Molecular Screening Analysis Of Plasma CfRNA Markers For Early Gastric Cancer

BackgroundGastric cancer(GC)is one of the five most common types of malignant tumors and ranks the fourth leading cause of cancer related death in the world.In the past decades,survival rates for patients with stomach cancer have been improved significantly in the past few decades,but patients with early-stage stomach cancer have no obvious symptoms and are usually diagnosed in advanced stages.The prognosis of most patients with gastric cancer is poor.Neoplastic transformation of the gastric mucosa is a cascade development process.Early diagnosis and treatment of patients can reduce the mortality associated with gastric cancer.In this way,it is necessary to use an effective screening method to detect precursor lesions and early gastric cancer(EGC),which can increase the 5-year survival rate of patients.In recent years,circulating cell-free RNA(cfRNA)and m6 A modified RNAs as molecular markers have shown potential in early diagnosis,personalized treatment and prognosis of cancer.Therefore,we will screen early gastric cancer biomarkers based on cfRNA and cfRNA fragments.At the same time,the m6 A methylation level of cfRNA in plasma was detected and analyzed to explore whether the change of m6 A modified RNA level could be used as a new biomarker for tumor diagnosis.Objective1.On the basis of previous research in our laboratory,six RNA molecules,including CEBPA-AS1,INHBA-AS1,AK001058,UCA1,PPBP and RGS18,are screened and analyzed in the plasma of EGC and precursor lesions,in order to obtain cfRNA candidate biomarkers for the diagnosis of EGC and precursor lesions.2.The expression levels of different fragments of lncRNA ENST0000568893.1 in plasma were analyzed to explore the feasibility of cfRNA fragmentation as diagnostic biomarkers for gastric cancer.3.The level of m6 A modified 28 S rRNA in plasma was detected,and the application value of m6 A modified RNA level as gastric cancer biomarkers was evaluated.Methods1.The qRT-PCR was utilized to detect the levels of six RNA molecules in the plasma samples of patients with precursor lesions and early gastric cancer and in the plasma samples of healthy persons.ROC analysis was used to evaluate the diagnostic accuracy of these six selected biomarkers.2.The lncRNA ENST00000568893.1 RNA full-length sequence was divided into3 distinct fragments,and PCR amplification primers corresponding to each RNA fragment is designed respectively.The q RT-PCR was utilized to detect the separate fragments of lncRNA in plasma samples of precursor lesions,EGC and healthy individuals.3.The recombinant YTHDF2 protein was used to enrich the m6 A modified RNA.The q RT-PCR was utilized to detect the level of m6 A modified 28 S rRNA in plasma samples of patients with EGC and healthy controls.Results and Conclusion1.Screening of cfRNA candidate biomarkers in plasma of EGCAmong the 6 RNA molecules,the levels of four lncRNAs,including CEBPA-AS1,INHBA-AS1,AK001058 and UCA1,were up-regulated in the plasma of patients with precursor lesions and EGC,the leves of two m RNAs,PPBP and RGS18 were down-regulated in the plasma of patients with precursor lesions and EGC.ROC analysis showed that a 4-RNA combination with the highest AUC value is constructed in precursor lesions,including INHBA-AS1,AK001058,UCA1 and RGS18.In EGC,a 6-RNA combination with the highest AUC value is constructed,including CEBPA-AS1,INHBA-AS1,AK001058,UCA1,PPBP and RGS18.2.Heterogeneity of lncRNA ENST000005688931.1 RNA fragments in plasma of EGCThe level of different fragments of lncRNA ENST00000568893.1 in the same type of plasma samples were different,and the level of the same fragment in different types of plasma samples are also different.These results suggested that lncRNA ENST00000568893.1 exists as fragments in plasma.The different combinations of different fragments of lncRNA ENST0000-0568893.1 may be more suitable as molecular markers for the diagnosis of EGC.3.Detection of m6 A modified 28 S rRNA in plasma of EGCThe recombinant YTHDF2 protein can enrich the m6 A modified RNA in the plasma,and the enriched RNA can be used to detect the level of m6 A modified 28 S rRNA.The level of m6 A modified 28 S rRNA in the plasma of patients with EGC was higher than that of healthy controls.The results of this study provide a basis for screnning cfRNA and m6 A modified RNA molecules for early diagnosis of gastric cancer.