The Study Of Mechanism And Role Of MiR-193b In The Process Of TGF-β1Enhancing Invasive And Metastatic Capacity

Gastric carcinoma (GC) is a malignant tumor with high morbidity and mortality in China. Invasion and metastasis are leading causes of death. It is important for clinical therapy to study the mechanisms of GC invasion and metastasi, look for new diagnostic markers, provide strong theoretical basis for the development of new treatment strategies.MicroRNAs (miRNAs) are non-coding RNAs (19-24nucleotides) that regulate gene expression by targeting mRNAs for translational repression, degradation, or both. Increasing lines of evidence indicate aberrant expression of microRNAs linked to cancer, and those miRNAs are thought to function as tumor suppressors, or oncogenes. The deep research of microRNA will provide a new perspective for gastric cancer metastasis and infiltrating mechanism research.Clinical studies have reported that an elevated serum level and overexpression of TGF-β1in primary gastric cancer correlated with lymph node metastasis and poor prognosis. Our previous studies have shown that treatment with TGF-β1could enhance metastasis and infiltration of gastric cancer cells in vitro. The expanded role of microRNAs suggests a new potential mechanism of regulating invasion and metastasis in gastric cancer. This study intends to discuss microRNA differentially expressed spectrum of gastric cancer cells with TGF-(31stimulation, the function and molecular mechanisms of miR-193b in invasion and metastasis of gastric cancer cells from a new perspective.ObjectiveTo detect the microRNA differentially expressed spectrum of gastric cancer cells with TGF-β1stimulation. To explore the function and mechanism of differentially expressed microRNA-miR-193b in invasion and metastasis of gastric cancer and provide the theoretical and experimental basis to GC gene therapy.Methods(1) The microRNA differentially expressed spectral of gastric cancer cell with TGF-β1stimulation was detected by microRNA microarray assay. Differentially expressed microRNAs were validated by Real-time RT-PCR in three different gastric cancer cells BGC823, SGC7901and MKN28.(2) Bioinformatics analysis on differentially expressed microRNAs using TargetScan, PicTar and miRanda software to predict which moleculars or signaling pathways the microRNA target or relate to.(3) miR-193b mimic or inhibitor, chemically synthesized and fluorescent marked, transfected to gastric cancer cells transiently. The number of intracellular fluorescent in each group was counted under inverted fluorescence microscope. The effect of transfection was confirmed by real time PCR.(4) Use the wound healing, transwell assays and liver metastasis in nude mice abdominal cavity forming assay to detect the effect of miR-193b mimic and inhibitor transient transfection on capacity of invasion and metastasis of gastric cancer cell.(5) Use real-time PCR, RT-PCR and Western blotting to detect the change and relationship of miR-193b, uPA mRNA and uPA protein levels before and after miR-193b mimic and inhibitor transfection, determine whether the uPA is a target of miR-193b in gastric cancer cells.Results(1) MicroRNA expression spectrum showed six differentially expressed microRNAs (differentially expressed before and after TGF-β1stimulation more than1.5times as a different miRNA) after TGF-β1stimulation for24h an36h in gastric cancer BGC823cells.Among them, the expression of miR-193b, miR-574-3p and miR-130b were downregulated, while miR-27a, miR-29b-1and miR-194were upregulated. These miRNAs should correlate with invasion and metastasis of gastric cancer BGC-823cell through TGF-β pathway. The three miRNAs with the most obvious changes (miR-193b, miR-130b and miR-27a) were subjected to real time RT-PCR verification. The real time PCR results confirmed microarray data obtained for BGC823and SGC7901. However, the similar results were not obtained in MKN28 cells.(2) Bioinformatics analysis finds there is a binding site for miR-193b in the uPA3'-UTR. Interestingly, uPA is a metastasis related gene.(3) miR-193b mimic and inhibitor transient transfection change miR-193b expression level in the gastric cancer BGC823cells obviously compared with negative transfection group and idling group. TGF-beta1stimulation could reduce miR-193b the expression level further after miR-193b inhibitor transfection in gastric cancer BGC823cells, attenuate the increased miR-193b expression level partly after miR-193b mimic transfection in gastric cancer BGC823cells.(4) miR-193b expression inversely correlated with uPA protein. No such changes were observed in cells transfected with inhibitors or mock controls. Interestingly, no significant difference could be seen in uPA mRNA levels between cells transfected with miR-193b mimics or inhibitors.(5) miR-193b mimic instantaneous transfection will significantly lower ability of migration, invasion and metastasis of gastric cancer BGC823cell, while, miR-193b inhibitor instantaneous transfection can promote migration, invasion and metastasis of gastric cancer BGC823cell.Conclusion(1) MicroRNA expression spectrum turned up six differentially expressed microRNAs.These miRNAs should correlate with invasion and metastasis of gastric cancer BGC-823cell through TGF-(3pathway.(2) TGF-β1reduces miR-193b expression and enhances migration, invasion, and metastasis in gastric cancer by targeting uPA.