Preliminary Research On The Regulation Mechanisms Of MiR-375/SHOX2 Axis In Invasion And Metastasis Of EsophagealSquamous Cell Carcinoma

Background and ObjectiveEsophageal squamous cell carcinoma and esophageal adenocarcinoma are two main pathological types of esophageal carcinoma, which is one of the eighth most common tumors in the world. Esophageal squamous cell carcinoma is prevalent and stays high fatality rate in south-east Asia. In Chinese eastern area, the incidence of esophageal squamous cell carcinomas is the fourth in all of malignant tumors. However, the molecular mechamisms of ESCC tumorigenesis and metastasis remain unclear. The therapy of the progressive esophageal squamous cell carcinoma just relied on radiotherapy and chemotherapy and the effect is poor. In addition, chemotherapy drug for esophageal squamous cell carcinoma is unsensitive. Therefore, actively searching for more effective treatments and specific targeted drugs to bring a new way for esophageal squamous cell carcinoma accurate treatment is imperative. MiRNAs are a sort of small molecule noncoding RNAs which have great regulated functions of post-transcraptopn. According to our previous gene chip analysis, miR-375 was low expressed in esophageal squamous cell carcinoma. Over expression of miR-375 inhibited the ability of proliferation, invasion and metastasis in esophageal squamous cancer cells. Blocking miR-375 expression could promote the ability of proliferation, invasion and metastasis in esophageal squamous cancer cells. The aim of this study was to explore the function and possible moleculor mechanism of miR-375 and its downstream target genes in invasion and metastasis of esophageal squamous carcinoma.Materials, Methods and ResultsPart Ⅰ miR-375 expression differences in human esophageal squamous cell carcinoma tissues and human nomal esophageal tissuesMethods:1. Collected 5 cases of esophageal squamous carcinoma tissue and corresponding normal epithelial tissue. Using microassay technology to detect the abnormal miRNA expression profiles.2. Verification of PCR assay in esophageal squamous cell carcinoma tissues and esophageal squamous carcinoma cells.3. Construction of miR-375 mimics, inhibitor and NC. Using MTT assay, healing assay, clone formation assay and transwell assay to analysis miR-375 on cell proliferation, invasion and metastasis in esophageal squamous carcinoma cells.Results:1. The gene chip analysis showed that there were 12 miRNAs in more than 50 times nomal differentially expressed compared with the corresponding nomal tissue. MiR-375 was the most obvious differences expressed in esophageal squamous cell carcinoma tissues.2. The confirmation of mRNA level in esophageal squamous carcinoma tissues and cells showed that miR-375 had a low expression level compared with normal esophageal tissues.3. Over-expressed miR-375 deduced the ability of proliferation, invasion and metastasis in esophageal squamous cancer cells; down-expressed miR-375 induced the ability of proliferation, invasion and metastasis in esophageal squamous cancer cells.Part Ⅱ SHOX2 negatively targeted by miR-375 expression and the function SHOX2 in vivo and vitroMethods:1.5 bioinformatics software Online (TargetScan, miRanda, PicTar, miRTarget2 and PITA) to predict downstream target genes of miR-375. Confirmation by luciferase, PCR and Western Blot assay.2. Constructed SHOX2 gene plasmid, and transfected it into esophageal squamous cancer cells. MTT assay, healing assay, clone formation assay and transwell assay were performed to analyze function of SHOX2.3. Select 100 cases of esophageal squamous cell carcinoma tissues and nomal esophageal tissues specimens collected by thoracic surgery general hospital of Nanjing military region between 2007 and 2013 and analysed SHOX2 expression in immunohistochemical staining.4. Using statistical methods to analyze SHOX2 expression relationship with the prognosis of patients in 100 cases of esophageal squamous cell carcinoma tissues with immunohistochemical staining.Results:1. The miRNA target gene prediction software showed that miR-375 could conbine with 3’SHOX2 UTR with only one conservative binding sites between 1156-1170 bp. The luciferase gene report, polymerase chain reaction and Western Blot suggested that miR-375 negatively targeted SHOX2 expression.2. MTT assay, healing assay, clone formation assay and transwell assay showed that over-expressed SHOX2 