Research On Function Of LOC401296, A Radiation-induced Early Response Gene

LOC401296was firstly found as a radiation‐induced early response gene in our laboratory，via cDNA microarray analysis of human peripheral blood exposed to γ rays. By now, no researcher has reported the function of LOC401296in any paper. In this study, an available antibody to LOC401296was obtained and it was confirmed that LOC401296played a vital role in cell proliferation and cell cycle regulation.First，we finished some researches on LOC401296in terms of homology, expression profile and subcellular location. After that, expression vectors of LOC401296were constructed and LOC401296‐6×His was expressed in E.coli for production of an antibody to LOC401296, considering investigation the function of it.Some essential information from NCBI revealed that LOC401296located in7p22.3, containing585bp and coding194AA. It was high homologous among primates: macaques81%, chimpanzees96%and bonobos97%, but no homology in mice. No conserved domains or nuclear localization sequences (NLS) were found. RT‐PCR demonstrated that LOC401296transcribed in several kinds of cells, including Hela, A549, MCF7, HEK293, U937, Molt4, K562, LO2and HepG2. Through laser confocal microscopy, GFP‐LOC401296was located in nucleus with increased intensity. And more important, it colocalized with PML‐Ⅳ. Subsequently, full‐length CDS of LOC401296was cloned into vector PET28B and vector pCMV‐Myc and expression vectors pET28B‐LOC401296and pCMV‐Myc‐LOC401296were constructed successfully. LOC401296‐6×His was expressed in E.coli induced by IPTG, then LOC401296‐6×His was purified and used to immune BALB/C mice for an available monoclonal antibody to LOC401296. Based on these studies, the function of LOC401296on proliferation, apoptosis, cell cycle progression was explored via gene silencing technology combined with flow cytometry, immunofluorescence and Western blot.According to our data, significant inhibition of cell proliferation was observed in Hela, A549, MCF7and HEK293, when LOC401296was down‐regulated. Besides, analysis of apoptosis proofed that LOC401296knockdown led to cells more susceptible to death: LOC401296knockdown group16.53%vs. control group9.26%in Hela cells; and LOC401296knockdown group7.06%vs. control group3.28%in HEK293cells. Furthermore, data from flow cytometry indicated that knockdown of LOC401296induced G2/M arrest in both Hela cells and HEK293cells; mitotic index increased significant in LOC401296knockdown group, in2or3days after cells transfected with siRNA‐LOC401296. More convincing evidences were that various types of aberrant spindles including displaced spindles, momople/multipole spindles, and spindles disappearanced, were observed in LOC401296knockdown group. Finally, to explain the mechanism of LOC401296knockdown on mitosis regulation, several critical molecules involved in G2/M regulation, cyclinB1, Cdk1, p‐Cdk1and Plk1were detected. The results of Western blot displayed a dramatical decline of Plk1. It gave a cue LOC401296maybe participate in the progression of mitosis through interaction with Plk1.Our research first studied the function of the undefined gene LOC401296, the results revealed that LOC401296was ubiquitously expressed in the cells indicated. It coded a protein located in nucleus, and it was a necessary protein for cells’ survival. Knockdown the expression of LOC401296induced G2/M arrest and spindle disorders and Plk1might be involved in this regulation.