A Novel 3’tRNA-derived Fragment TRF-Val Promotes Gastric Cancer Progression By Targeting EEF1A1

BackgroundGastric cancer(GC)is one of the most common digestive tract tumors in China.Because of its high morbidity and mortality,it is particularly urgent and important to find new molecular targets for the occurrence,progression and treatment of GC.tRFs&tiRNAs are new small non-coding RNA(ncRNA)molecules discovered in recent years.At present,it has been found that tRFs&tiRNAs were involved in the progression of many kinds of malignant tumors,but the research in GC is still rare.Objectives1)To identify the highly expressed tRFs&tiRNAs molecules in GC.2)To screen out candidate carcinogenic tRFs and verify their clinicopathological correlation and biological function in GC cells.3)To explore the molecular mechanism of target tRFs in the progression of GC,and provide a new theoretical basis and therapeutic target for the occurrence and progression of GC.Methods1.Expressions of tRF-Val in GC and its correlation with clinicopathological features1)Four pairs of GC tissues and paired normal tissue samples were collected for tRFs&tiRNAs high-throughput sequencing analysis,and the five highly expressed tRFs with the highest fold change were selected as candidate molecules.2)The five candidate tRFs were verified by qRT-PCR in 10 pairs of GC tissues and paired normal tissues.Among them,tRF-60:76-Val-CAC-2(hereinafter referred to as tRF-Val)was the most significant tRF.3)qRT-PCR verification and clinical correlation analysis were conducted in 65 pairs of GC tissues and paired normal tissues.4)qRT-PCR was conducted to vertify the expressions of tRF-Val in 4 GC cell lines MKN-45,MKN-28,HGC-27,AGS and immortalized gastric mucosal epithelial cell GES-1.2.Biological functions of tRF-Val in GC cells1)tRF-Val knockdown and overexpression models were constructed in GC cells,and the transfection efficiencies were verified by qRT-PCR.2)Cell biology experiments in vitro were carried out;CCK-8 assay was used to detect cell proliferation ability,colony formation assay was used to detect cell colony formation ability,flow cytometry was used to detect cell apoptosis,and Transwell assay was used to detect cell invasion ability.3)Cell biology experiments in vivo were carried out;the effect of tRF-Val knockdown on tumor growth was detected by subcutaneous tumor transplantation assay in nude mice.3.tRF-Val binds to EEF1A1 protein and promotes its transport into the nucleus1)tRF-Val biotin probe and antisense chain control probe were synthesized in vitro.RNA pulldown assay,mass spectrometry assay,silver staining assay,and RNA immunocoprecipitation(RIP)assay were used to detect and verify tRF-Val interaction proteins in GC cells.2)qRT-PCR assay,nucleocytoplasmic proteins isolation assay,western blot(WB)assay,immunofluorescence(IF)assay and fluorescence in situ hybridization(Fish)assay were used to detect the regulatory effects between tRF-Val and EEF1A1 in GC cells.4.EEF1A1 promote GC progression by enhancing the effects of MDM21)The knockdown and overexpression models of EEF1A1 were constructed in GC cells,and the transfection efficiencies were verified by WB.2)The effects of tRF-Val overexpression and at the same time EEF1A1 knockdown on the growth of GC cells were detected by colony formation assay.3)The effects of EEF1A1 knockdown and overexpression on the growth and apoptosis of GC cells were detected by colony formation assay and flow cytometry.4)Co-immunoprecipitation(Co-IP)experiment was used to verify the binding of EEF1A1 and MDM2-p53 complex,and WB was used to verify the effects of EEF1A1 knockdown and overexpression on the phosphorylation of MDM2 at Ser166.5)Nucleocytoplasmic protein isolation assay and WB were used to verify the effects of EEF1A1 knockdown and overexpression on the nuclear localizations of MDM2 and p-MDM2.6)Ubiquitination assay was used to verify the effect of EEF1A1 overexpression on p53 ubiquitination.7)WB was used to verify the effects of EEF1A1 knockdown and overexpression on p53 and its downstream proteins p21,Bax,and Bcl2.5.tRF-Val regulates the MDM2/p53 pathway by promoting the interaction between EEF1A1 and MDM21)IF assay was used to verify the effects of tRF-Val on the co-localization of EEF1A1 and MDM2;Co-IP assay was used to verify the effects of tRF-Val transfection concentration on the binding of EEF1A1 and MDM2.2)Nucleocytoplasmic protein isolation assay and WB were used to verify the effects of tRF-Val knockdown and overexpression on the nuclear localization of MDM2 and p-MDM2.3)Ubiquitination assay was used to verify the effect of tRF-Val overexpression on p53 ubiquitination.4)WB rescue experiments were used to verify the effects of tRF-Val knockdown,overexpression and at the same time EEF1A1 overexpression,knockdown on p-mdm2,p53,p21,Bax and Bcl2 proteins in GC cells.5)CCK-8 and clony formation rescue experiments were used to verify the effects of tRF-Va overexpression and at the same time EEF1A1 knockdown on cell