| Objective: Gastric Cancer is the fifth most common cancer.and the third most common cause of cancer death worldwide.It is one of the most serious threats to human health.It's also one of the leading causes of morbidity and mortality in China.Recently,although the surgical technique,other adjuvant therapy and targeted therapy have been improved,the clinical efficacy of gastric cancer is still unsatisfactory.The five-year survival rate for gastric cancer is still lower than for some cancers,such as breast cancer and prostate cancer.Therefore,to explore the molecular mechanism of gastric cancer and to find more effective diagnostic markers or therapeutic targets have become urgent problem in basic and clinical research of gastric cancer.Traditionally,transfer RNA(tRNA)has been thought to be involved in cell protein synthesis.tRNA-derived fragment(tRF)is a new type of small non-coding RNA,which is derived from the specific splicing of precursor or mature tRNA.Recently,a growing number of studies have shown that these tRFs are deregulated frequently in human cancers.Some tRFs can affect tumor cell proliferation,apoptosis,migration and invasion in a variety of cancers.With the development of research,new tRNA derivatives have been found and are considered as new biomarkers and targets for cancer therapy.In this study,we demonstrated that tRF-3017 A,a tRNA-derived fragment,is up-regulated in gastric cancer than in non-cancerous adjacent tissue,and its high expression is associated with lymph node metastasis in gastric cancer.In addition,cell function experiments demonstrated that tRF-3017 A could promote migration and invasion of gastric cancer cells.At the same time,this study explored the mechanism of tRF-3017 A affecting the metastasis of gastric cancer,and provided a new idea and target for the treatment of gastric cancer.Methods:In the first part,we used high-throughput PCR array to analyze differential expression of tRFs in 10 gastric cancer tissues and paired non-cancerous tissues from the first affiliated hospital of the China Medical University.In the result of array analysis,tRFs were selected based on P < 0.05 and fold change > 2.We examined the expression of various candidate tRFs in 87 pairs of gastric cancer tissues and analyzed the correlation between their expression level and clinicopathologic data.We selected tRNA-derived fragment,tRF-3017 A,which was significantly associated with lymph node metastasis in gastric cancer,for further study.In order to explore the value of tRF-3017 A in the diagnosis of lymph node metastasis of gastric cancer,we used receiver operating characteristic(ROC)to evaluate the diagnostic efficacy,sensitivity and specificity of tRF-3017 A.Next,we analyzed the expression of tRF-3017 A in gastric cancer cell lines(AGS,HGC-27,MGC-803,MKN-45 and SNU-16)by qRT-PCR.In the second part,the effects of tRF-3017 A on migration,invasion and proliferation of gastric cancer cells were investigated by Transwell assay,scratch healing and CCK8 proliferation assay.mi Randa and Target Scan databases were used to predict the downstream target genes of tRF-3017 A.qRT-PCR and Western blot were used to determine whether the expression of target gene was influenced by the expression of tRF-3017 A.Direct association between tRF-3017 A and the downstream target gene was verified by double luciferase reporter assay.RIP assay was performed to verify whether tRF-3017 A was associated with Ago2 and the target gene to form RISC complex.Function experiments were performed to investigate the effect of knock-down or over-expression target genes on migration and invasion of gastric cancer cells.Transwell assay and scratch-healing test were used to verify whether knock-down or over-expression target gene can restore the effect of tRF-3017 A on the migration and invasion of HGC-27 and AGS cells,and to determine whether the target gene is involved in tRF-3017A-induced metastasis in gastric cancer.Results:?.Expression of tRF-3017 A in gastric cancer and its correlation with clinicopathologic features:1.The expression of tRF-3017 A was higher in 87 gastric cancer tissues than in matched non-cancer tissues(62/87,p < 0.001).2.High expression of tRF-3017 A was significantly correlated with lymph node metastasis(p = 0.016),there was no significant correlation between the TNM stage,age(p = 0.144),sex(p = 0.126),tumor size(p = 0.411),pathological grade(p = 0.807),T stage(p = 0.558),TNM stage(p = 0.064).3.The AUC of tRF-3017 A in all patients with gastric cancer was 0.6103(p < 0.05),and the AUC of tRF-3017 A in patients with/without lymph node metastasis was 0.6465(p <0.05).4.The expression of tRF-3017 A in gastric cancer cell lines SNU-16,AGS and HGC-27 was significantly higher than that in normal gastric epithelial cells.?.Effect of tRF-3017 A on biological function of gastric cancer cells:1.After transient transfection,the overexpression of tRF-3017 A was detected by qRT-PCR in HGC-27 cells(60.89 ± 6.049,p < 0.001)and AGS cells(46.88 ± 7.669,P =0.0012)2.Overexpression of tRF-3017 A could promote migration and invasion of gastric cancer cells,but had no effect on cell proliferation.3.tRF-3017 A knock-down significantly inhibited the migration and invasion of gastric cancer cells.?.Prediction and validation of the downstream target gene of NELL2 directly regulated by tRF-3017A:1.Based on mi Randa & Target Scan database,it is predicted that the NELL2 may be the target gene directly regulated downstream of tRF-3017 A.2.After overexpression or knock-down of tRF-3017 A,qRT-PCR and Western Blot showed that the expression of NELL2 was negatively correlated with tRF-3017 A.3.qRT-PCR showed that the expression of NELL2 was significantly lower in gastric cancer tissues than that in non-cancer tissues(p < 0.001),and the expression of NELL2 was negatively correlated with the expression of tRF-3017A(r =-0.41,p < 0.001).4.In gastric cancer,the protein expression level of NELL2 was negatively correlated with tRF-3017 A.5.The dual-luciferase reporter assay show that tRF-3017 A can bind directly to the 3'UTR region of the NELL2 m RNA.6.RIP assay demonstrated that tRF-3017 A may form RISC complex with NELL2 via Ago2 protein.?.Explore whether NELL2 involved in tRF-3017A-induced metastasis of gastric cancer:1.The ability of migration and invasion of gastric cancer cells was enhanced by silencing NELL2,and the migration and invasion of gastric cancer cells were inhibited by overexpression of NELL2.2.In Transwell assay and cell scratch experiments,overexpression of the NELL2 gene restored the cell migration and invasion promoted by overexpression of tRF-3017 A,and the cell migration and invasion inhibited by knock-down of tRF-3017 A can be restored by NELL2 knock-down.Conclusion: 1.tRF-3017 A is highly expressed in gastric cancer tissues and cells.2.High expression of tRF-3017 A in gastric cancer is associated with lymph node metastasis.3.Overexpression of tRF-3017 A promoted the migration and invasion of HGC-27 and AGS cells,but had no significant effect on proliferation.4.tRF-3017 A knockdown inhibits the migration and invasion of HGC-27 and AGS cells.5.The expression of NELL2 was generally down-regulated in gastric cancer cells,and the expression of NELL2 in gastric cancer was negatively correlated with tRF-3017 A.6.tRF-3017 A promotes migration and invasion of gastric cancer cells by targeting it's downstream target gene NELL2 through a mi RNA-like mechanism. |
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