MicroRNA-19a-3p Regulates The Invasion And Migration Of Esophageal Squamous Cell Carcinoma Cells By Targeting CDH11

Part one:Correlation between miR-19a-3p,miR-17-3p,miR-18b-5p and lymph node metastasis of esophageal squamous cell carcinomaBackground and objectiveWith an improvement of people’s living standards and awareness of health,the incidence of esophageal cancer(EC)has declined,while the mortality rate remains high in recent years.Esophageal cancer is an aggressive and lethal malignancy,ranking seventh in terms of incidence and sixth in terms of mortality globally in 2020.Esophageal squamous cell carcinoma(ESCC)is the main histological subtype of esophageal cancer in China,accounting for nearly 90%of EC.Current therapies have limited efficacy due to local invasion and lymphatic metastasis.Tendency toward extensive regional lymph node metastasis(LNM)is an important clinical characteristic of ESCC and differs greatly between patients.Therefore,the search for appropriate molecular biomarkers to predict regional LNM of ESCC has become the focus at present.microRNAs(miRNAs)are not only involved in the invasion and metastasis of ESCC,but also associated with lymph node metastasis.We performed a microarray analysis of miRNA and quantitative reverse transcription polymerase chain reaction(qRT-PCR)based on LNM status to identify tumor-specific miRNAs closely associated with LNM in ESCC.Methods58 ESCC tumor samples were enrolled.Four pairs of ESCC tumor tissues with or without LNM were selected for microarray analysis to identify differentially expressed miRNAs,and another 50 samples were selected to verify the difference of the screened miRNAs expression with qRT-PCR.The relations between candidate miRNAs and clinicopathologic features were analyzed to verify their association with lymph node metastasis in ESCC.Receiver operating characteristic(ROC)curve was used to evaluate the ability of miRNA to predict lymph node metastasis in this study.Results1.According to the results of the comparative microarray analysis,62 miRNAs showed obvious differential expression(|fold change|>2.0,p-value<0.05)in ESCC samples with LNM compared to those without LNM.Among them,19 miRNAs were upregulated and 43 miRNAs were downregulated in ESCC samples with LNM compared to those without LNM.2.Of these,19 miRNAs were screened for qRT-PCR verification in 50 ESCC samples,and three miRNAs(miR-19a-3p,miR-17-3p,and miR-18b-5p)were significantly upregulated in ESCC samples with LNM compared to those without LNM.3.The three miRNAs were significantly correlated with TNM stage(p<0.01)and lymph node metastasis in 50 ESCC patients.4.ROC curves suggested that miR-19a-3p(AUC=0.694,p=0.018),miR-17-3p(AUC=0.730,p=0.005)and miR-18b-5p(AUC=0.778,p=0.001)may have predictive power for lymph node metastasis of ESCC.ConclusionIn summary,the present microarray analysis of miRNAs based on the LNM status of ESCC identified three tumor-specific miRNAs(miR-19a-3p,miR-17-3p,and miR-18b-5p)for the prediction of regional LNM in ESCC,which significantly correlated with regional lymph node metastasis of ESCC.Part two:Downregulation of miR-19a-3p inhibits esophageal squamous cell carcinoma cells invasion and migration by upregulating CDH11Background and objectivemicroRNAs regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression.miR-19a-3p plays a dual role in cancer,either acting as a cancer-promoting factor,or behaving as a tumor suppressor.To date,there are no published studies reporting in ESCC of miR-19a-3p,which has been widely reported in other cancers.Here,in vitro experiments were firstly performed to confirm the effect of miR-19a-3p on ESCC cells and to search for the underlying downstream target gene.MethodsAfter miRNA knockdown(KD)and negative control(NC)lentiviral vectors transfection into KYSE-150 and TE-1 cell lines,the migration capacity was detected by Celigo scratch assay,and the difference of miRNA expression was detected by qRT-PCR and western blot(WB)analysis.CCK-8 was used to investigate cell proliferation,and Transwell assay was utilized to analyze ESCC cells invasion and migration.Bioinformatics method was combined with qRTPCR to predict target messenger RNAs(mRNAs)for miR-19a-3p.The dual luciferase reporter gene system was used to detect whether miR-19a-3p could directly target the 3’-untranslated region(3’UTR)of cadherin 11(CDH11)mRNA.The overexpression(OE)of CDH11 mRNA and protein was analyzed after CDH11 OE/NC transfection,and cell invasion and migration were detected by Transwell assay.Finally,the difference of CDH11 expression was detected after CDH11 siRNA/si-NC and miR-19a-3p KD/NC cotransfection into ESCC cells,and cell invasion and migration were assessed.In vitro experiments were performed in triplicate.Results1.The migration ability was significantly reduced after LV-miR-19a-3p-inhibition transfection into KYSE-150(p<0.001)and TE-1 cells(p<0.001),consequently,miR-19a-3p was selected as the target miRNA for subsequent experiments.2.The miR-19a-3p expression was significantly decreased in KD group compared with NC group after transfection(p<0.001),and its downregulation suppressed the invasion and migration but not proliferation of ESCC cells in vitro.3.The expression of CDH11 mRNA(p<0.001)and protein(p<0.01)was significantly upregulated in KD group compared with NC group after transfection.4.The dual luciferase reporter assay confirmed that 3’UTR of CDH11 mRNA was a target of miR-19a-3p,p<0.01.5.Overexpression of CDH11 could significantly upregulate the expression of CDH11 mRNA and protein(p<0.001)in ESCC cells,and suppress the invasion and migration of KYSE-150(p<0.01)and TE-1 cells(p<0.001).6.Compared with KD group,CDH11 expression level was significantly decreased in KD+siRNA group(p<0.001),the invasion and migration was significantly increased(p<0.001).And KD+siRNA group was not significantly different from NC group(p>0.05).ConclusionsInhibition of miR-19a-3p could suppress ESCC cells invasion and migration.CDH11 was a possible downstream target gene of miR-19a-3p.CDH11 siRNA weakened the effect of miR19a-3p inhibition on cell invasion and migration.We firstly reported that miR-19a-3p at least partially regulates ESCC cells invasion and migration via CDH11,which merits further investigation.