The Molecular Mechanisms And Biological Function Research Of Micro RNAs Regulating PLK1、BMI1 And DAB2 Gene In Thoracic Cancers

Objective:Lung cancer and esophageal cancer are becoming a hot spot of thoracic cancer research. It has been high incidence in our country, especially the incidence rate of lung cancer has increased dramatically in recent years, including non-small cell lung cancer(NSCLC) accounts for more than 85%. Esophageal cancer is a primary esophageal malignant tumor, mainly composed of esophageal squamous cell carcinomas(ESCC). Early symptoms of lung cancer and esophageal cancer are not obvious, most of patients were in median or advanced stage when they were diagnosed. So the lack of early diagnostic markers is a major cause of the high mortality of lung cancer and esophageal cancer.Polo-like kinase1(PLK1) is a kind of cell cycle cytokines which play an important role in the process of mitosis. BMI1 as a key oncogene belongs to comb gene family which participates in cell self-renewal and cell proliferation, and has an important role in regulates epithelial mesenchymal transformation and promotes tumor metastasis. DAB2 protein is a widely expressed endocytosis adapters. It plays an important role in a variety of cellular signal transduction. DAB2 is found down-regulated or depleted in various types of cancers which plays an important role in cancers occurrence and development.Micro RNAs(mi RNAs) are a kind of short noncoding RNAs and regulate gene expression at post-transcription levels. Many studies have showed that mi RNAs play an important role in tumor occurrence and development of NSCLC and ESCC. This research aims to explore the mechanism that mircro RNAs regulated PLK1,BMI1 and DAB2 in thoracic cancer and provide effective basis for the early diagnosis and treatment of thoracic cancer.Methods:Part One. mi R-296-5p directly targets PLK1 and inhibits cell proliferation in NSCLC.1. Using bioinformatics to predict and verify the micro RNAs that directly regulated PLK1.Using bioinformatics software such as Target Scan, Pic Tar and mir Base to predicte the mirco RNAs that directly regulated PLK1. Mi R-296-5p mimics was synthesized and transiently transfected in A549 cells. Western blot was used to detect the PLK1 protein expression after transfection. Using luciferase assay to verify whether mi R-296-5p directly targeted PLK1-3’UTR.2. mi R-296-5p influence biological functions of NSCLC cells through targeting PLK1.PLK1 m RNA and mi R-296-5p relative expression were detected by using real-time quantitative PCR(q PCR) in NSCLC tissues and adjacent non-tumor tissues. PLK1 protein expression were measured by Western blot in HBE cells and NSCLC cells(A549, 95 C, 95 D, LTEP-α-2 and H1299 cells) and mi R-296-5p relative expression was measured by q PCR. NSCLC cells were transfected by mi R-296-5p mimics and PLK1-si RNA respectively, and then its proliferation was detected using CCK-8 and Ed Uassays.Part Two. mi R-203 directly targets BMI1 and inhibits cell proliferation in NSCLC.1. Using bioinformatics to predict and verify the micro RNAs that directly regulated BMI1.Using bioinformatics software such as Target Scan and Pic Tar and mir Base to predicte the mirco RNAs that directly regulated BMI1. Mi R-203 mimics was synthesized and transiently transfected in A549 and LTEP-α-2 cells. BMI1 protein expression was detected by Western blot. Using luciferase assay to verify whether mi R-203 directly targeted BMI1-3’UTR.2. mi R-203 influences biological functions of NSCLC cells through targeting BMI1.BMI1 m RNA and mi R-203 relative expression were detected by using q PCR in NSCLC tissues and adjacent non-tumor tissues. BMI1 protein expression was measured by Western blot in HBE cells and NSCLC cells(A549, H1650, H226, H460 and LTEP-α-2 cells). NSCLC cells were transfected with mi R-203 mimics and BMI1-si RNA respectively and its proliferation were detected using CCK-8 and Ed U assays. NSCLC cells were transfected by mi R-203 mimics and BMI1-si RNA respectively, and the invasion effects were measured by using transwell assays. NSCLC cells were transfected by mi R-296-5p mimics and BMI1-si RNA respectively, and the apoptosis effects were measured by using flow cytometric