| BackgroundEsophageal squamous cell carcinoma(ESCC)is a malignant tumor of the digestive tract and the sixth most common cause of cancer death worldwide.The most common pathological classification of esophageal cancer in China is esophageal squamous cell carcinoma(ESCC),which accounts for half of the global population of esophageal squamous cell carcinoma patients.ESCC has a relatively high incidence rate and mortality.The early symptoms of ESCC are not obvious.Most patients are in the late stage when they are diagnosed,and the five-year survival rate is only about 20%.Therefore,exploring the pathogenesis and development of esophageal squamous cell carcinoma,seeking new specific biomarkers,is particularly important for early diagnosis,individualized treatment,and prognosis prediction of ESCC patients.MicroRNAs(miRNAs)are a highly conserved group of endogenous non coding small RNAs widely present in eukaryotes,with tissue specificity and involvement in regulating various pathological and physiological processes in the human body.Research has shown that miRNAs are involved in the occurrence and development of various tumors,playing a promoting or inhibiting role in the invasion,metastasis,and angiogenesis of cancer cells.Previous studies have shown low expression of miR-149 in esophageal squamous cell carcinoma tissue,but the role and mechanism of miR-149-3p in esophageal squamous cell carcinoma tissue are still unclear.Apelin is the only known endogenous ligand of APJ receptor and a member of G protein coupled receptor family.Apelin and APJ are widely distributed in the body.Studies have found that Apelin is overexpressed in tumor tissue and can promote tumor cell proliferation,metastasis,and angiogenesis,playing an important role in the occurrence and development of tumors.Therefore,Apelin is expected to serve as a biomarker for early diagnosis,treatment,and prognosis monitoring of cancer.However,the role and mechanism of Apelin in the occurrence and development of esophageal squamous cell carcinoma are not fully understood.ObjectiveThis study combines clinical esophageal squamous cell carcinoma tissue samples and esophageal squamous cell carcinoma ECA109 and ECA9706 cell lines to explore the mechanism of miR-149-3p in ESCC cell proliferation,apoptosis,metastasis,and invasion,and clarify whether Apelin is the target of miR-149-3p.To provide new theoretical basis for studying the etiology and development process of esophageal squamous cell carcinoma,and to provide new research directions and treatment targets for its clinical prevention and treatment.Methods1.Detect the expression of miR-149-3p and Apelin in esophageal squamous cell carcinoma tissue and adjacent tissues.Taking tissue samples from patients with squamous cell carcinoma of the feeding tube,the expression levels of miR-149-3p and Apelin in cancer and adjacent tissues were detected by qRT PCR and immunohistochemistry.2.Use miRNA bioinformatics technology and double Luciferase reporter gene to detect whether Apelin is the target gene of miR-149-3p.Transfecting NC and miR-149-3p mimetics into ECA109 and ECA9706 cells,qRT-PCR,Western blot,and immunofluorescence were used to detect the expression levels of miR-149-3p,Apelin mRNA,and protein.3.ECA109 and ECA9706 cells were transfected with NC and miR-149-3p mimetics,and the cell cycle arrest of esophageal squamous cell carcinoma was detected by cell flow cytometry.Colony forming ability of esophageal squamous cell carcinoma cell lines was detected by colony cloning assay.Immunofluorescence,qRT PCR,and Western blot were used to detect the expression levels of PCNA,Cyclin D1 mRNA,and protein.4.ECA109 and ECA9706 cells were transfected with NC and miR-149-3p simulants,and the migration and invasion ability of esophageal squamous cell carcinoma cell migration were detected by scratch test and Transwell cell invasion test.Phalloidin staining was used to observe the changes of cytoskeleton microtubule filaments and cell morphology.Western blot and qRT PCR were used to detect the expression levels of MMP9 protein and mRNA associated with invasion of esophageal squamous cell carcinoma.5.Detection of apoptotic protein related expression by flow cytometry and Western blot.Observe cell apoptosis using Tunel staining under an inverted fluorescence microscope.6.In vivo experiment: After transfecting esophageal squamous cell carcinoma cell line ECA109 with NC and miR-149-3p mimetics,tumor growth was observed in the armpit of nude mice.Western blot and qRT PCR were used to detect the expression of tumor Apelin,MMP9,PCNA mRNA and related proteins.Results1.The immunohistochemical staining results of human esophageal squamous cell carcinoma tissue samples showed that compared with adjacent tissues,there was an increase in brown particle deposition and a significant increase in Apelin expression in esophageal squamous cell carcinoma tissue.The qRT PCR results showed that the expression level of Apelin mRNA in esophageal squamous cell carcinoma tissue was significantly increased compared to adjacent tissues,indicating high expression of Apelin in tumor tissue.2.