| BACKGROUND Gastric cancer is a common malignant tumor ofdigestive system, Seriously affect the health of human being. Despite itssteady declining trend worldwide, gastric cancer is still a leading cause ofcancer-related death in the world, ranking second in males and fourth infemales in frequency. Surgical resection is currently the most effectivetherapy available for early-stage cancer. However, the majority of patientswith gastric cancer are detected and diagnosed at a late stage of cancerprogression, the prognosis is poor. The survival rate remains not satisfieddespite combined with chemotherapy and other treatment measures. Inrecent years, based on research on tumor related genes and pathways,tumor prevention and treatment has made great progress and this will bethe future direction of cancer research.Grainyhead-like2(Grhl2), a mammalian homologue of Drosophilagrainyhead, belongs to grainyhead-like (Grhl) transcription factor family.Studies have show that members of the family play an important role in thegrowth and development, neural tube closure and epithelial cell differentiation etc. With the in-depth study, the role of Grhl family inoccurrence and development of tumor has received increasing attention.Grhl2promote progression of many malignant neoplasms, such as breastcancer, oral squamous cell carcinoma and hepatocellular carcinoma. On theother hand, Grhl2could also inhibit EMT as well as tumor progression ofhuman breast cancer. However, to our knowledge, expression of Grhl2andits roles in gastric cancer have not been reported, therefore it has very highresearch value.OBJECTIVES The aim of this study is to detect the expression ofGrhl2in cell lines and tissues of gastric cancer, and then evaluated its rolein occurrence and development of gastric cancer according to theexpression of Grhl2in gastric cancer. Combined with the results of ourstudy and related literature, we further explore the potential molecularmechanism of Grhl2influencing the development of gastric cancer. In aword, we hope this study can provide a new clue for the prevention andtreatment of gastric cancer.METHODS (1) Immunohistochemistry was performed to explore theexpression of Grhl2in48pairs of gastric cancer and surroundingnon-tumor tissues. qPCR and Western-blot were used to detect theexpression of Grhl2in gastric cancer cell lines and Normal gastricepithelial cell line. Finally, differences between these groups wereanalyzed. (2)Construction of a lentiviral vector over-expression Grhl2.Transfected it into gastric cancer cell line and establish a stable strain.Thegastric cancer cell line which low expression of Grhl2was selected for thenext experiment. After transfection, the changes of cell proliferation,apoptosis, invasion and migration ability were tested by MTT, flowcytometry and Transwell invasion experiments, respectively. Xenograftmodel of nude mouse was established to observe the effect of Grhl2over-expression on tumor growth.(3) Western-blot was used to detect the changes of c-myc, Bcl-2andMMP2protein expression after Grhl2over-expression. LY364947, aninhibitor of TGF-β signaling pathway, was used to inhibit the TGF-βsignaling, and then the expression of Grhl2was detected by Western-blot.TGF-β1, a TGF-β signaling agonists, was added to Grhl2over-expressioncell line, then the effects of Grhl2on TGF-β signaling pathway andepithelial-mesenchymal transition (EMT) were evaluated by Western-blot.RESULTS (1) Grhl2was significantly downregulated both in gastriccancer tissues and cell lines, compare with corresponding surroundingnon-tumor tissues and normal gastric epithelial cell line. The difference wasstatistically significant. Moreover, down-regulation of Grhl2in gastriccancer was significantly associated with lymph node metastasis and TNMstaging.(2) Lentiviral vector expressing Grhl2was constructed and transfected into SGC7901cells. Finally, stable strain was established successfully.Over-expression of Grhl2can not only significantly inhibit cellproliferation, invasion and metastasis, promote apoptosis in vitro, but alsoinhibit the growth of transplanted tumors in nude mice in vivo.(3)The protein expression level of c-myc, Bcl-2and MMP2wasdown-regulated after Grhl2over-expression. In addition, Inhibition of TGFβ signaling pathway in gastric cancer cells can restore the expression ofGrhl2. Furthermore, over-expression of Grhl2blocked TGF beta signalingpathway, inhibition of TGF β induced EMT.CONCLUSIONS (1) Grhl2was significantly down-regulated ingastric cancer. Over-expression of Grhl2can inhibit the biologicalcharacteristics of gastric cancer. Grhl2may exert its inhibitory effects ontumor by down-regulation the expression of c-myc, Bcl-2, MMP2andinhibition of TGF-β signaling pathway as well as TGF-β induced EMT. |
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