analysis.Part Three. mi R-93 directly targets DAB2 and promotes cell proliferation and invasion in ESCC.1. Using bioinformatics to predict and verify the micro RNAs that directly regulated DAB2.Using bioinformatics software such as Target Scan to predicte the mirco RNAs which directly regulated DAB2. Mi R-93 mimics was synthesized and transiently transfected in EC109 cells. DAB2 protein expression was detected by Western blot after transfected. Using luciferase assay to verify whether mi R-93 directly targeted DAB2-3’UTR.2. mi R-93 influences biological functions of ESCC cells through targeting DAB2.DAB2 m RNA and mi R-93 relative expressions were detected in ESCC tissues and adjacent non-tumor tissues by q PCR. ESCC cells were transfected by mi R-93 mimics and DAB2-si RNA respectively, and detected the proliferation using CCK-8 and Ed U assays. ESCC cells were transfected by mi R-93 mimics and DAB2-si RNA respectively, and measured the cell cycle using flow cytometric analysis. ESCC cells were transfected by mi R-93 mimics and DAB2-si RNA respectively, and measured the invasion effects using transwell assays.Part Four. mi R-218 directly targets BMI1 and suppresses cell proliferation and invasion and promotes cell apoptosis in ESCC.1. Using bioinformatics to predict and verify the micro RNAs that directly regulated BMI1.Using bioinformatics software such as Target Scan and PicTar and mirBase to predicted the mirco RNAs that directly regulated BMI1. Mi R-218 mimics was synthesized and transiently transfected to EC109 cells. BMI1 protein expression was detected by Western blot after transfected. Using luciferase assay to verify whether mi R-218 directly targeted BMI1-3’UTR.2. mi R-218 influences biological functions of ESCC cells through targeting BMI1.BMI1 m RNA and mi R-218 relative expressions were detected in ESCC tissues and adjacent non-tumor tissues using q PCR. Mi R-218 and BMI1 m RNA expression were measured by q PCR in HEEC cells and ESCC. ESCC cells were transfected by mi R-218 mimics and BMI1-si RNA respectively, and detected its proliferation using CCK-8 and Ed U assays. ESCC cells were transfected by mi R-218 mimics and BMI1-si RNA respectively, and measured the migration and invasion effects using transwell assays. ESCC cells were transfected by mi R-218 mimics and BMI1-si RNA respectively, and measured the apoptosis effects using flow cytometric analysis. Synthesized BMI1 overexpression vector(without 3’UTR), constructed ESCC cell which overexpressed BMI1 and transfected with mi R-218 mimics, then detected BMI1 protein expression and the biological functions in ESCC cells.Results:Part One.mi R-296-5p directly targets PLK1 and inhibits cell proliferation in NSCLC.1. mi R-296-5p directly targets PLK1-3’UTR and regulates PLK1 protein expression.After transfected with mi R-296-5p mimics in NSCLC cells, the significantly decreased of PLK1 protein expression was detected by Western blot. Mi R-296-5p was predicted to regulate PLK1 by using bioinformatic software. Dual luciferase reporter assay demonstrated that mi R-296-5p could directly targeted PLK1 m RNA 3’UTR.2. mi R-296-5p influences NSCLC biological functions through targeted regulation of PLK1 expression.In NSCLC tissues, PLK1 m RNA expression increased significantly compared with adjacent non-tumor tissues(P < 0.01), while mi R-296-5p expression decreased significantly compared with adjacent non-tumor tissues(P < 0.01). Compared with HBE, PLK1 protein expression increased significantly in NSCLC cells, while mi R-296-5p expression decreased significantly in NSCLC cells. After transfected with mi R-296-5p mimics and PLK1-si RNA in NSCLC cells, the results showed the upregulation of mi R-296-5p and downregulation of PLK1 expression could suppress NSCLC cell proliferation.Part Two. mi R-203 directly targets BMI1 and inhibits cell proliferation in NSCLC.1. mi R-203 directly targets BMI1-3’UTR and regulates BMI1 protein expression.After transfected with mi R-203 mimics NSCLC cells, the significantly decreased of BMI1 protein expression was detected by Western blot. Mi R-203 was predicted to regulate BMI1 by using bioinformatic software. Dual luciferase reporter assay demonstrated that mi R-203 could directly