The Study Of OLFM4mRNA Interacting With Nucleolin Protein

Objective: Olfactomedin4(OLFM4) gene also known as GW112andhGC-1is highly expressed in many tumors, studies have shown thatOLFM4plays a very important role in the development of cancer, butresults are not consistent, indicating a cell or tissue type-dependent role ofOLFM4function. The aim of this study is to explore the interactionbetween OLFM4mRNA and nucleolin (C23or NCL) protein, and theregulation of nucleolin protein on the transcriptional level of OLFM4.Methods: Expression nucleolin plasmid GC146-Ncl with the negativecontrol plasmid GV-142and knock down nucleolin plasmidpGenesil-1.1-Si with the negative control plasmid pGenesil-1.1-HK wereconstructed, and they were transfected into SGC-7901and293-T cellsrespectively, the expression of C23protein were measured by Western blot.The above plasmids were transfected with OLFM4reporter plasmid, theactivity of nucleolin effecting on the OLFM4were analysised. Thenuclear chromatin of SGC-7901were collected, and incubated the nuclearchromatin with nucleolin protein, after protein-RNAco-immunoprecipitation experiment, extracted the RNA, The type of mRNA which combined with nucleolin were detected with RT-PCT.Results: Expression plasmid GC146-Ncl with the negative controlplasmid GV-142and knock down plasmid pGenesil-1.1-Si with thenegative control plasmid pGenesil-1.1-HK were transfected into cellsrespectively, the fluorescence rate of48h was reached60%. Compared tothe negative control group, the levels of nucleolin protein were upregulatedin nucleolin overexpression group, and were reduced in the nucleolin knockdown group by western blot. Dual-luciferase reporter assay showed that therelative luciferase activity of overexpressing plasmid GC146-Ncl wasabout1.15times as much as the control plasmid（P<0.05），and the relativeluciferase activity of the knock down plasmid pGenesil-1.1-Ncl was about0.8times as much as the control plasmid（P<0.05）in SGC-7901cells; therelative luciferase activity of expression plasmid GC146-Ncl is about1.7times compared with the control plasmid(P<0.05), and the relativeluciferase activity of knock down of plasmid pGenesil-1.1-Ncl is about0.5times compared with the control plasmid(P<0.05) in293-T cells. Thesuitable conditions of ultrasonic fragmentation were determined: that theoutput power of40%,30pules, every pule continued to1s, with a30-second rest. After protein-RNA immunoprecipitation, the RT-PCRresults showed,490bp fragment of the OLFM4gene and153bp fragmentof Bcl-2gene were amplified.Conclusion: OLFM4directly interacted with the nucleolin protein, and nucleolin stabilizes the OLFM4mRNA by increasing OLFM4transcription and extending OLFM4mRNA half-life.