proliferation in GC cells.6)WB and immunohistochemistry(IHC)assay were used to verify the changes of p53,p21,Bax and Bcl2 proteins after tRF-Val knockdown in nude mice.Results1.Expressions of tRF-Val in GC and its correlation with clinicopathological features1)High throughput sequencing showed that tRFs&tiRNAs were differentially expressed in 4 pairs of GC and control normal tissues,and tRF-3a was the main up-regulated tRFs type in GC.2)Five tRF-3a molecules with the highest fold change were selected for qRT-PCR verification in 10 pairs of GC tissues.tRF-Val was the most significantly up-regulated tRF(P=0.002).3)tRF-Val was significantly overexpressed in 65 GC tissues and 4 GC cell lines.4)Clinicopathological analysis showed that the expression level of tRF-Val was significantly positively correlated with tumor size and depth of invasion(P=0.040 and P=0.004).2.Biological functions of tRF-Val in GC cells1)tRF-Val had the highest expression in AGS and MKN-45 cells.tRF-Val overexpression and knockdown models were constructed.qRT-PCR results showed that the expression of tRF-Val increased and decreased significantly.2)tRF-Val overexpression significantly promoted the proliferation and invasion of GC cells and inhibited their apoptosis;tRF-Val knockdown significantly inhibited the proliferation,invasion and apoptosis of GC cells.3)tRF-Val knockdown inhibited the growth of GC in nude mice.3.tRF-Val binds to EEF1A1 protein and promotes its transport into the nucleus1)The results of RNA pulldown and mass spectrometry showed that tRF-Val targeted binding to EEF1A1;RIP experiment reversely verified the combination of EEF1A1 and tRF-Val.2)qRT-PCR results showed that tRF-Val did not affect the expression of EEF1A1 at the mRNA level.3)qRT-PCR results showed that EEF1A1 knockdown and overexpression did not affect the expression of tRF-Val.4)WB results showed that tRF-Val did not affect the expression of EEF1A1 at the total protein level,but tRF-Val overexpression significantly increased the expression of nuclear protein EEF1A1 and decreased the expression of cytoplasmic protein EEF1A1.5)The results of IF assay showed that tRF-Val overexpression significantly enhanced the fluorescence intensity of EEF1A1 in the nucleus.6)The FISH assay confirmed that tRF-Val overexpression also mainly enhanced its fluorescence in the nucleus,which was the same trend as EEF1A1.4.EEF1A1 promote GC progression by enhancing the effects of MDM21)The proliferation promoting effect of tRF-Val overexpression on GC cells could be reversed by the knockdown of EEF1A1.2)EEF1A1 overexpression significantly promoted the growth of GC cells and inhibited their apoptosis;EEF1A1 knockdown significantly inhibited the growth of GC cells and promoted their apoptosis.3)The Co-IP result showed that EEF1A1 could bind to MDM2-p53 complex and promote the phosphorylation of MDM2 at ser166 in GC cells.4)EEF1A1 overexpression could significantly increase the expression of MDM2 and p-MDM2 in the nucleus of GC cells.5)EEF1A1 overexpression significantly increased the ubiquitination level of p53 in GC cells.6)EEF1A1 overexpression significantly decreased the expression of p53,p21 and Bax proteins and increased the expression of Bel2 protein in GC cells,EEF1A1 knockdown showed the opposite result.5.tRF-Val regulates the MDM2/p53 pathway by promoting the interaction between EEF1A1 and MDM21)IF assay and Co-IP assay results showed that tRF-Val promoted the nuclear entry of EEF1A1 and its binding to nuclear localized MDM2.2)tRF-Val overexpression could significantly increase the expression of MDM2 and p-MDM2 in the nucleus of GC cells.3)tRF-Val overexpression significantly increased the ubiquitination level of p53 in GC cells.4)WB rescue experiment showed that tRF-Val overexpression significantly increased the expression of p-MDM2 and Bcl2 in GC cells and decreased the expression of p53,p21 and Bax,tRF-Val knockdown showed the opposite results.In addition,the regulation of MDM2/p53 pathway by tRF-Val knockdown or overexpression could be reversed by EEF1A1 overexpression and silencing,respectively.5)CCK-8 rescue assays and colony formation rescue assays indicated that the growth-promoting effect of tRF-Val overexpression on GC cells could be reversed by the knockdown of MDM2.Conclusions1)tRF-Val was significantly overexpressed in GC tissues and GC cell lines,and its expression level was significantly positively correlated with tumor size and depth of invasion(T stage).2)tRF-Val significantly promoted the proliferation and inhibited apoptosis of GC cells both in vivo and in vitro.3)Mechanismly,tRF-Val targeted EEF1A1 and mediated its transport into the nucleus,thus promoting the interaction between EEF1A1 and nuclear localized MDM2,enhancing the regulatory effect of MDM2 on p53 ubiquitination,inhibiting the function of p53 downstream molecules,and finally promoting tumor progression.4)This study provided a new molecular mechanism and therapeutic target for the occurrence and progress of GC.