The Role Of MiR-483 In The Development Of Esophageal Cancer

Background Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer-related mortality. Now, there are more than 450,000 people worldwide and the incidence is rapidly increasing. The earlier the esophageal cancer patients are diagnosed, the better outcomes they will get. However, most of the patients are diagnosed at the advanced stage for lack of effective means of early diagnosis. The overall 5-year survival of esophageal cancer patients ranges from 15% to 25%. So, it is necessary to explore applicable molecular markers and targets of esophageal cancer, so as to provide clues for the early diagnosis and treatment of esophageal cancer. Micro RNAs(mi RNAs), a class of small, endogenic and evolutionarily conserved, single-stranded non-coding RNAs of 18–25 nucleotides in length, mainly affect the body’s a variety of physiological and pathological processes by inhibiting the translation of target genes. Recently, by detecting the differentially expressed molecules in 25 cases of esophageal squamous cell cancer(ESCC) tissues and the corresponding adjacent tissues through mi RNA chip technology, we have firstly found and reported an esophageal cancerrelated mi RNAs group. Among them, mi R-483 is upregulated in esophageal cancer tissues. However, the impacts of mi R-483 on a variety of malignant phenotypes of esophageal cancer and the molecular mechanism has not been elucidated. Here we will start our research on these problems.Aims 1. To detect the expression of mi R-483-3p in ESCC cell lines and the normal esophageal squamous epithelium cell line. 2. To construct the esophageal cancer cell lines which can upregulate or downregulate the expression of mi R-483-3p, and study the effects of mi R-483-3p on the biological behaviors of esophageal cancer cells through in vivo and in vitro experiments. 3. To study the molecular mechanism of mi R-483-3p regulating the development of esophageal cancer.Methods 1. Detecting the expression of mi R-483-3p in ESCC cell lines and the normal esophageal squamous epithelium cell line by quantitative real-time PCR. 2. Establishing the esophageal cancer cell models overexpressing mi R-483-3p and the control cell models by transfecting cells with mi R-483-3p lentivirus vector and the control lentivirus vector. 3. Establishing the esophageal cancer cell models with decreasd expression of mi R-483-3p and the control cell models by transfecting cells with mi R-483-3p inhibitor and inhibitor NC. 4. Studying the role of mi R-483-3p in regulating the proliferation of esophageal cancer cell by MTT experiment. 5. Observing the role of mi R-483-3p in regulating the migration of esophageal cancer cell by transwell experiment. 6. Detecting the role of mi R-483-3p in regulating the cell cycle of esophageal cancer cell by flow cytometry. 7. Detecting the role of mi R-483-3p in regulating the cell apoptosis of esophageal cancer cell by flow cytometry.8. Studying the role of mi R-483-3p in regulating the drug sensitivity of esophageal cancer cell by MTT experiment. 9. Screening target genes of mi R-483-3p through united forecast of bioinformatics and whole-genome expression profile chip. 10. Identifing the target genes of mi R-483-3p by q PCR, Western blot and dual- luciferase reporter gene assay.Results 1. mi R-483-3p is overexpressed in ESCC cell lines EC109, EC9706 and TE-1 as compared with the normal esophageal squamous epithelium cell line Het-1A. 2. We have successfully established the esophageal cancer cell models with increased or decreased expression of mi R-483-3p. 3. Upregulation of mi R-483-3p in esophageal cancer cells leads to increased cell proliferation and migration, promoted transformation of cell cycle from G1 phase to G2 phase, decreased ADR induced cell apoptosis, and reduced cell sensitivity to chemotherapy drugs. 4. Downregulation of mi R-483-3p in esophageal cancer cells results in decreased cell proliferation and migration, G1 arrest, increased ADR induced cell apoptosis, and enhanced cells sensitivity to chemotherapy drugs. 5. Seven potential mi R-483-3p target genes(EI24, KLHL12, GRSF1, MKRN1, LEPROTL1, CREBL2 and MAPKAPK2) are screened through united forecast of bioinformatics and whole-genome expression profile chip. 6. Upregulation of mi R-483-3p in esophageal cancer cells doesn’t influence the m RNA level of the seven target genes. 7. Upregulation of mi R-483-3p in esophageal cancer cells inhibits the protein level of the tumor suppressor EI24.Conclusion 1. The expression of mi R-483-3p was higher in ESCC cell lines as compared with the normal esophageal squamous epithelium cell line.2. Upregulation of mi R-483-3p can promote proliferation and migration of esophageal cancer cells by regulating cell cycle, and can increase the drug resistance of cancer cells. And vice versa. 3. mi R-483-3p may play an important role in the development of esophageal cancer by regulating EI24.