| Research background:Gastric cancer is one of the most common gastrointestinal tumors in China.It has high incidence,low early detection rate,high degree of malignancy,poor treatment effect,short survival time and poor prognosis.In addition,in recent years,the incidence of gastric cancer has an obvious younger trend.Gastric cancer has become one of the major public health problems that seriously threaten the health of the Chinese people.At present,the population structure of our country has changed a lot.With the extension of life expectancy,the aging phenomenon is increasing gradually.Environmental pollution,unhealthy diet and lifestyle,and the accumulation of many risk factors such as chronic infection of Helicobacter pylori,the situation of prevention and control is very serious.Gastric cancer has attracted more and more attention.The formation of gastric cancer is a dynamic process,including a variety of gene activation or inactivation,multi-step,multi-stage completion,with different components and genes involved in different stages of its development.Different components are involved in the different stages and processes of gastric carcinogenesis.Studying the possible molecular or signaling pathways involved is beneficial for us to better understand the pathogenesis and progression of gastric cancer.MicroRNA(miRNA)is a kind of single-stranded small RNA,which is widely found in a variety of organisms.Its sequence length is about 20?25 nucleotides,and it does not encode protein.Mature microRNA s can negatively regulate the expression of target genes by recognizing and completely or incompletely binding to the 3 ' non-coding region(UTR)of specific target genes,and ultimately inhibiting the synthesis of proteins or inducing the degradation of target genes.microRNA s are involved in various life processes,such as embryonic development,cell proliferation,differentiation,and apoptosis.Studies have found that the microRNA expression profile of tumor cells is significantly different from that of normal cells and it is speculated that it may play an important role in the formation of tumors?microRNA can be used as an oncogene or tumor suppressor gene involved in the whole process of tumor occurrence and development,but also affect the outcome of tumor treatment and affect the prognosis.Therefore,we speculate that the study of microRNA may provide a new target for the diagnosis,treatment and prognosis of tumors.The miR-17-92 oncogene is located in the intron of C13orf25 on chromosome 13q31,a typical highly conserved gene cluster containing a polycistronic promoter,which encodes seven microRNA s including miR-17-5p,miR-17-3p,miR-18a,miR-19a,miR-20a,miR-19b-1,and miR-92-1?The miR-17-92 gene cluster was found to be abnormally expressed in a variety of tumors and closely related to tumor proliferation and metastasis.It also has dual functions of oncogene and tumor suppressor gene.MiR-19a/b is the most important microRNA in the miR-17-92 gene cluster,and miR-19a/b has been found to increase the proliferation?invasion and metastasis of cancer cells?NF-kB(nuclear factor-kB)is a transcription factor,which exists widely in many types of cells.It has been found that a large number of cellular immune emergency responses are involved in its regulation,such as inflammatory response,cellular immune response,and the transcription of genes related to the anti-apoptotic effect of cells.In recent years,microRNAs have been recognized as important regulators of the NF-kB signaling pathway.Our study mainly focused on the effect of miR-19a onNF-kB activation and the effect of miR-19a on gastric cancer cell proliferation.Methods:Part I:Expression of miR-19a in gastric cancer cells and organizations,and the correlation between the expression of miR-19a with clinical parametersObjective:To study the expression of miR-19a in three gastric cancer cell lines,gastric cancer tissues and paracancerous tissues,and to study the relationship between its expression and clinicopathological parameters.Methods:Gastric cancer cell lines MGC-803,BGC-823 and SGC-7901 were cultured.The RT-qPCR method was used to detect the expression of miR-19a in three gastric cancer cells compared with the GES-1 immortalized gastric epithelial cell line.The expression of miR-19a was also detected byRT-qPCR in 30 cases of gastric cancer and its adjacent tissues,and the correlation between the expression and the clinicopathologicalparameters was analyzed.Results:miR-19a levels are upregulated in human gastric cancer cells.Relative levels of miR-19a in human gastric cancer cells were measured by RT-qPCR.Compared with GES-1 immortalized