| Part?The expression of ZNF139 in gastric carcinoma tissues andmetastatic lymph nodesObjective: Gastric cancer is one of the most common malignant gastrointestinal tumors with highest mortality.Early gatric cancer patients have no obvious symptoms.Therefore,most of gastric cancer patients have been diagnosed at moderate or advanced stages.Because of the poor sensitivity of ganstric cancer cells to chemotherapeutic drug,five year overall survival rate of gastric cancer is less than 30%.The development of gastric cancer are affected by many factors,including invasion,metastasis,adhension,extracellular matrix degradation and tumor angiogenesis.It has been reported that Zinc finger proteins are associated with the pathogenesis and MDR of tumors.ZNF139 belongs to Kruppel family.However,the role of ZNF139 in pathogenesis and MDR of gastric cancer remains unknown.In the present study,we provided insight into the effect of ZNF139 on the pathogenesis and development of gastric cancer and novel ideals for gastic cancer treatment.Methods: Gastric carcinoma tissue specimens,matched normal gastric tissue and matched adjacent tissues were obtained from twenty-one patients who were identified as aggressive gastric adenocarcinoma at the Fourth Affiliated Hospital of Hebei Medical University from February 2011 to September 2011.And 21 specimens of lymph nodes were collected.The histologic diagnosis of these tissue samples was confirmed by a pathologist.Real-time PCR and immunohistochemical stainning were used to determine the expression of ZNF139.The correlation between the level of ZNF139 and clinicopathological parameters of patients were analyzed.SPSS 13.0 software was applied to analyze the data,and significance was assessed in all experiments as a probability value of P<0.05.Results:1 Compared with adjacent normal tissues,the expression of ZNF139 was significantly increased in gastric carcinoma tissues(1.416±0.032 vs 2.021±0.054,P<0.5).It was suggested that ZNF139 might be involved in the pathogenesis of gastric cancer.2 Compared with normal lymph nodes,the expression of ZNF139 was increased in metastatic lymph nodes(1.00±0.144 vs 2.227±0.249,P<0.5),which suggested that ZNF139 might be related with metastsis.3 ZNF139 was related with differentiation,invasion,clinical TNM stage and metastisis of gastric cancer.The level of ZNF139 mRNA and protein were increased in poor differentiated,invasive,advanced staged and metastic gastric carcinama tissues.Conclusions:1 The level of ZNF139 was increased in gastrica carcinoma tissues and metastic lymph nodes.2 ZNF139 was correlated with diffrentiation,invasion,clinical TNM stage and metastisis of gastric carcinoma tissues.Part II The effect of ZNF139 on 5-FU sensitivity of garstric cancerObjective: chemotherapy is an important method in the comprehesive treatment for gastric cancer(GC).However,the presence of multi-drug resistance(MDR)of GC often leads to the failure of chemotherapy,which is a main cause of death in patients with GC.Therefore,reversing MDR of GC would be of great significance in enhancement of the effect of chemotherapy and improvement for prognosis.In this study,we explored the role of ZNF139 on 5-FU sensitivity of GC.It might provide novel ideals of the treatment of GC.Methods:1 To investigate the relationship between ZNF139 level and 5-FU chemosensitivity,the chemosensitivity of GC cells to 5-FU was tested with MTT assay;2 The expression of ZNF139 and MDR related genes,such as MDR1/ P-gp,MRP1,BAX and Bcl-2 were analyzed by real-time PCR in 5-FUresistant GC tissues;3 In order to determine the effect of ZNF139 on 5-FU resistance of GC cells,the siRNA-ZNF139 was transfection into SGC7901 cells for 48 h.Methods:1 Prooly 5-FU sensitivite GC tissues were much more sensitive to 5-FU.2 Lower levels of ZNF139,MDR1/P-gp,MRP1 and Bcl-2 and high level of bax were found in 5-FU sensitivite GC tissues.3 In SGC7901 cell transfected with siRNA-ZNF139 for 48 h,the expression of ZNF139 was decreased and the 5-FU sensitivity was increased.4 The mRNA and protein expression of ZNF139 were reduced in SGC7901 cell transfected with siRNA-ZNF139 for 48 h.And in SGC7901 cells transfected with siRNA-ZNF139,the mRNA and protein levels of MDR1/P-gp,MRP1 and Bcl-2 were significantly decreased,while the expression of Bax was increased.Moreover,the chemosensitivity of SGC7901 cells to 5-FU was enhanced by transfection with siRNA-ZNF139.Conclusions:1 In 5-FU-sensitive garstric tissues,the expression of ZNF139 was decreased.2 In 5-FU-sensitive garstric tissues,the expression of P-gp,MRP1 and Bcl-2 were decreased,while the expression of BAX was incresed.3 Silencing of ZNF139 could revers the 5-FU resistance of GC cells.Part III Down-regulation of ZNF139 inhibits the proliferation of humangarstric cancer cellObjective: Gastric cancer is one of common malignant cancer with high morbidity and mortality in our country.It has been reported that many genes regulate the pathogenesis and development of gastric cancer.However,the role of ZNF139 in pathogenesis and MDR of gastric cancer remains unknown.In the present