Establishment Of TRNA Fragment Detection Method And Its Application In Gastric Cancer Cellstolerance

tRNA is an important linker molecule in protein synthesis and is involved in cellular communication and reverse transcription.Studies have shown that under physiological conditions,a large number of tRNA fragments exist in mouse sperm,and intracellular tRNA is cleaved under various stress conditions,producing a large number of tRNA fragments,which have important biological functions.In this study,the bacterial-derived tRNA extraction technology was optimized for mouse tissues and cells,and combined with qPCR technology,a method for pre-processing of samples with rapid detection and high-throughput detection of tRNA fragments in tissue cells was established.It provides an efficient method for the detection and analysis of tRNA fragments and their functions.This method was used to screen for cancer markers and to investigate the effects of DNMT2 and polysaccharides on cellular stress tolerance and tRNA stability.The details are as follows:1.Establishment of tRNA fragment detection method?1?tRNA extraction method adjustmentIn this field,the method of extracting bacterial tRNA is optimized,and the optimized method can remove the total tRNA obtained from the 28S rRNA hybrid band without obvious towing phenomenon,which can satisfy the next work of this study..?2?Detection of tRNA fragments in mouse spermThe tRNA extracted from mouse sperm,testis and liver,linked to the 3'end,was reverse transcribed into cDNA.Using 5.8 sRNA as an internal reference,primers for tRNA^Glu,tRNA^Gly,tRNA^Asp,and 3'linker and 5.8sRNA were designed and detected by qPCR technique.The results showed that the expression of tRNA fragments in mouse sperm was much higher than that in liver and testis,which was consistent with the literature on the large amount of tRNA fragments in mouse sperm.2.Expression levels of tRNA fragments in gastric mucosal epithelial cells GES-1and gastric cancer cells SGC7901Extraction of tRNAs from SGC7901 cells and GES-1 cells This method was used to detect tRNA fragments of tRNA^Glu,tRNA^Gly,tRNA^Asp,tRNA^Val,tRNA^Cys in SGC7901 cells and GES-1 cells.It was found that the relative expression of tRNA fragments in cancer cells is much lower than that of normal cells,and tRNA^Glu,tRNA^Gly,and tRNA^Asp have the potential to become new cancer markers.3.The expression of tRNA fragment under stress conditions and the effect ofoverexpression of DNMT2 on cellular tRNA stress toleranceThree kinds of cellular stress models were established to establish high glucose stress,hypoxia stress and oxidative stress,and the expression of tRNA fragments in model cells was detected by established methods.It was found that the expression levels of tRNA fragments in normal cells and cancer cells were increased under stress conditions,which was consistent with the report that the tRNA in cells was sheared into specific small RNA fragments under stress conditions.And as stress conditions persist,intracellular tRNA fragments are reduced,probably because of increased stress tolerance in cells.Subsequently,DNMT2 was overexpressed in the cells,which confirmed that DNMT2 can increase the stress tolerance of the cells and protect tRNA from being sheared under stress conditions.4.Effects of different polysaccharides on the tolerance of tRNA stress in cellsPolysaccharide treatment was added to the stress cell model to detect the effects of polysaccharide on cell proliferation,tRNA fragment expression and DNMT2expression.It was found that polysaccharides PGA4-3b,CFAA-1 and LJW0F1 had no significant effect on cell proliferation under stress conditions.Polysaccharide WSS25inhibited cell proliferation under stress conditions.The four polysaccharides had no significant effect on the expression of intracellular tRNA fragments under stressconditions,and had no significant effect on the expression of DNMT2 protein in cells.