Functional Study Of TRF-Val-CAC-010 In The Development Of Lung Adenocarcinoma

Background:Lung cancer is a cancerous growth that poses a major threat to human health.Lung cancer currently has a poor overall prognosis,a low clinical cure rate,and a lack of efficient screening methods.Because the origin of lung cancer is so complex and unknown,it’s critical to understand the molecular mechanisms of lung cancer pathogenesis and develop efficient early diagnostic markers and targeted therapy locations.Small RNAs(ts RNAs)generated from t RNA have gotten a lot of attention as possible regulatory agents.Furthermore,hypoxia is a typical hallmark of many solid tumors and is one of the major reasons contributing to clinical radiotherapy’s decreased efficacy.Studies have confirmed that cells under stress induce the production of t RNA-derived fragments,which are especially elevated under hypoxic or other stress conditions,and that these fragments participate in the stress response and regulate apoptosis.As a result,we investigated the molecular mechanism of its development in lung adenocarcinoma under hypoxia in order to provide new ideas and targets for lung adenocarcinoma treatment.Propose:The goal of this project is to identify tRF target molecules linked to lung adenocarcinoma,investigate their impact on lung adenocarcinoma phenotypic function,and identify new molecular markers and therapeutic targets for lung adenocarcinoma treatment.Methods:In the first part,we used high-throughput sequencing technology to examine aberrantly expressed t RNA-derived fragments in three cases of lung adenocarcinoma and paracancerous non-cancerous tissues at Yunnan Province’s First People’s Hospital,and we used the Next Seq system to determine the differential expression levels of tRF and ti RNA in lung adenocarcinoma and adjacent percancerous tissues at P<0.05 and absolute value of the difference expression multiplier.Bioinformatics has been used to investigate possible target genes in considerable depth.The expression levels of differently expressed ts RNAs in lung adenocarcinoma tissues were verified by real-time fluorescence quantitative PCR,and we chose the t RNA derivative with the largest differential expression ploidy: tRF-Val-CAC-010.The expression levels of tRF-Val-CAC-010 in lung adenocarcinoma cell lines were then verified using real-time fluorescence quantitative PCR.In the second part,combining the target genes screened by bioinformatics analysis in the first part,tRF mimic and tRF inhibitor were designed to overexpress tRF-Val-CAC-010 in normal lung epithelial cell line Beas-2B and silence tRF-Val-CAC-010 in lung adenocarcinoma cell lines A549 and PC9 to observe the effect of the target molecule on the cell phenotype of each group: using q PCR to detect the transfection efficiency of target molecules,CCK8 kit to observe the growth curve of cells,cell scoring assay to observe cell migration,Transwell assay to observe the change of cell invasion ability,and flow cytometry combined with PI single staining and Annexin-V double staining to detect cell cycle and apoptosis.A series of experiments were carried out to see if target gene overexpression or inhibition could restore the effect of tRF-Val-CAC-010 on the phenotypic function of lung adenocarcinoma cells.Results:1.Screening of t RNA derivatives in lung adenocarcinogenesis and development(1)Analysis based on tRF&ti RNA high-throughput sequencing results.Comparison with paracancerous tissues revealed that 17 tRF&ti RNAs were abnormally expressed in the molecules obtained by this sequencing.The clustering analysis showed that there were 34 different isoforms of molecules in both lung adenocarcinoma and paraneoplastic groups,of which 20 were up-regulated and 14 were down-regulated.And the t RNA-derived fragments with P<0.05 and absolute value of the difference expression multiplier ≥ 1 were analyzed bioinformatically,and tRF-Val-CAC-010 with significant differential expression fold was selected as the target molecule.tRF-Val-CAC-010 was significantly highly expressed in lung adenocarcinoma tissues(P<0.05)and lung adenocarcinoma cell lines A549 and PC9(P<0.01).(2)tRF-Val-CAC-010 was predicted to have 420 target genes by mi Randa and Target Scan.GO analysis showed that most of the target genes were enriched in "positive regulation of mast cell activation"(biological process),"cell membrane cavity-like invagination"(cellular component)and "binding scaffold protein"(molecular function).In the KEGG pathway analysis,the top ten significantly enriched pathways associated with tRF-Val-CAC-010 target genes were identified(p<0.05),the most significant of which was the "Hedgehog signaling pathway".2.Effect of tRF-Val-CAC-010 on the phenotypic function of lung adenocarcinoma cells(1)The expression level of tRF-Val-CAC-010 mimic was significantly increased after overexpression of tRF-Val-CAC-010 in BEAS-2B cells(P<0.001).(2)The expression level of tRF-Val-CAC-010 inhibitor was significantly decreased after inhibition of tRF-Val-CAC-010 in A549 and PC9 cells(P<0.001).(3)Overexpression of tRF-Val-CAC-010 molecule promoted proliferation,migration and invasion of normal lung epithelial cells(BEAS-2B)(P<0.05).However,there was no effect on cell cycle and apoptosis.(4)Inhibition of tRF-Val-CAC-010 molecule inhibited proliferation,migration and invasion and promoted apoptosis of lung adenocarcinoma cells(A549)(P<0.05).However,there was no effect on the cell cycle.(5)Inhibition of tRF-Val-CAC-010 molecule inhibited proliferation,migration and invasion of lung adenocarcinoma cells(PC9)(P<0.05).However,it had no effect on cell cycle and apoptosis.Conclusion:High-throughput sequencing was used to screen the relevant t RNA-derived fragment tRF-Val-CAC-010 in fresh tissue samples from lung adenocarcinoma patients and their paraneoplastic tissue samples;tRF-Val-CAC-010 has a discriminatory value for lung adenocarcinoma tissues and cell lines,as well as paraneoplastic tissues and normal cells;tRF-Val-CAC-010 was predicted to have 420 target genes using mi Randa and Target Scan.GO analysis showed that most of the target genes are enriched in "positive regulation of mast cell activation"(biological process),"cell membrane cavity-like invagination"(cellular component)and "binding scaffold protein "(molecular function);KEGG pathway analysis identified the main signaling pathways involved in tRF-Val-CAC-010 target genes,most notably the "Hedgehog signaling pathway";tRF-Val-CAC-010 promotes proliferation,migration,and invasion of lung adenocarcinoma cells,and no regularity was found for cell cycle and apoptosis at present.