could strengthen the ability of proliferation, invasion and metastasis in esophageal squamous cancer cell. Down-expressed SHOX2 could weaken the ability of proliferation, invasion and metastasis in esophageal squamous cancer cell.3. Compared with the matched nomal esophageal tissues, SHOX2 had high expression in esophageal squamous cell carcinoma tissues.4. The Kaplan-Meier survival curve analysis showed that high SHOX2 expression was associated with poor prognosis in patients (p= 0.019). SHOX2 expression was correlated with tumor range, lymph node metastasis and pathological staging (= 0.006, p< 0.001 or< 0.001). Proportional hazards method analysis showed that high SHOX2 expression was an adverse prognostic factor followed with tumor range and pathological stage (p< 0.001,= 0.033).Part Ⅲ The interaction of miR-375 and SHOX2 on EMT course in esophageal squamous cell carcinomaMethods:1. Over-expressed miR-375 in the esophageal squamous carcinoma cells and used PCR, Western Blot and ICC assay to detect the specific markers of epithelial (E-cadherin, β-catenin) and mesenchymal (Vimentin and N-cadherin) expression.2. Over-expressed SHOX2 in the esophageal squamous carcinoma cells and used PCR, Western Blot and ICC assay to detect the specific markers of epithelial (E-cadherin, β-catenin) and mesenchymal (Vimentin and N-cadherin) expression.3. Co-transfected with miR-375 mimics and SHOX2 plasmid in the esophageal squamous carcinoma cells and used PCR, Western Blot and ICC assay to detect the specific markers of epithelial (E-cadherin, β-catenin) and mesenchymal (Vimentin and N-cadherin) expression.4. Inoculation of stable transfected with over-expressed SHOX2 of esophageal squamous cancer cells, injected subcutaneously into nude mice and analyzed by PCR, Western Blot and ICC assay for the specific markers of epithelial (E-cadherin, β-catenin) and mesenchymal (Vimentin and N-cadherin) expression.Results:1. Result from PCR, Western Blot and ICC assay showed that over-expression of miR-375 strengthened markers of epithelial (E-cadherin, β-catenin) expression, but weakened markers of mesenchymal (Vimentin and N-cadherin) expression in esophageal squamous cell casinoma.2. Result from PCR, Western Blot and ICC assay showed that over-expression of SHOX2 weakened markers of epithelial (E-cadherin, β-catenin) expression, but strengthened markers of mesenchymal (Vimentin and N-cadherin) expression in esophageal squamous cell casinoma.3. Result from PCR, Western Blot and ICC assay showed that Co-transfected with miR-375 mimics and SHOX2 plasmid in the esophageal squamous carcinoma cells weakened markers of epithelial (E-cadherin, β-catenin) expression, but strengthened markers of mesenchymal (Vimentin and N-cadherin) expression.4. Result from PCR and Western Blot by tumors of nude mice showed that over-expressed SHOX2 of tumors weakened markers of epithelial (E-cadherin, P-catenin) expression, but strengthened markers of mesenchymal (Vimentin and N-cadherin) expression.Conclusions1. MiR-375 was down-expressed in esophageal squamous cell carcinoma tissues and miR-375 could inhibit the proliferation, invasion and metastasis of esophageal squamous casinoma cells.2. SHOX2 was downstream target genes of miR-375. SHOX2 could promote the proliferation, invasion and metastasis of esophageal squamous casinoma cells and its expression was negatively correlated with the poor prognosis of patients.3. MiR-375 directly targeted SHOX2 and participated in epithelial- mesenchymal transformation process of esophageal squamous cell carcinoma.4. To sum up, this study preliminarily revealed the relationship of miR-375, SHOX2 in EMT of esophageal squamous cell carcinomas. MiR-375 was down-expressed in esophageal squamous cell carcinomas tissues, and its downstream target gene SHOX2 was negatively regulated by miR-375. Down-expression of miR-375 resulted in up-regulation of SHOX2 and induced proliferation, invasion and metastasis of esophageal squamous casinoma cells and high SHOX2 expression was negatively correlated with poor clinical prognosis. Interaction between miR-375 and SHOX2 was involved in epithelial- mesenchymal transformation process of esophageal squamous cell carcinoma. Therefore, it was concluded that SHOX2 could be viewed as a new prognostic indicators and targeting miR-375/SHOX2 axis might be a novel strategy for the treatment of esophageal squamous cell carcinomas in the future.