| Esophageal cancer(EC)is one of the most common malignant upper gastrointestinal tract tumors.According to the global cancer statistics in 2020,there were 604,000 new cases of esophageal cancer around the world,accounting for 3.1%of all malignant tumor incidences,ranking eighth while there were 544,000 new deaths of esophageal cancer,accounting for 5.5%of all malignant tumors death,ranking sixth.If the incidence rate is stable,it is estimated that there will be 957,000 new cases and 880,000 deaths in 2040.China has always been a country with high incidence of esophageal cancer,the incidence and death of esophageal cancer overcome 50%in the world.The EC seriously threatens our people’s health.Because the invasion and metastasisof EC is strong,coupled with the absence of typical symptoms in the early stage and of effective early tumor markers,many patients are diagnosed at advanced stages with poor prognosis.Although the treatments and diagnosis methods are constantly improving,the therapeutic effect is still unsatisfactory.The fundamental reason is that the pathogenesis of EC is complex and uncertain.MicroRNAs(miRNAs)are a class of highly conserved,endogenous singlestranded non-coding small molecule RNAs with about 21-25 nucleotides(nt)in length,which can regulate the expression of genes.Studies have proved that miRNA is closely related to the occurrence,progression and prognosis of tumors,and can be used as the target for disease treatment or a marker related to diagnosis and prognosis.Therefore,studying the pathogenesis of miRNA in EC and its influence on prognosis plays an important role in early diagnosis,advanced treatment and improvement of prognosis.Objective:In this study,machine learning algorithm was used to screen miRNA with independent prognostic value in public databases,that can be used as molecular markers for predicting the prognosis of EC.In order to provide scientific basis for the pathogenesis and clinical diagnosis of EC,the biological effect of this miRNA and its related mRNA on the development of EC cells was explored by vitro experiments.Method:1.The common differentially expressed miRNAs in EC from GSE97051,GSE67269 and TCGA databases were screened by biological information analysis,and the different expression of these miRNAs were verified in GSE122497 data set.The prognosis-related miRNAs were obtained by Lasso regression algorithm,and the miRNAs with independent prognostic value were analysis by Cox proportional hazard regression,and the target miRNA(miR-378c)was determined according to the accuracy in predicting the survival probability of EC.Cox proportional hazard regression was used to ensure the independent prognostic value of miR-378c when it combined with the demographic characteristics,clinical characteristics.2.RT-qPCR method(fluorescence quantitative reverse transcription-polymerase chain reaction)was used to detect the expression of target miRNA in human EC cells and esophageal epithelial cells(HEsEpiC),as well as in serum of patients with EC(83 cases)and control group without tumor(83 cases),to verify their differential expression and analyze the relationship between the expression of miR-378c and clinical features in EC.3.The effect of miR-378c on the proliferation of EC cells was verified by CCK-8 and cell clone formation experiments.Flow cytometry was used to verify its effect on apoptosis of EC cells.4.Prediction,screening and verification of mRNA related to miR-378c.The possible related mRNAs to miR-378c were preliminarily predicted by bioinformatics analysis.Based on the information of TCGA database,the expression of each mRNA in EC were analyzed,and the correlation between mRNA and miRNA378c was analyzed as well.RT-qPCR was used to observe the expression of SKP2 in patients’ serum,then the correlation analysis was used to verify the relationship of SKP2 and miRNA-378.RT-qPCR was used to detect the expression of SKP2 in EC cells and normal esophageal epithelial cells.5.EC cells were transfected with miR-378c mimics and miR-378c mimics+SKP2 overexpression plasmids.The effect of overexpression of miR-378c and SKP2 on the biological function of EC cells were verified by cell proliferation and apoptosis experiments.6.The relationship between the expression of SKP2 in EC and its clinical features as well as prognosis value were analyzed.Results:1.Screening the study miRNA of EC.There were 29 common differentially expressed miRNAs(17 down-regulated