrate Of Protein Kinase CK2

Cancer is a leading cause of mortality worldwide. Invasion and metastasis is themain biological characteristics of cancer cells, and the major cause of death inpatients with cancer. The investigation concerning tumor cell invasion andmetastasis has become the focus of intense researches. However, the molecularmechanism of the progression of cancer cell invasion and metastasis has not beenfully elucidated.Previously, in order to identify genes that may be responsible for cancerinvasion and metastasis, we obtained an unknown cDNA fragment, which wasnamed as OPB7-1, expressed differently in two human lung cancer cell lines withdifferent metastasis potential using differential display RT-PCR. Then we mapped itto human chromosome1p34.3according to bioinformatic retrieves. Bioinformaticmethods, RACE （Rapid Amplification of cDNA Ends） and sequencing wereperformed to obtain the3′and5′ends of the gene from normal human lung tissuesubsequently. BLASTN results revealed that this cDNA sequence was homologouswith Homo Sapiens Chromosome1ORF109（c1orf109, GenBank accession number:NM₀17850.1）. However, no functional study on c1orf109has been reported yet.In the present study, to further investigate the biological function of c1orf109inthe cell, we analyzed the regulation mode of the gene, the expression pattern andsubcellular localization of the predicted protein in the cell, and its role involving incell proliferation and cell cycle modulation. Moreover, we also performed aninteractive molecules screening.Dual-luciferase reporter assay, protein-DNA cross linking and EMSA wereemployed to analysis the basal transcriptional requirements of the predictedpromoter regions. We found two cis-acting elements within the crucial region ofc1orf109promoter, one TATA box and one CAAT box, are required for maximaltranscription of the c1orf109gene. The5’ flanking region of the c1orf109gene couldbind specific transcription factors and Sp1may be one of them.Bioinformatic analysis indicated that C1ORF109was a solvable protein. Wealso found that C1ORF109was a phosphoprotein in vivo, and could bephosphorylized by the protein kinase CK2in vitro. The protein C1ORF109was mainly located in the nucleus and cytoplasm using immunofluorescence and cellularfractionation. The phosphorylation of the protein C1ORF109by protein kinase CK2could ont influent its subcellular localization.Moreover, employing western blot analysis and quanstitive real-time PCR, theendogenetic C1ORF109protein was identified in cells and upregulated expression ofc1orf109was detected in multiple cancer tissues and cancer cell lines. Exogenousexpression of C1ORF109in breast cancer Hs578T cells induced increased colonynumber and the rate of cell proliferation. Concomitant rise in levels of PCNA（proliferating cell nuclear antigen） and cyclinD1expression were observed.Overexpression of c1orf109promoted the invasiveness of Hs578T cells. Meanwhile,depletion of c1orf109by siRNA in breast cancer MDA-MB-231cells confirmed therole of c1orf109in proliferation and cell cycle. Furthermore, depletion of c1orf109expression significantly decreased cancer cell invasiveness. Taken together, ourfindings suggest that C1ORF109may be the down stream target of protein kinaseCK2and involved in the regulation of cancer cell proliferation and invasion.A GST-pull down assay was performed to screen potential interactive proteinsof C1ORF109. In addition, the interaction between C1ORF109with MARVELD1was identified by coimmunoprecipitation and was affected by the phosphorylationststus of C1ORF109.In conclusion, we identified the novel gene, c1orf109, encoding a potentialprotein kinase CK2substrate, and involved in the modulation of cancer cellproliferation and invasion. More work will be required to identify the molecularmechanisms by which C1ORF109affects the cancer cell proliferation and invasion.