study,we provided insight into the effect of ZNF139 on the growth of gastric cancer cells and novel ideals for gastic cancer treatment.Methods:1 SGC7901 cells were transfected with siRNA-scramble and siRNAZNF139 for 48 h.The transfection efficiency was assessed by number of cells expressed green fluorescent protein.And real-time PCR and Western blot were used to determine the levels of ZNF139 mRNA and protein.2 The EdU staining assay and MMT assay were performed to measure the proliferation of SGC7901 cells transfected with siRNA-scramble and siRNA-ZNF139 for 48 h.3 The levels of Bcl-2 and survivin were analyzed by real-time PCR and Western blot in SGC7901 cells transfected with siRNA-scramble and siRNAZNF139 for 48 h.4 In order to determine the effect of ZNF139 on the proliferation of SGC7901 cells in vivo,the human gastric carcinoma nude mouse orthotopic transplantation model was established.Results:1 The transfection efficiencies of siRNA-scramble and siRNA-ZNF139 reached about 85% in SGC7901 cells.2 Compared with transfection with siRNA-scramble,transfection with ZNF139 could dramatically decrease ZNF139 mRNA and protein expression.3 Silencing of ZNF139 inhibited the proliferation of SGC7901 cells.Down-regulation of ZNF139 decreased the levels of Bcl-2 and survivin mRNA and protein.4 In the human gastric carcinoma nude mouse orthotopic transplantation model,silencing of ZNF139 significantly down-regulated the mRNA and protein levels of of Bcl-2 and survivin mRNA and protein.Conclusions:1 Silencing of ZNF139 inhibited the proliferation of SGC7901 cells,accompanied by decreased expression of Bcl-2 and survivin.2 In the human gastric carcinoma nude mouse orthotopic transplantation model,silencing of ZNF139 decreased the expression of Bcl-2 and survivin.Part miR?-195-5p regulates multi-drug resistance of gastric cancer cellsvia targeting ZNF139Objective: Gastric cancer is one of the most common malignant tumors with a high mortality rate.There are no specific clinical symptoms or signs in the early stage of gastric cancer.The presence of multi-drug resistance of gastric cancer cells in the advanced stage leads to the failure of chemotherapy,which is a main contributor to mortality rates in patients with gastric cancer.In the part II of our study,the results showed that ZNF139 could regulate the multi-drug resistance of gastric cancer cells.However,up-stream regulation mechanism of ZNF139 was still unknown.MicroRNAs are a group of small,non-coding RNA,which are 21-23 nucleotides in length.MicroRNAs play important biological function,which can negatively regulate downstream gene expression as post-transcriptional regulators by binding at 3'-UTR of target gene mRNA.In this study,the effects and molecular mechanism of miR-195-5p in regulating the multi-drug resistance were investigated.Our study provided a new therapeutic target of gastric cancer.Methods:1To investigate the relationship between miR-195-5p level and chemosensitivity of 5-FU and L-OHP,the chemosensitivity of GC cells to 5-FU and L-OHP was tested with MTT assay;2 The expression of ZNF139 and MDR related genes,such as p-gp,mrp1 and bcl-2 were analyzed by real-time PCR in gastric cancer tissues;3 In order to determine the effect of ZNF139 on 5-FU resistance of GC cells,the protein levels of ZNF139 and MDR related genes,such as P-gp,MRP1 and Bcl-2 were analyzed by Western blot in MNK28 cells transfected with miR-195-5p mimic or miR-195-5p inhibitor for 48 h.4 Bio-informatic database predicted that ZNF139 was a target of miR-195-5p.Luciferase assay indicated that miR-195-5p could negatively regulate ZNF139 protein level by directly binding to 3'-UTR of its mRNA.5 In order to determine the effect of ZNF139 on miR-195-5p-induced multi-drug resistance,the protein levels of ZNF139 and multi-drug resistance related genes,such as P-gp,MRP1 and Bcl-2 were analyzed by Western blot in MNK28 cells co-transfected with miR-195-5p inhibitor and ZNF139-specific siRNA for 48 h.Results:1 The sensitivity to 5-FU and L-OHP was high in poorly differentiated gastric cancer tissues.2 Compared with poorly differentiated gastric cancer cells,mRNA levels of ZNF139 and multi-drug resistance related genes,such as p-gp,mrp1 and bcl-2 were decreased in well differentiated gastric cancer cells.3 The protein levels of multi-drug resistance related genes,such as P-gp,MRP1 and Bcl-2 were decreased in MNK28 cells transfected with miR-195-5p mimic.And the protein levels of MDR related genes,such as P-gp,MRP1 and Bcl-2 were increased in MNK28 cells transfected with miR-195-5p inhibitor.4 ZNF139 was a target of miR-195-5p.Luciferase assay indicated that miR-195-5p could negatively regulate ZNF139 protein level by directly binding to 3'-UTR of its mRNA.5 Silencing of ZNF139 could reverse the effect of miR-195-5p inihibitor on multi-drug resistance of gastric cancer cells.Conclusions:1 miR-195-5p could regulate multi-drug resistance of gastric cancer cells.2 miR-195-5p regulated multi-drug resistance of gastric cancer cells via targeting ZNF139. |
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