| Objective: Gastric cancer is the fourth most common cancer in the world and the second leading cause of death in cancer worldwide.In order to reduce the mortality rate of gastric cancer,one of the problems to be solved is to discover new biomarkers specific to early gastric cancer,achieve the diagnosis of gastric cancer in early stage,attain the goal of early surgical treatment,and fundamentally solve the problem of high mortality of gastric cancer.Our previous study found that peroxiredoxin 4(PRDX4)has the potential for using as a biomarker of early gastric cancer.By methods such as establishing cell transformation model,overexpression in transformed cells,etc,the purpose of this study was to explore the role of the PRDX4 in transformation of cells and to reveal that the PRDX4 reduces the amount of reactive oxygen species(ROS)in transformed cells,making the cells stay in a microenvironment that is beneficial for the growth and proliferation of cells,thus to promote the malignant transformation of cells,that is,to promote the generation and development of gastric cancer.Methods and results:1.Establishment of GES-1 transformation model and detection of tumor related functional indicators in transformed cells(1)Establishment of a malignant transformation cell lineage: The transformation was induced by N-methyl-N-vitro-N-nitrosoguanidine(MNNG)in normal gastric epithelial cells GES-1 for 0h,4h,8h,12 h,16h,20 h and 24 h,respectively,and then was cultured for 24 h.(2)Observation and analysis of the morphological changes of GES-1 cells: It was found that with the prolonged duration of MNNG induction,the morphology of the transformed cells were changed from spindle-shaped and closely-arranged cells to round,irregular,stacked cells,confirming the successful malignant transformation of GES-1 cells.(3)Results of tumor-related cellular functional indicators: The results of scratch test,soft agar colony formation test,and MTT test showed that the migration ability,colony formation ability,and proliferation ability of the transformed cells were increased with the prolonged duration of MNNG induction,in which the proliferation ability of MTC-16 cells was the strongest,suggesting that the transformation degree of the transformed cells was gradually increased with the prolonged duration of induction.Based on the above experimental results,MTC cells(malignant transformed cell)were almost completely transformed after induction of 16 hours.Therefore,MTC-16 cells were selected as the recipient cells for subsequent transfection of overexpression plasmid.2.Detection of ROS levels in transformed cells at different duration of induction confirms that ROS promotes the tumorigenesis of gastric cancerThe DCFH-DA fluorescent probe was used to detect the cellular ROS levels at different time after induction.Compared with the results of GES-1 cells cultured at the same time,it was revealed that the fluorescence intensity in transformed cells showed a trend of first rising and then decreasing with the prolonged duration of transduction.When inducted from 0-12 h,the fluorescence intensity in the transformed cells was increasing gradually.When inducted from 12-16 h,the fluorescence intensity in the transformed cells was increased to 51092.92 ? 99.54,reaching the highest level.When inducted from 16-24 h,the fluorescence intensity of the transformed cells was slightly decreased,which was 49652.14 ? 429.15 and 47558.16 ? 956.53,respectively.The fluorescence intensity in transformed cells of different induction time was significantly higher than that of GES-1 cells(42231.99 ? 1064.91).The results showed that ROS could promote the cell transformation processes with the prolongation of induction time.After basic transformation of cells(16 hours after induction),the ROS levels were gradually decreased with the increase of transformation degree to prevent the apoptosis of transformed cells,thus to provide a benign microenvironment for the growth and proliferation of cells.3.Measurement of PRDX4 levels in cells at different induction time confirmed the role of PRDX4 in promoting the generation and development of gastric cancer.The expression levels of PRDX4 mRNA and protein in different tested cells were detected by RT-PCR,Western Blot and ELISA.The results revealed that the PRDX4 levels had no significant change(2.70 ? 0.47ng/m L in GES-1 cells)during 0-12 h of induction.From 12 h to 16 h of induction,the PRDX4 levels were increased to3.51 ? 0.08 ng/mL.The expression of PRDX4 in cells was gradually decreased from16 to 24 hours.These results demonstrated that the PRDX4 did not remove ROS in cells in time at 0-12 h,which is one of the contributors to cause the rapid increase in intracellular ROS.It indirectly promoted cell transformation,which is promoted the generation of gastric cancer.At 16 h to 24 h of induction,the PRDX4 levels were increased,and the intracellular ROS were removed,which make the cells in a microenvironment conducive to the growth and proliferation of transformed cells,thereby promoting the malignant transformation of cells,that is,promoting the development of gastric cancer.4.The establishment of MTC-16 cell lineage with PRDX4 overexpression confirms the role of PRDX4 in tumor progression(1)Establishment of MTC-16 cell lineage with PRDX4 overexpression: The recombinant constructed overexpression vector GV230-PRDX4 was transfected into the MTC-16 cells using liposome transient transfection method.The transfection efficiency was observed by fluorescence microscopy.On the basis of confirming the successful transfection,the PRDX4 protein levels in the PRDX4 overexpression system were verified by western blot.Results confirmed that the PRDX4 overexpression was successfully established.(2)Results of indicators of tumor-associated cell function: The migration ability,colony formation ability and proliferation ability were significantly higher in the MTC-16-PRDX4 group than that in the MTC-16 group and GES-1 group,confirming that PRDX4 promotes the development of early gastric cancer.5.Detection of ROS and PRDX4 protein levels in the MTC-16 cell system with PRDX4 overexpression confirms the cellular tumor-promoting mechanism of PRDX4(1)ELISA was used to detect the PRDX4 protein levels in GES-1,MTC-16,MTC-16-GV230,and MTC-16-PRDX4 cells.The results showed that the PRDX4 protein level in MTC-16-PRDX4 cells was 3.75 ? 0.08 ng/mL,which was significantly higher than that in the GES-1(2.20 ? 0.12 ng/mL),MTC-16(3.14 ? 0.06ng/mL)and MTC-16-GV230(3.05 ? 0.08 ng/mL)cells.(2)The ROS levels in GES-1,MTC-16,MTC-16-GV230 and MTC-16-PRDX4 cells were detected by DCFH-DA fluorescent probe.The results showed that the ROS levels in MTC-16-PRDX4 group(45271.78 ? 1736.2)was significantly lower than that in the early gastric cancer model cells(51092.92 ? 810.99),which were only transfected with the empty vector,further confirming that PRDX4 protects cells against apoptosis by eliminating ROS to make the cells in a microenvironmentconducive to growth and proliferation,thus to play a role in promoting the development of gastric cancer.Conclusion:1.The tumor-related functional indicators(migration ability,colony formation ability and proliferation ability)of transformed cells are elevated with the increase of induction time.The transformed cells(MTC-16)were basically completed transformation after 16 hours of induction.2.Before the basic transformation was completed,the PRDX4 could not promptly eliminate ROS and thus indirectly promote the generation of gastric cancer.After the basic transformation was completed,the PRDX4 can clear the ROS in the tumor cells to prevent the cellular apoptosis due to high levels of ROS,making the cells in a microenvironment conducive to cell growth and proliferation,thereby promoting the development of gastric cancer. |
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