Screening Of Specific RNA Markers In Gastric Cancer Plasma And Cloning And Expression Of The M~6A Modified RNA Binding Protein YTHDF2

Objective:Gastric cancer is one of the most common malignant neoplasms in the world.Due to its lack of precise early diagnosis,most patients are diagnosed with the risk of advanced tumors and their metastasis,leading to its morbidity and mortality ranking first in the world,At present,the diagnosis of gastroscope and traditional gastric cancer tumor markers used in clinic has some limitations and lack of strong accuracy,which brings difficulties in the discovery and treatment of gastric cancer.On the one hand,this study aimed to screen RNA molecules differentially expressed in the plasma of patients with gastric cancer and normal people to evaluate whether they could be used as candidate markers for the diagnosis of gastric cancer,On the other hand,in this study,the recombinant expression vector of m6A-modified RNA binding protein YTHDF2was constructed and its activity was determined.,Using its ability to bind to the modified m6A RNA.Methods:1)According to the result of GeneChip^?Human Transcriptome Array 2.0?HTA2.0?^[1],We next validated the six differential expression in a new training set of81 GC and 77 healthy volunteer plasma samples using RT-qPCR.2)We further validated the best candidate RNA biomarkers in a testing set of 36 GC patients and 34healthy volunteers,and ROC curve analyses were performed,while AUC values,sensitivity and specificity values are obtained.3)we determined the relationships between expression levels and patient clinicopathological data to assess the diagnostic value of these biomarkers in patients with GC.4)The coding region of YTHDF2 gene was amplified by RT-PCR.5)The recombinant plasmid pET-28a-YTHDF2 was constructed and expressed in E.coli BL21?DE3?,it was purified by Ni²⁺-NTA resin affinity chromatography.6)the fusion protein activity was analyzed by Ni²⁺-NTA magnetic spheres.Results:The levels of six candidate RNAs,including RGS18?Regulator of G-protein signaling 18?,ITM2B?Integral membrane protein 2B?,PPBP?Pro-platelet basic protein?,NAP1L1?Nucleosome assembly protein1-like 1?,n324674,and ENST00000442382,We found that expression levels of noncoding RNAs n324674?P=0.0069?and ENST00000442382?P=0.3994?were significantly elevated,while those of mRNAs RGS18?P<0.0001?,ITM2B?P=0.0078?,PPBP?P<0.0001?,and NAP1L1?P=0.0237?were reduced,while AUCs of the ROC curves of 0.735,0.635,0.721,0.629,0.639,and 0.597 in Training set of plasma of GC patients.RGS18?P<0.0001?,PPBP?P<=0.0012?,and AUCs of the ROC curves of 0.811 and 0.710 in Test set of plasma of GC patients,Relative PPBP expression in gastric cancer plasma samples and its clinical significance,it was significantly lower in females with GC In addition,The recombinant YTHDF2 protein was expressed in E.coli BL21?DE3?and purified,it was capable of binding RNA with m6A-modification.Conclusion:Combined with differential expression levels of RGS18 and PPBP in gastric cancer plasma and diagnostic sensitivity and specificity as a diagnostic marker for gastric cancer,RGS18 and PPBP have potential value as molecular markers of gastric cancer diagnosis.YTHDF2 fusion protein was capable of binding RNA with m6A-modification,it provides an essential tool that researching the biological function of RNA with m6A-modification.