TRNA/pre-miRNA-derived Fragments Inhibit Colorectal Cancer Tumorigenesis

Background and Objevtive:Transfer RNA-derived RNA fragments?t RFs?belong to a family of short non-coding RNAs?nc RNAs?,which were first confirmed in the 1970 s in human urine,many studies have shown that those t RFs are present in most organisms.We found a special 17 bp small RNA fragment that was known as mi R-1280,but recent it has been shown that the RNA sequence,which has been called mi R-1280,is a non-coding fragment of t RNA^Leu.the fragment is the t RFs.Therefore,some researchers have suggested that mi R-1280 is removed from the small RNA library,but there is no relevant literature to support that mi R-1280 is not a precursor structure from mi R-1280,and that lack of potent experimental data on its origin.Interestingly,we found that at the 17 bp small RNA from the finally t RNA of leucine(t RNA^Leu)and mi R-1280 precursor?Pre-mi R-1280?,during the study of this small RNA fragment,we named this fragment as t RF/mi R-1280.In our earlier study,we found that the expression of t RF/mi R-1280 in human colorectal cancer?CRC?was significantly lower than that in normal tissues,meanwhile those cells with high expression of t RF/mi R-1280 in human colon cancer cell lines HCT116 and HCT15,the proliferation,migration capacity of the cells was significantly reduced.This may indicate that t RF/mi R-1280 has a modulatory effect on the development of colon cancer.In this study,the effect of t RF/mi R-1280,a t RNA / pre-mi RNA-derived fragment,on the development of tumor in colon cancer was studied for the first time,and the direct target gene was discussed.Methods:we identified a 17 bp fragment,which is the human mi R-1280 and t RNA^Leu shared mature sequence,in order to explore this fragment we designed three DNA probes that can separately detect the expression of this mature fragment,pre-mi R-1280 and t RNA^Leu.By Northern blot analysis we found this fragment derived from t RNA^Leu and pre-mi RNA,So we named this fragment t RF/mi R-1280.t RF/mi R-1280 expression in human CRC tissues was confirmed by RT-PCR.We constructed high/low expression of t RF/mi R-1280 human colorectal cancer cell line HCT116 and HCT15 by the Lentiviral vector,meanwhile t RF/mi R-1280 express was confirmed by RT-PCR.In vitro studies such as cell proliferation and colony formation and we dected the tumour volume,weight and HE staining in vivo studies to learn about the function of t RF/mi R-1280.Then in order to expiore t RF/mi R-1280 directly targets we used m RNA target-predicting algorithms?mi Randa,Target Scan,Target Rank?,we selected four genes?JAG2,SMO,PMM2,and AGK?.Then we cloned the 3'UTR of key target genes into a dual-luciferase UTR vector to found that t RF/mi R-1280 correlation with the 3'UTR of JAG2.JAG2 was used as a t RF/mi R-1280 downstream target gene by double luciferase reporter assay and Western blot analysis.Results:The successful construction of t RNA^Leu and mi R-1280 overexpressing CRC cell line and Control cell line,meanwhile,reduce the express of Dicer.Northern blot analyses find that the small RNA fragments in this study changed with the expression of t RNA^Leu and mi R-1280 in the cells.The results showed that the expression of Dicer enzyme affected the expression of this small RNA fragment,indicating that there was a relationship between the fragment and Pre-mi R-1280 and t RNA^Leu.So we named it t RF/mi R-1280.RT-PCR results showed that t RF/mi R-1280 expression in human colorectal cancer tissues was lower than that in normal tissues.In vitro experiments,we found that the proliferation ability,cell colony formation ability and cell migration ability of colorectal cancer cells with high expression of t RF/mi R-1280 were lower than that of the control group.In contrast,the ability of colorectal cancer cells to proliferate,cell colony formation and cell migration were significantly higher than those in control group at low t RF/mi R-1280.Subsequently,In vivo,we used colorectal cancer cells with high expression of t RF/mi R-1280 and its control group,low expression of t RF/mi R-1280 colon cancer cells and their control group subcutaneous tumor test in nude mice,The results showed that the experimental group injected with colorectal cancer cells with high expression of t RF/mi R-1280 had a significant decrease in tumor volume and weight compared with the control group.In contrast,in the experimental group injected with colorectal cancer cells with low expression of t RF/mi R-1280,Group tumor volume and weight were significantly increased.On the other hand,the results of mouse liver metastases and lung metastases showed that liver metastases and lung metastases were significantly associated with colorectal cancer cells with high expression of t RF/mi R-1280 in the spleen of nude mice compared to the control group reduce.After a series of in vitro and in vivo phenotypic experiments,we explored the possible target genes downstream of t RF/mi R-1280.The four candidate target genes of t RF/mi R-1280 were predicted by m RNA targeting prediction software?mi Randa,Target Scan,Target Rank?.JAG2,SMO,PMM2.JAG2 was found as t RF/mi R-1280 downstream direct target gene by double luciferase reporter assay and Western blot analysis.Conclusion:The above results indicate that the small RNA fragments,which have been called mi R-1280,are derived from both Pre-mi R-1280 and t RNA^Leu,hence the name t RF / mi R-1280,and t RF / mi R-1280 inhibits colonization both in vivo and in vitro Cancer cell growth and metastasis.At the same time we also found t RF / mi R-1280 possible downstream target JAG2,the above results suggest that t RF / mi R-1280 may be effective in inhibiting the development of colon cancer.