Identification Of The Small RNA Of FHIT

The fragile histidine triad (FHIT) gene, encompassing the fragile site, FRA3B, at human chromosome 3p14.2. The FHIT gene spans about 2 Mb in the genome and consists of 10 small exons separated by large introns; and exons 5 to 9 encode the Fhit protein. FHIT mRNA is only 1.1 kb in length and encodes a 146 amino acid chain that forms a protein of 16.8 kDa. The FHIT gene plays an important role in maintaining genomic stability, regulating the cell cycles, inducing cell apoptosis, preventing and treating tumors and using as the biomarker. The deletion and methylation of FHIT gene and expression reduction of Fhit protein occurred in about 70% of human epithelial tumors, particularly in those tumors resulting from exposure to environmental carcinogens.Fhit is a tumor suppressor confirmed by cell biology, cancer molecular biology, transgenic and gene knockout. Fhit induces apoptosis and inhibits tumor cell proliferation through specific pathways. It is clear that Fhit inhibits cancer by Fhit-substrate complex, more and more research results show that Fhit has a good prospect in cancer prevention and treatment.Small RNA is non-coding RNA with the length of 19-25 nucleotides, which include small interfering RNA (siRNA) and microRNA (miRNA). RNA interfering means post-transcriptional gene silencing, which makes cellular homologous mRNA degradation by double-stranded RNA, and blocking or reducing homologous genes expression. microRNAs (miRNAs) are single-stranded RNAs (ssRNAs) of 19-25 nucleotides in length that are generated from endogenous hairpin-shaped transcripts. miRNAs function as guide molecules in post-transcriptional gene silencing by base pairing with target mRNAs, which leads to mRNA cleavage or translational repression.We previously reported that Fhit-/- cells showed more resistance to DNA damage inducers. To further study the function of FHIT gene in DNA damage response, we here identify the small RNA of FHIT. FHIT-siRNA vectors were constructed, and two FHIT-siRNAs which repress Fhit expression in 293T cells were obtained, then the expressions of RPA70 and CT45 were tested, we found that Fhit expression was repressed by siRNAs, the expressions of RPA70 and CT45 were decreased, which revealed that Fhit may have functional link with RPA70 or CT45.Here we predicted the candidate miRNAs targeting FHIT by TargetScan and used the dual luciferase reporter plasmid to search microRNAs targeting FHIT, we found that miR-143 directly targets FHIT and miR-143 can repress the translation of FHIT gene in 293T cells and A549 cells by western blotting. The results show that introduction of miR-143 expression in human cells resulted in significant G2 phase arrest following ionizing radiation (IR) by the flow cytometry, and overexpression of miR-143 protects cells from DNA damage induced killing in 293T and A549 cells. All these results proved that miR-143 improved cell tolerance to DNA damage by repressing the expression of Fhit.We get two FHIT-siRNAs and a microRNA targeting FHIT, which provide a basis on the further study of the function of FHIT in DNA damage response. The studies of the function of miR-143 also provide a new thinking on the prediction and treatment of cancer.