(1)The results of double Luciferase reporter gene experiment showed that miR-149-3p could regulate the wild type Apelin 3 ’-UTR region and inhibit the transcription of Apelin compared with the mutant group.(2)After 72 hours of transfection of miR-149-3p mimetics and NC in esophageal squamous cell carcinoma cell lines ECA109 and ECA9706,qRT-PCR and Western blot results showed that compared with the control group,the expression level of miR-149-3p mRNA in the transfected miR-149-3p mimetics ECA9706 group was significantly increased by about 30 times,while the expression level of Apelin mRNA and protein in the ECA109 miR-149-3p mimetics group was significantly increased by about 400 times.The immunofluorescence results showed that compared with the control group,the Apelin fluorescence signal of cells became weaker after transfection with miR-149-3p mimetic.These results suggest a negative regulatory effect between miR-149-3p and Apelin.3.The results of colony cloning experiment showed that compared with the control group,after overexpression of miR-149-3p mimic,the number of colony clones was reduced,the cell cycle was blocked in G0/G1 phase,and the proliferation and replication ability of cells became weaker.The results of qRT-PCR and Western blot showed that compared with the control group,transfection with miR-149-3p simulant significantly reduced the expression levels of PCNA,Cyclin D1,Ki67 mRNA and protein,weakened cellular immunofluorescence signals,and decreased protein expression.These results suggest that miR-149-3p can inhibit the proliferation of esophageal squamous cell carcinoma.4.Exploring whether overexpression of miR-149-3p inhibits the migration and invasion of esophageal squamous cell carcinoma cells.The results of cell scratch test showed that compared with the control group,the migration ability of cells to the middle was significantly weakened after transfection of miR-149-3p simulant,the number of cells passing through the matrix glue of Transwell chamber was reduced,phalloidin staining showed that the cytoskeleton was smaller,the distribution of microtubules and microfilaments of cells was reduced,and the fluorescence signal was weakened.The results of qRT PCR,Western blot,and immunofluorescence showed that compared with the control group,the transfection of miR-149-3p mimetics resulted in a decrease in MMP9 mRNA and protein levels,and a weakened fluorescence signal.These results suggest that overexpression of miR-149-3p inhibits the migration and invasion of esophageal squamous cell carcinoma cells.5.In esophageal squamous cell carcinoma cell lines ECA109 and ECA9706,cell apoptosis was detected by flow cytometry after transfection with miR-149-3p mimetics and NC.The results showed that compared with the control group,the group transfected with miR-149-3p mimetics showed an increase in early and late cell apoptosis.Western blot experiments showed a significant increase in the expression of cleared caspase-3 and cleared caspase-8 proteins,as well as a significant increase in the fluorescence intensity and quantity of cell Tunel staining,These results suggest that transfection of miR-149-3p mimetics promotes apoptosis in esophageal squamous cell carcinoma cells.6.In vivo experimental results showed that compared with the control group,the tumor volume of cells transfected with miR-149-3p simulant was significantly smaller.qRT-PCR results showed a decrease in Ki67,Cyclin D1,MMP9,PCNA mRNA and expression levels.Western blot and immunohistochemical staining showed that compared with the control group,the protein levels of Apelin,PCNA,and MMP9 in the transfected miR-149-3p simulant group decreased,and the deposition of brown particles in Apelin,Cyclin D1,and Ki67 was significantly reduced.These results suggest that cells pretreated with miR-149-3p can inhibit tumor growth in vivo.Conclusion1.In vitro,express miR-149-3p inhibits esophageal cancer cell proliferation,migration and invasion,promote cell apoptosis.2.In vivo,overexpression of miR-149-3p inhibits tumor growth in esophageal squamous cell carcinoma cells.3.The expression of miR-149-3p and Apelin is negatively correlated,and miR-149-3p can directly target Apelin,providing a theoretical basis for clinical treatment of esophageal squamous cell carcinoma. |
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