| Objective:There is a high incidence of gastric cancer(GC)in China.Due to the lack of specific clinical symptoms in the early stage and the low specificity and sensitivity of tumor markers used in gastric cancer screening,most patients have progressed to the advanced stage when they are diagnosed.It is particularly important to find new tumor markers for GC early diagnosis.LncRNA(Long non-coding RNA)have a wide range of biological functions,and its abnormal expression is an important factor affecting the occurrence and development of tumors.In this study,we used RNA-seq(RNA sequencing)technique to screen the differential co-expression lncRNA in chronic non-atrophic gastritis and low/high grade gastric intraepithelial neoplasia(precancerous lesions)to gastric cancer and explored tumor markers for GC early diagnosis.Methods:1.We collected 45 cases of gastric lesions samples confirmed by the Department of Pathology of the affiliated Hospital of Qingdao University from September 2019 to September 2020,including 22 cases of chronic non-atrophic gastritis,14 cases of gastric intraepithelial neoplasia(including 9 cases of low-grade intraepithelial neoplasia tissues,and 5 cases of high-grade intraepithelial neoplasia tissues)and 9 cases of gastric cancer.Taking the OD260/280 between 1.8 and 2.0,the concentration of RNA≥100 ng/μl,the total amount of RNA≥1μg and RIN≥6.5 as the standard of quality inspection,five samples with better quality were selected from each group for bioinformatics analysis.2.RNA-seq technique was used to analyze all transcripts of 15 samples,then,screening the differentially expressed lncRNA among each group by edge R package and finding the differentially expressed lncRNA which were co-expressed among each group.The co-expressed m RNA of different lncRNA among groups was analyzed by Pearson correlation coefficient method.Furthermore,we speculated the biological significance of the combination of lncRNA and targeted m RNA.3.RNA of 3 groups samples were extracted and reverse transcribed into c DNA,the expression level of differential co-expressed lncRNA in 3 groups samples were detected by q RT-PCR.Results:1.We used RNA sequencing to find that there were 666 lncRNAs screened out between chronic non-atrophic gastritis and gastric cancer.Compared with chronic non-atrophic gastritis group,there were 188 lncRNAs up-regulated and 478 lncRNAs down-regulated in gastric cancer group.Compared with low/high grade gastric intraepithelial neoplasia group,6 lncRNAs expression was up-regulated and 7 lncRNAs expression was down-regulated in gastric cancer group.There were 6 differential co-expressed lncRNAs(lncRNA H19,LINC00895,lnc-SRGAP2C-16,lnc-HLA-C-2,lnc-APOC1-1 and lnc-B3GALT2-1)in three groups.2.According to the results of q RT-PCR test: compared with chronic non-atrophic gastritis,the expression level of lncRNA H19(n= 10,p < 0.01),LINC00895(n= 10,p <0.001),lnc-SRGAP2C-16(n=10,p<0.01),lnc-HLA-C-2(n=10,p<0.001)and lncAPOC1-1(n= 10,p < 0.0001)were significantly higher in GC group,but the expression level of lnc-B3GALT2-1(n= 10,p<0.001)were significantly lower in GC group.Compared with gastric intraepithelial neoplasia group,the expressions of lncRNAH19(n=10,p<0.05),LINC00895(n=10,p<0.001),lnc-SRGAP2C-16(n=10,p<0.001),lnc-HLA-C-2(n=10,p<0.01)and lnc-APOC1-1(n=10,p<0.0001)in GC group were significantly higher,while the expression level of lnc-B3GALT2-1(n=10,p<0.01)were significantly lower in GC group.3.Pearson correlation coefficient showed that three differential lncRNAs may have co-expressed m RNA,lncRNA H19 co-expressed with LRRC38,LINC00895co-expressed with BTN1A1 and lnc-SRGAP2C-16 co-expressed with ZNF729.Conclusion:1.In this study,we found and verified the differential lncRNAs among chronic non-atrophic gastritis,low/high grade gastric intraepithelial neoplasia and gastric cancer.There were 6 co-expressed differential lncRNAs between chronic non-atrophic gastritis,gastric low/high grade intraepithelial neoplasia and gastric cancer.Compared with chronic atrophic gastritis and gastric low/high grade intraepithelial neoplasia lncRNA H19,LINC00895,lnc-SRGAP2C-16,lnc-HLA-C-2 and lnc-APOC1-1 were up-regulated but the expression of lnc-B3GALT2-1 was down-regulated in GC tissue,these 6 lncRNAs are expected to become molecular markers for GC early diagnosis.2.There is a targeting correlation between 3 different lncRNAs and m RNA:lncRNA H19 and LRRC38,lnc00895 and BTN1A1,lnc-SRGAP2C-16 and ZNF729.The mechanism of action of three lncRNAs in gastric cancer needs to be further studied. |
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