| Background and objectiveGastric cancer is the fifth most common malignant tumor in the world.The mortality rate of gastric cancer is in the fourth place in the world.The incidence and mortality of gastric cancer in China are in the first place in gastrointestinal cancer.At present,active surgery or postoperative adjuvant chemotherapy is a common treatment,but the prognosis of many gastric cancer is still poor,the main reason may be that early gastric cancer is not easy to diagnose.At present,the most commonly used method of gastric cancer detection is still invasive and expensive pathological examination,so we urgently need to find new biomarkers for early diagnosis and more comprehensive understanding of the molecular mechanism of gastric cancer occurrence and development,so as to overcome this devastating disease.In 2011,the competitive endogenousRNA（Cerna）hypothesis was first proposed.This new regulatory mechanism between ncRNA and coding messengerRNA has completely broken the traditional understanding ofRNA.That is to say,on the basis of the traditional mode of miRNA toRNA transformation,there is still a certain reverse conversion mode between the two,and Cerna plays a key role in tumor development and other pathological processes.Therefore,it is of great significance to screen and identify gastric cancer-related Cerna molecules.This paper proposes to select the lnc-ARHGEF26-AS1/miR-373 route which is studied in this experiment,to verify whether there is differential expression of the target lncRNA in gastric cancer tissue/paracancerous tissue and gastric cancer cells/normal gastric mucosa cells,to explore the significance of target lncRNA expression in gastric cancer and its potential as a new molecular marker,and to verify the possible regulatory relationship between target lncRNA and target miRNA expression in gastric cancer cells,by download miRNA andRNA Seq from normal and gastric cancer tissues through TCGA database,screen the differential lncRNA and miRNA by bioinformation analysis,and then obtain the lncRNA/miRNA pathway map related to gastric cancer,and also by reading a lot of relevant literature.Method1.From March 2019 to August 2019,a total of 40 pairs specimens of gastric cancer tissues and their paracancerous tissue,which were fixed with formalin and embedded by paraffin,were collected from Ningbo clinical pathological diagnosis center for the study.And 1cm for each specimens were immediately immersed in-80℃RNA preservation solution for preservation and use.2.In this paper,it also designed and synthesized miRNA primer,U6 internal reference primer,si-ARHGEF26-AS1,mimics-miR-37,negative control group NC（negative control）and lnc-ARHGEF26-AS1 primer.3.Normal gastric mucosal cells（GES）,gastric cancer cell lines（BGC and SGC）and gastric cancer cell lines（BGC）transfected by NC,mimics-miR-373 and si-ARHGEF26-AS1 were cultured.5.FFPERNA kit was used to extract tissue totalRNA from formalin fixed paraffin embedded samples,and to extract cell totalRNA by using Trizol method.6.ComplementaryDNA（cDNA）was synthesized by methods of Go Script reverse transcription（RT）system,Hairpin-itTMmiRNA quantitative and U6 calibration kit.7.The cDNA was amplified by quantitative,reverse transcription polymerase reaction technology（qRT-PCR）,each sample was repeated three times to eliminate random errors,and finally,confirmed the QRT PCR product by sequencing.8.The expression level of lnc-ARHGEF26-AS1 in gastric cancer and its adjacent tissues,gastric cancer cell lines and corresponding normal gastric mucosa cells was detected,and the difference of expression level were analyzed by qRT-PCR.9.The expression level of lnc-ARHGEF26-AS1 was detected by qRT-PCR in the negative control group and gastric cancer cell line primed with mimics-miR-373,and then analyzed the difference of expression level.10.The expression of miR-373 in the negative control group and si-ARHGEF26-AS1transfected gastric cancer cell line was detected by qRT-PCR,and then analyzed the difference of expression level.11.The relationship between the expression level of lnc-ARHGEF26-AS1 and clinicopathological factors was analyzed and drew a figure by using Graph Pad Prism 8.0 software.12.The diagnostic value of lnc-ARHGEF26-AS1 was analyzed and evaluated by constructing the receiver operating characteristic（ROC）;Result1.Compared with paracancerous tissue,lnc-ARHGEF26-AS1 increased significantly in gastric cancer（P<0.0001）;the expression level of lnc-ARHGEF26-AS1 in SGC（P=0.0491）and BGC（P=0.0101）,which were significantly higher than that in normal GEC（P<0.0001）;2.In BGC,the expression of miR-373 increased（P=0.0013）after down-regulation of lnc-ARHGEF26-AS1,and the expression of lnc-ARHGEF26-AS1 decreased（P=0.025）after up-regulation of miR-373,which suggested that there was a reverse regulation relationship between lnc-ARHGEF26-AS1 and miR-373 in gastric cancer cells.3.The expression of lnc-ARHGEF26-AS1 was significantly correlated with age（P=0.0291）and depth of infiltration（P<0.0001）,which were obtained by the statistical analysis of clinicopathological data.4.The area under the ROC curve（AUC）was 0.64,which indicated that lnc-ARHGEF26-AS1had predictive value in the diagnosis of gastric cancer.ConclusionIt has been confirmed that the up-regulation of lnc-ARHGEF26-AS1 expression in gastric cancer may have certain significance in the growth and differentiation process of gastric cancer and the diagnosis and treatment of gastric cancer,and lnc-ARHGEF26-AS1 and miR-373 may constitute the lnc-ARHGEF26-AS1/miR-373 pathway in the role of ceRNA to participate in the development of gastric cancer cells. |
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