genes and 12 up-regulated genes)in GSE97051,GSE67269 and TCGA databases,and the expression of the above genes was verified in GSE122497 data set.18 miRNAs have the prognosis value were screened by Lasso regression algorithm.According to Cox proportional hazard regression,miR-378c,miR-26b-5p,miR-99a-5p,miR-143-5p and miR-935 had independent prognostic value,among pf them,miR-378c predicting the 1 year and 3 years overall survival probability of EC most precisely,therefore,it was selected as the study gene.The clinical independent prognostic value of miR-378c was analyzed by Cox proportional hazard regression which showed that ICD-10,pathological T-stage and miRNA-378c could be independent prognostic factors of EC,and miRNA-378c was still an independent prognostic risk factor when combining analysis with clinical indicators.2.Differential expression of miR-378c at ECThe expression of miR-378c in serum of EC patients was lower than that in control group without tumor(P<0.05).miR-378c was poorly expressed in EC cells(TE-1 and KYSE150)(P<0.05).The EC patient with lower expression of miR-378c would have shorter survival time.3.The effect of miR-378c on EC cells.After overexpression and silencing of miRNA-378c in TE-1 and KYSE150 cells,CCK-8 experiment and cell clone formation experiment showed that compared with the control group,the proliferation and clone ability of TE-1 and KYSE150 cells which overexpressed miRNA-378c were significantly reduced(P<0.01),and the proliferation and clone ability of EC cells were showed the opposite results after silencing the expression of miRNA-378c.The results of flow cytometry showed that the apoptosis rate of up-regulated miRNA-378c cells was significantly higher than that of the control group(P<0.01),and the apoptosis rate of silent miRNA-378c cells was significantly lower than that of the control group(P<0.01).4.Prediction,screening and verification of miR-378c related genes.Different databases were used to predict the related genes of miR-378c.The common ones were SKP2,PIM2,MAPK1 and SOBP.The gene expression analysis in TCGA database showed that there was no significant difference in the expression of MAPK1 and PIM2 between EC and adj acent tissues(P>0.05),while SOBP was lower in cancer tissues(P<0.05)and SKP2 was higher in cancer tissues(P<0.05).Spearman analysis was used to analyze the correlation between the expression of miRNA-378c and SKP2 in cancer tissues,the results indicated that SKP2 was negatively correlated with the expression of miRNA378c(r=-0.295,P<0.001).The expression of SKP2 in serum of patients with EC was verified by RT-qPCR.The results showed that SKP2 was highly expressed in serum of patients(P<0.05),and its expression in serum was negatively correlated with miRNA-378c(r=-0.674,P<0.001).RT-qPCR method showed that the expression of SKP2 in esophageal cancer cells was higher than that in esophageal epithelial cells(P<0.05).The expression of SKP2 in EC cells was down-regulated after overexpression of miR-378c,and up-regulated after silent expression of miR-378c(P<0.05).These results suggest that SKP2 might be the related gene of miR-378c.5.Study of the relationship between miR-378c and SKP2The expression of SKP2 in EC was higher(P<0.05)than that in control group.Spearman analysis showed that the expression of SKP2 and miR-378c were negatively correlated(r=-0.674,P<0.001).Overexpression of SKP2 could reverse effect of miR378c promoting the apoptosis and proliferation and inhibiting the proliferation.6.The relationship between SKP2 and clinical features or prognosis valueThe expression of SKP2 was differently expressed in age group,pathological Grade and pathological T stage.In addition,the survival prognosis of patients with high expression of SKP2 was poor.Conclusions:1.The miR-378c was significantly down-regulated in EC.EC patients with downregulated miR-378c had poor prognosis.miR-378c can be a molecular marker to predict the prognosis for esophageal cancer.2.miR-378c inhibited the proliferation and induced apoptosis in EC cells.miR378c might act the role as tumor suppressor in EC.3.The expression of miR-378c and SKP2 was negatively correlated.SKP2 might involve in the regulation of miR-378c on EC cells.4.SKP2 was highly expressed in EC.Patients with higher expression of SKP2 had shorter survival time,indicating that SKP2 might be a sign of poor prognosis for EC patients. |
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