| Background:Esophageal cancer is one of the deadliest types of cancer in the digestive system.It is one of the common human tumors.It is also a common malignant digestive system tumor.About 300 thousand people worldwide are killed by esophageal cancer every year.The incidence and mortality of esophageal cancer in China are the highest.More than 90%of the pathological types of esophageal cancer are squamous and adenocarcinoma.In rare cases,the esophagus may also have other cancers such as melanoma,leiomyosarcoma and lymphoma.Esophageal squamous cell carcinoma is common in East Asia and Central Asia.It is derived from dysplasia squamous cells.It usually occurs on the top 2/3 of the esophagus.Adenocarcinoma of the esophagus occurs mainly in the distal esophagus.Continuous gastroesophageal reflux esophagitis can transform the squamous epithelium into a columnar intestinal epithelium and then develop into a esophageal adenocarcinoma by a low or high level of dysplasia.H19 is the first lncRNA discovered by human beings.It is abnormal expression in many cancers.LncRNA H19,a long chain,2.3 KB long,non coded RNA gene,is located on the human chromosome 11p15.5,including 5 exons and 4 introns,and is one of the earliest identified imprinted genes.H19 has the paternal imprinting characteristics and selectively expresses the parent allele,which is mainly regulated by methylation of CpG Island rich in the methylation area at the 4000 bp upstream of its upstream.In recent years,a large number of studies have found that H19 has abnormal expression in many kinds of tumors.It has been widely involved in the process of tumor cell differentiation,angiogenesis and invasion and metastasis.It plays a vital role in the development of tumor.Therefore,to understand the biological functions of H19 in different tumors,and to provide a theoretical basis for the early diagnosis of H19,the target of gene therapy and the prognosis of the tumor.H19 is overexpressed in malignant tumors,such as esophageal cancer,gastric cancer,lung cancer,pancreatic cancer,breast cancer,ovarian cancer and so on,showing the function of oncogene.The main functions are accelerating cell growth and deterioration,enhancing metastasis and enhancing the function of oncogenes.MicroRNAs(miRNAs)is a non-coded small RNA with a length of about 20-24 NT,which regulates the expression of polar shadows in many tumors and can affect the stability and translation of mRNA.H19 was also used as a precursor of miR-675 by Cai and X.et al.Experimental studies confirmed that the experimental study showed that miR-675 was formed by H19 in the cell by shear,and that many experiments also proved that miR-675 was expressed in the tumor,for example,in cancer cells and tissues such as gastric cancer,liver cancer and other cancers,the expression of miR-675 was similar.Normal cells or tissues are up-regulated.Objective:To investigate the expression of H19/miR-675-3p in the tissues and cells of esophageal cancer,and to compare the expression of H19/miR-675-3p with the normal esophageal tissue in the same condition,and to analyze the expression of esophageal cancer tissue with the clinical data of the patients,and to determine whether or not it can provide guidance for the clinical practice of tube cancer;The physiological activity of esophageal cancer cell tumor was tested by cell biological activity,and the effect of H19/miR-675-3p on the physiological activity of cancer cells in esophageal cancer cells was discussed.The cycle detection of esophageal cancer cells,or H19/miR-675-3p interfered cells by flow cytometry and predicting the corresponding microRN related to the cycle were predicted.The A target is validated by experiments,and the influence of H19/miR-675-3p on the cell cycle of esophageal cancer and the possible mechanism are discussed.Method and materials:The expression of H19 and miR-675-3p in the tissue specimens of patients with esophageal cancer was measured by fluorescence quantitative PCR.The results were calibrated by beta-actin,and the data obtained were related to the pathological information of the patients.Through cell transfection,H19siRNA and miRNA inhibition were transferred to the esophageal cancer cells to reduce the level of H19/miR-675-3p expression in esophageal cancer cells,and then detected by the CCK-8 test enzyme scale.The expression level of H19/miR-675 in esophageal cancer was down regulated,and the adherent cells were marked by scratch test and in 0h.12h,24h took a photo record;the Transwell experiment made glue or non-paving treatment on the chamber.Then,the number of cells transfected well and the H19/miR-675-3p cells were assigned to the chamber.After 24h,the cells on the membrane were fixed and stained,and the results were analyzed;the Elisa experiment and the protein were analyzed.Western blot assay was used to detect the expression of proteins that affect the migration and invasion ability of cells,and to identify the primary mechanisms that affect the migration and invasion of esophageal cancer cells.The effect of H19/miR-675-3p on the cell cycle of esophageal cancer was detected by flow cytometry.The study will turn the esophageal cancer cells with the expression level of H19 and miR-675 down,and after PI staining,the cell cycle of miR-675-3p gene is detected by flow cytometry.The target was used to predict the results.The recombinant plasmid,H19si and miR-675 inhibition were cotransfected to Kyse-150 and Te-1 of the esophageal cancer cells.Then the cells were incubated with the drug for 48 h,and the fluorescence intensity was detected by the multi-function enzyme scale to determine fluorescein.Enzyme activity,which clearly predicts whether the target is a direct target for miR-675-3p,thus clarifying the possible mechanism that affects the cycle characteristics of esophageal cancer cells through H19/miR-675-3p Axis.Results:We identified that H19 and miR-675-3p were over-expressed in ESCC tissues compared with the normal tissues,and also has a highly expression level in ESCC cells compared with the HEEC.The results showed that expression of H19 and miR-675-3p was significantly higher in cancer tissues than normal tissues(P<0.001).A significant correlation was observed between H19 and miR-675-3p expression in ESCC tissues(R=0.5086,P<0.001).The results of scratch and transwell assays revealed a predominant up-regulation of cell migration and invasion ability in high expression H19 and miR-675-3p.We have demonstrated that H19 and miR-675-3p promoted ESCC cell proliferation,migration and invasion ability.In addition,the effects of H19 siRNA and miR-675-3p inhibiton on ESCC cell lines were eliminated by H19 siRNA or miR-675-3p inhibitor and miR-675-3p.mimic co-transfection.The results indicated that H19/miR-675-3p involved in the progression of ESCC through regulating ESCC cell migration and invasion capacity via modulating MMP2 and MMP9.Transfection with H19 siRNA and miR-675-3p inhibition induced G2 arrest.After target prediction in Targetscan,luciferase assays and western blots further verified CDK13 was a direct target of miR-675-3p. |
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