Effect Of TRNA-derived Small RNA On The Occurrence Of Gastric Cancer And Its Diagnostic Value

Background and ObjectivetRNA-derived small RNAs(tsRNAs)belong to small non-coding RNA(nc RNA)family.Current researchers have shown that they are ubiquitous in most organisms.In past,tsRNAs were considered as the product of random degradation of tRNAs;however,recent studies have found that tsRNAs have many important biological functions,especially participating in the occurrence and development of different types of tumors.Therefore,it is believed that tsRNAs have a very broad prospect in the diagnosis and treatment of tumors.tsRNAs mainly include tRNA-derived RNA fragments(tRFs)and tRNA halves(tiRNAs).Most of tiRNAs are between 31 nt-40 nt in length.Based on whether 5’or 3’ sequence of the anticodon cleavage site is included,tiRNAs can be divided into two subclasses: 5’ tiRNAs and 3’ tiRNAs.5’ tiRNAs start from the 5’ end of mature tRNAs to the end in the anticodon loop,whereas 3’ tiRNAs start from the anticodon loop to 3’ end of mature tRNAs.The length of tRFs is almost between 14 nt-30 nt.They can be roughly divided into five categories.tRF-3s are derived from the cleavage in the 3’ end of the mature tRNAs.tRF-5s are produced from cleavage in the 5’ end of mature tRNAs and in the nucleus.RNase Z produces tRF-1s from the tail sequence of the precursor tRNA.In addition to the three main types of tRFs mentioned above,there are two types of relatively rare tRFs.Among them,tRF-2s is not classified because it is not suitable for a general classification scheme in terms of its source and/or length.Another type of i-tRFs come from the internal region of mature tRNA.World widely,gastric cancer is a malignant tumor with high morbidity and mortality.However,in China,the average diagnosis rate of early gastric cancer is less than 10%;and the vast majority of patients with gastric cancer have reached middle-late stages when they seek treatment.If patients with early gastric cancer can be treated immediately,their 5-year survival rate can rise to more than 90%.Nonetheless,the sensitivity and specificity of traditional tumor markers such as Carcinoembryonic antigen(CEA)and Carbohydrate antigen 19-9(CA19-9)are not high enough.Therefore,in the diagnosis of gastric cancer,especially in early diagnosis,it plays a very important role that if we can find an ideal gastric cancer biomarker.In this study,we explored the value of tsRNAs in the diagnosis of gastric cancer after screening and identifying related tsRNAs.Furthermore,we also studied the role of gastric cancer-related tsRNAs in the occurrence of gastric cancer by up-regulating and down-regulating them.Methods1.To screen the target gastric cancer-related tsRNAs,we firstly sequenced three gastric cancer patients’ plasma samples and three normal human plasma samples.Then the tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP were identified as the research objects in this study.2.The matching primers of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP were designed by consulting the database;and the accuracy of the primers was verified by RNA electrophoresis and TA cloning sequencing.3.Collecting 89 pairs of plasma samples from one day before operation and seven days after operation of patients with gastric cancer,and 98 healthy human plasma samples.4.After modified and spliced the RNAs extracted from plasma samples,we used the quantitative reverse transcription-polymerase chain reaction(q RT-PCR)technology to detect the expression levels of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP.Combined with clinical pathological data of gastric cancer patients,area under the receiver operating characteristic curve(AUC)was used to analyze its value of clinical diagnosis.5.Designed mimics and inhibitors of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP,and used the transfection reagent Lipofectamine 2000 to transform into cells,respectively.To verify whether the mimics and inhibitors were designed successfully,we used q RT-PCR to detect the expression of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP in gastric cancer cells.6.After cells were transfected with mimics of tRF-19-3L7L73JD or mimics and inhibitors of tRF-33-P4R8YP9LON4VDP,the cell migration ability was detected by cell scratch experiments and Trans-well experiments.Cell counting reagent-8(Counting Kit-8,CCK-8)and clone formation experiments were used to detect the cell proliferation ability.Flow cytometry was used to detect apoptosis and cell cycle changes.Results1.The results showed that a single peak was formed after performing q RT-PCR on the reverse transcribed c DNA with the designed amplification primers.The results of electrophoresis of the PCR products were in line with expectations.The length and number of bases of the PCR products after TA cloning and sequencing were in line with expectations.2.The results in q RT-PCR experiments showed that compared with healthy human plasma samples,tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP were low-expressed in the plasma of gastric cancer patients.Apart from this,compared with normal gastric epithelial cells,tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP were also low-expressed in four gastric cancer cell lines.As well,the AUC of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP were 0.6230 and0.5877,respectively.Combining with the analysis of clinicopathological data,it was found that the expression level of tRF-19-3L7L73JD was related to the tumor size(P = 0.0051).There was also a certain relationship between tRF-33-P4R8YP9LON4VDP and tumor marker CA19-9(P = 0.0124).Therefore,we thought that both tsRNAs have certain diagnostic values.3.The results of cell scratch experiments and Trans-well experiments showed that after upregulation of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP,the cell migration ability of experimental groups decreased compared with Negative Control(NC)groups.After tRF-33-P4R8YP9LON4VDP was down-regulated,the cell migration ability of experimental groups was higher than that of NC groups.The results of CCK-8 experiment and clone formation experiment showed that after up-regulation of tRF-19-3L7L73JD,up-regulation and down-regulation of tRF-33-P4R8YP9LON4VDP,compared with the NC groups,the cell growth and proliferation ability of experimental groups were decreased and increased correspondingly.Flow cytometry assays of cell cycle and apoptotic experiments showed that the apoptotic rate increased and decreased,respectively.The cell cycle was blocked at G0/G1 and G2/M phases,respectively.Conclusion1.The results of TA clone sequencing of PCR products were consistent with the original tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP sequences and their corresponding amplification primer sequences.2.The expression of tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP had a certain relationship with the clinicopathological factors of gastric cancer patients.tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP had certain diagnostic value and had the potential to be a new biomarker of gastric cancer.3.Biological function experiments showed that tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP had a tumor suppressive effects.These results help us to further study how tRF-19-3L7L73JD and tRF-33-P4R8YP9LON4VDP affecting the occurrence of gastric cancer.