Role Of MiR-210 In The Development Of Esophageal Squamous Cell Carcinoma

[Background]Esophageal cancer(EC)ranks seventh in terms of incidence and sixth in cancer deaths worldwide.Currently,there are more than 570,000 patients with EC in the world,and the incidence rate is still increasing year by year.Due to the lack of effective early diagnostic approaches and therapy methods,the five-year survival rate of EC patients is extremely low.Therefore,discovering new diagnostic biomarkers,establishing effective treatment strategies,and improving patients' quality of life are urgently needed.microRNAs(miRNAs)are a group of small non-coding RNA(18-25 nucleotides in length),which function as negative gene regulators at the post-transcriptional level via the inhibition of RNA translation or degradation of target mRNAs.miRNAs are widely participated in the physiological and pathological processes of cells.Previously,our team detected the expression levels of miR-210 in 25 esophageal squamous cell carcinoma(ESCC)tissues and paired adjacent normal tissues,and we found that miR-210 was significantly downregulated in ESCC tissues.Moreover,patients with lower expression of miR-210 had poorer prognosis.However,the role and mechanisms of miR-210 in ESCC have not been elucidated.The current study aims to explore the key functions and regulatory mechanisms of miR-210 in ESCC,and finally provide in-depth understanding of miR-210 for cancer research and management.[Aims] 1.To detect the expression level of miR-210 in ESCC cells and tissues.2.To determine the roles of miR-210 in regulating the malignant biological characteristics of ESCC.3.To explore the underlying mechanisms of miR-210 in ESCC development.[Methods] 1.qPCR was used to detect the expression levels of miR-210 in ESCC cell lines and one esophageal epithelial cell line.Samples from ESCC tissues and matched adjacent normal tissues were also collected and used for detecting miR-210 expression by qPCR.2.miR-210 overexpression and inhibition lentiviral vectors were constructed,and miR-210 mimic and inhibitor were also synthesized.Then,ESCC cell models with increased or decreased miR-210 expression were established either by stable infection or by transient transfection.3.The roles of miR-210 in ESCC proliferation,metastasis and apoptosis were studied using the CCK8 assay,colony formation assay,flow cytometry,Transwell migration and invasion assay,nude mice xenograft assay and tail vein metastatic assay.4.The target genes of miR-210 were screened through the combination of bioinformatics analysis and microarrays.Then,the target genes of miR-210 were verified by qPCR,Western Blot and dual-luciferase reporter gene assay.5.The molecular mechanisms of miR-210 in regulating the metastasis of ESCC were also explored.[Results] 1.Compared with the esophageal epithelial cell line Het-1A,miR-210 was significantly downregulated in ESCC cell lines.Similarly,compared with adjacent normal tissues,miR-210 was also decreased in ESCC tissues.2.miR-210 expression was successfully modulated either by stable transfection using lentiviral infection or by transient transfection using miR-210 mimics and inhibitors.CCK8 assay and colony formation assay revealed that miR-210 inhibited the in vitro growth and proliferation of ESCC.Cell cycle analysis by flow cytometry revealed that miR-210 inhibited cell cycle progression in ESCC cells.Cell apoptosis detection by flow cytometry also revealed that miR-210 could promote the chemotherapy-induced apoptosis in ESCC cells.Transwell migration and invasion assays indicated that miR-210 inhibited the migration and invasion of ESCC cells.Nude mice xenograft assay revealed that miR-210 could inhibit the in vivo growth of ESCC cells.Tail vein metastatic assay demonstrated that miR-210 had the capacity to inhibit ESCC cells from forming metastatic sites in the liver and lung.3.ATG7 was identified as a direct and functional target of miR-210 via microarrays,bioinformatics analysis,qPCR,Western Blot,dual-luciferase reporter gene assay and other methods.4.Functional and molecular experiments confirmed that miR-210 could inhibit autophagy by downregulating ATG7.Through rescue experiments,it has been found that miR-210 could inhibit the invasion and metastasis of ESCC through ATG7/Wnt/?-catenin signaling pathway.[Conclusion]This study confirmed that miR-210 was downregulated in ESCC cells and tissues,and revealed that decreased miR-210 expression might be related to the occurrence and development of ESCC.Functional studies indicated that miR-210 could inhibit cell proliferation,cell migration and metastasis.Moreover,miR-210 could promote chemotherapy-induced apoptosis in ESCC.Importantly,we also found that miR-210 could regulate autophagy in ESCC,and our results suggested that miR-210 could inhibit autophagy by downregulating ATG7 expression.Finally,our study proposed and demonstrated that miR-210 could inhibit the invasion and metastasis of ESCC through the ATG7/Wnt/?-catenin signaling pathway.This study provides a basis for understanding the role of miR-210 in regulating the malignant phenotypes of ESCC.Thus,miR-210 is expected to be a novel target for ESCC treatment.