gastric epithelial cell lines,MGC-803,BGC-823 and SGC-7901 human gastric cancer cell lines had significantly increased miR-19a levels(>1 fold),Among them,theexpression level of miR-19a was the highest in MGC-803 cells?miR-19a expression levels were normalized by U6(P<0.05).Based on these results,the level of miR-19a was significantly increased in human gastric cancer cells.The expression level of miR-19a in gastric cancer was significantly higher than that in adjacent tissues.The expression level of miR-19a was not related to the age,gender,tumor size,differentiation degree and tumor infiltration degree of patients,but related to tumor stage and lymph node metastasis.Part ?:miR-19a promotes the proliferation and metastasis of gastriccancer cellsObjective:To study the effect of miR-19a on the proliferation of gastric cancer cell line MGC-803Methods:MGC-803 cells with the highest miR-19a expression were selected and transfected with miR-19a mimic,inhibitor or negative control for 24,48 or 72 h,respectively.The MTT assay was used to examine the effect of miR-19a on cell viability.Results:When microRNA-19a mimics was transfected into human gastric cancer cell line MGC-803,the cell proliferation ability increased after 24,48 and 72 hours,and increased by 23%and 35%respectively at 48 and 72 hours.On the contrary,when the expression of microRNA-19a wasinhibited,the cell proliferation decreased,In contrast,when theexpression of miR-19a was inhibited,cell viability decreased by 17 and 36%at 48 and 72 h,respectively.These results indicated that miR-19a could increase MGC-803 cell viability.Part ?;TNF-? can promote the expression of miR-19a,miR-19a activates NF-kappa B signaling pathway and promotes the proliferation of gastric cancer cellsObjective:To study the effect of TNF-alpha on the expression of miR-19a,and to observe whether miR-19a can play a role by activating the NF-kappa B signaling pathway.Methods:1.After MGC-803 cells were treated with 10 ng/ul TNF-a for 48 h,the relative levels of miR-19a were detected by RT-qPCR,and the changes of p-NF-kB and NF-kB protein levels were detected by western-blot.2.To examine whether miR-19a contributes to NF-kB activation,we transfected miR-19a mimics into MGC-803 cells and applied western-blot to detect changes in the protein levels of p-NF-kB,NF-kB,VCAM,ICAM,and MCP-1.Changes in NF-kB protein expression by immunofluorescence 3.To study the effect of NF-kB on cell proliferation,small interfering(si)RNA was selected to target NF-kB,siNF-kB and siNF-kB and miR-19a mimics were co-transfected into MGC-803 cells,respectively,and the changes in the protein levels of NF-kB,VC AM,I CAM and MCP-1 were detected by western-blot;the changes in cell viability were detected by MTT.Results:1.MGC-803 gastric cancer cells were treated with 10 ng/ul TNF-a for 48 h,and the relative levels of miR-19a were increased approximately 4-fold by RT-qPCR analysis.At the same time,Western-blot was used to detect the expression of p-NF-kappa B and NF-kappa B.It was found that the expression of p-NF-kappa B and NF-kappa B increased compared with the control group.2.After MGC-803 gastric cancer cells were transfected with miR-19a mimic,Western-blot analysis revealed that the phosphorylated NF-kB/NF-kB ratio was increased more than 1 fold,and vascular cell adhesion molecule(VCAM),intercellular adhesion molecule(ICAM)and monocyte chemoattractant protein-1(MCP-1)protein levels were increased compared with the negative control,and their relative levels were all increased to 1 fold.Immunofluorescence of cells showed that:after transfection with miR-19a mimics,the expression of NF-kB increased.3.After transfection of si-RNA targeting NF-kB,cell viability was significantly reduced even in cells transfected with miR-19a mimic.MTT assay showed that the viability of cells transfected with si-NF-kB was significantly reduced,and after co-transfection,the viability of cells was increased,but still lower than that of the blank control group.Similarly,the protein expression of NF-kB,VCAM,ICAM,and MCP-1 was detected by Western blot,and the level was lowest in the si-NF-kB group transfected alone and increased in the cotransfected group,but it was still lower than that in the control group.Conclusion:The expression of miR-19a is increased in gastric cancer cells,and its signaling mediates the proliferation of gastric cancer cells through the activation of NF-kB pathway.The expression of miR-19a in gastric cancer tissues was higher than that in adjacent tissues,and was negatively correlated with tumor stage and lymph node metastasis. |
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