MicroRNA-188 Targets SIX1 Gene And Nhibites Cell Proliferation And Invasion In Oral Squamous Cell Carcinoma Through Pregulating ERK Signal Pathway

[Objective] We evaluated SIX1 and miR-188 expression and clinical relevance in human oral squamous cell carcinoma tissues,and investigated their role on biological behavior and the underlying molecular mechanisms. [Method]Primary tumor specimens were researched under immunohistochemistry;cell culture and transfection;Quantitative real-time PCR (SYBR Green method); colony formation assay;MTT assay;flow cytometry for cell cycle analysis;matrigel invasion assay;validation for interaction of miRNAs and Target Genes using Luciferase Reporter Assays. [Result] 1. the expression of SIX 1 in oral squamous cell carcinoma:We did not found positive staining for SIX1 in normal oral mucosa epithelial tissues. In 32 of 80 cancer tissues, SIX1 showed positive nuclear and cytoplasmic immunostaining.2. Clinical significance of SIX1 in oral squamous cell carcinom:SIX1 overexpression significantly correlated with advanced TNM stage, tumor status and nodal metastasis.3. SIX1 promotes OSCC cell proliferation and invasion:Detroit 562 cell line has high endogenous SIX1 expression and KB cell line has low endogenous expression. SIX1 plasmid significantly upregulated its expression at both protein and mRNA levels. SIX1 transfection facilitated cell proliferation rate in KB cell line, while its siRNA blocked proliferation in Detroit 562 cell line. Significant reduction of invading ability was found in cells with SIX1 depletion while SIX1 plasmid upregulated invading ability.4. SIX1 regulates cyclin D1 and MMP-2:SIX1 transfection upregulated cyclin D1 and MMP-2 expression while SIX1 depletion decreased these proteins.5.SIX1 regulates MMP-2 through ERK signaling:SIX1 overexpression significantly upregulated ERK phosphorylation. The change of JNK and p38 phosphorylation was not significant.6. SIX1 regulates epithelial-mesenchymal transition:SIX1 overexpression downregulateed E-cadherin in KB cell line while SIX1 depletion slightly upregulated E-cadherin expression. SIX1 also positively regulated Snail and Twist protein expression.7. miR-188 expression was downregulated in oral squamous cell carcinoma:The level of miR-188 was lower in oral squamous cell carcinoma tissues than in normal oral mucosa tissues.8. expression of miR-188 in vitro:Detroit 562 has relative low miR-188 expression and FaDu has relative high expression. The miR-188 mimic significantly upregulated miR-188 in Detroit 562 cell and inhibitor downregulated miR-188 expression in FaDu cells.9.miR-l 88 regulates cell proliferation and invasion:colony number of Detroit 562 cells transfected with a mimic was significantly decreased, while colony formation ability of FaDu transfected with miR-188 inhibitor was increased.The number of invasive cells increased significantly for FaDu when transfected with miR-188 inhibitor.10. miR-188 regulates cell cycle.treatment of miR-188 mimic inhibited G1-S transtion, while miR-188 inhibitor treatment facilitated cell cycle progression.11. miR-188 upregulates expression of cyclin D1, cyclin E and MMP9:miR-188 mimic transfection could downregulate cyclin D1, cyclin E and MMP9 expression, while treatment with miR-188 inhibitor upregulated these protein.12. miR-188 negatively regulates SIX1 oncogene:miR-188 negatively regulated p-ERK expression. Overexpression of miR-188 significantly decreased SIX1 mRNA.13. SIX1 was a direct target of miR-188:miR-188 mimic reduced the fluorescence intensity in Detroit 562 cells transfected with a vector containing wild-type SIX1 3’-UTR, while no significant change was observed in cells transfected with vector containing mutant binding site. miR-188 mimic reduced the fluorescence intensity in Detroit 562 cells transfected with a vector containing wild-type SIX1 3’-UTR, while no significant change was observed in cells transfected with vector containing mutant binding site. [Conclution]1.SIX1 is overexpressed in human oral squamous cell carcinoma.2.SIX1 overexpression contributes to cell proliferation and invasion.3.SIX1 promotes invasion through ERK-MMP-2 signaling pathway and epithelial-mesenchymal transition(EMT).4.miR-188 is a downregulated miRNA in oral squamous cell carcinoma, serves as a tumor suppressor.5.miR-1 88 inhibits OSCC proliferation and invasion by targeting SIX1.