targeted BMI1 m RNA 3’UTR.2. mi R-203 influences NSCLC biological functions through targeted regulation of BMI1 expression.In NSCLC tissues, BMI1 m RNA expression increased significantly compared with adjacent non-tumor tissues(P < 0.01), while mi R-203 expression decreased significantly compared with adjacent non-tumor tissues(P < 0.01). Compared with HBE, BMI1 protein expression increased significantly in NSCLC cells. After transfected with mi R-203 mimics and BMI1-si RNA in NSCLC cells, the results showed the upregulation of mi R-203 and downregulation of BMI1 expression could suppress NSCLC cell proliferation. Flow cytometric analysis indicated that cell apoptosis were increased by transfected with mi R-203 mimics or BMI1-si RNA. Transwell assay demonstrated that the metastatic ability of NSCLC cells was inhibited by transfected with mi R-203 mimics or BMI1-si RNA.Part Three. mi R-93 directly targets DAB2 and promotes cell proliferation and invasion in ESCC.1. mi R-93 directly targets DAB2-3’UTR and regulates DAB2 protein expression.After transfected with mi R-93 mimics in NSCLC cells, the significantly decreased of DAB2 protein was detected by Western blot. Mi R-93 was predicted to regulate DAB2 by using bioinformatic software. Dual luciferase reporter assay demonstrated that mi R-93 could directly targeted DAB2 m RNA 3’UTR.2. mi R-93 influences ESCC biological functions through targeted regulation of DAB2 expression.In ESCC tissues, DAB2 m RNA expression decreased significantly compared with adjacent non-tumor tissues(P < 0.01), while mi R-93 expression increased significantly compared with adjacent non-tumor tissues(P < 0.05). After transfected with mi R-93 mimics and DAB2-si RNA in ESCC cells, the results showed the upregulation of mi R-93 and downregulation of DAB2 expression could promote NSCLC cell proliferation. Flow cytometric analysis indicated that cell cycle promoted by transfected with mi R-93 mimics or DAB2-si RNA. Transwell assay demonstrated that the metastatic ability of ESCC cells was inhibited by transfected with mi R-93 mimics or DAB2-si RNA.Part Four. mi R-218 directly targets BMI1 and suppresses cell proliferation and invasion and promotes cell apoptosis in ESCC.1. mi R-218 directly targets BMI1-3’UTR and regulates BMI1 protein expression.After transfected with mi R-218 mimics in ESCC cells, the significantly decreased of BMI1 protein expression was detected by Western blot. Mi R-218 was predicted to regulate BMI1 by using bioinformatic software. Dual luciferase reporter assay demonstrated that mi R-218 could directly targeted BMI1 m RNA 3’UTR.2. mi R-218 influences ESCC biological functions through targeted regulation of BMI1 expression.In ESCC tissues, BMI1 m RNA expression increased significantly compared with adjacent non-tumor tissues(P < 0.01), while mi R-218 expression decreased significantly compared with adjacent non-tumor tissues(P < 0.01). Using mi R-218 mimics and BMI1-si RNA transfected ESCC cells and cell proliferation was detected by CCK-8 and Ed U assays. The results showed that upregulation of mi R-218 or downregulation of BMI1 could suppress ESCC cell proliferation. Flow cytometric analysis indicated that cell apoptosis increased by transfected with mi R-218 mimics or BMI1-si RNA. Transwell assay demonstrated that the metastatic ability of NSCLC cells was inhibited by transfected with mi R-218 mimics or BMI1-si RNA. Using mi R-218 mimics to transfected ESCC cells which overexpressed PLK1 but not contained 3’UTR. Western blot showed that PLK1 protein was downregulated by transfected with mi R-218 mimics, but that this could be restored by transfection with PLK1 plasmid. Using cell migration and invasion assays, we observed that overexpression of PLK1 expression significantly rescued mi R-218-induced cell growth migration and invasion inhibition.Conclusion:1. mi R-296-5p suppressed NSCLC cell proliferation by directly targeting PLK1.2. mi R-203 suppressed NSCLC cell proliferation, invasion and promoted apoptosis by directly targeting BMI1.3. mi R-93 directly targeted DAB2 and promoted cell proliferation and invasion in ESCC.4. mi R-218 suppressed ESCC cell proliferation, invasion and promoted apoptosis by directly targeting BMI1.