| Background and objectiveLung cancer is one of the most common malignant tumors in the world with the highest morbidity and mortality,and is one of the most serious threats to human health and life.As one of the most important pathological types of lung cancer,the diagnosis and treatment of lung adenocarcinoma(LUAD)is a difficult problem.Although therapeutic advances have significantly improved the prognosis of specific subgroups of LUAD patients,the overall survival rate of LUAD patients remains very low.It is very important to find a new and reliable method for the diagnosis and treatment of lung adenocarcinoma.In recent years,the role of miRNAs has received wide attention in the research of tumor.On one hand,miRNA can be found significantly expressed in a variety of tumors including liver cancer,lung cancer,gastric cancer,esophageal cancer,colorectal cancer,breast cancer and so on;on the other hand,miRNA could play an essential role in regulating cell growth,migration,invasion,apoptosis,differentiation and cell adhesion in various cancers.miRNA is involved in almost all of the basic signal transduction pathways in vivo,including the expression of many important tumor associated genes.miR-519 d was reported to be a tumor suppressor in various cancers,while its biological functions in LUAD are still unclear.Eukaryotic translation initiation factor 4(eIF4H)has played an essential role in the translation process of protein;it can stimulate more than 93% of the tumor associated protein translation,promoting tumor cell growth and invasion.In this study,we explored the expression of miR-519 d and e IF4 H in lung adenocarcinoma tissues and cells;analyzed the influence of aberrant expression of miR-519 d and e IF4 H on the biological behavior of LUAD;explored possible mechanisms by which miR-519 d works.The study is divided into six chapters.Chapter 1 The expression of miR-519 d and e IF4 H in lung adenocarcinoma tissuesMethods 1 Collecting tissues.69 cases of lung adenocarcinoma and matched adjacent normal tissue specimens were collected from The First Affiliated Hospital of Zhengzhou University.The collection meets ethical committee standards.2 The expression of miR-519 d and e IF4 H in 69 cases of lung adenocarcinoma and matched adjacent normal tissues were detected by q RT-PCR.3 Statistical analysis,Pearson correlation was used to compare the correlation between miR-519 d and e IF4 H.The relationship among the expression of miR-519 d and e IF4 H and gender,age,pathological type,TNM staging and lymph node metastasis of the patients were also analysised.Results1 Compared with the adjacent tissues,the expression of miR-519 d was significantly down regulated in lung adenocarcinoma(P<0.05),and the expression of e IF4 H m RNA was upregulated in lung adenocarcinoma(P<0.05).2 There was a negative correlation between the expression of miR-519 d and e IF4 H in lung adenocarcinoma,R2=0.563(P<0.05).3 The expression of miR-519 d and e IF4 H was not associated with gender,age,pathological type,TNM staging(P>0.05),but was associated with lymph node metastasis in patients with lung adenocarcinoma(P<0.05).Chapter 2 Effect of overexpressed miR-519 d on proliferation of lung cancer cellsMethods1 A549 and H1299 lung cancer cell lines were well cultured.2 Cells transfected with miR-519 d agomir were regarded as miR-519 d agomir groups;Cells transfected with miR-519 d scramble were regarded as miR-519 d scramble groups;Cells transfected with nothing were regarded as blank groups.3 q RT-PCR was used to detect the expression of miR-519 d and e IF4 H in miR-519 d agomir groups,miR-519 d scramble groups and blank groups 4 CCK-8 assay and colony formation assay were performed to exam the effect of miR-519 d expression on the proliferation ability of each group cells 5 Statistical analysis,experimental data obtained in the chapter were analyzed using statistical software SPSS21.0,the mean and standard deviation(Mean + SD)of experimental data were used to process statistical analysis with inspection level ?=0.05.Results1 The expression of miR-519 d was significantly upregulated in cells of miR-519 d agomir groups compared with the miR-519 d scramble groups and blank groups(P<0.05).2 CCK-8 assay showed that the number of cell proliferation in miR-519 d agomir groups was significantly lower than that in miR-519 d scramble groups and blank groups(P<0.05).3 The results of clone formation assay showed that the number of cell clone formation in miR-519 d agomir groups was significantly decreased(P<0.05).Chapter 3 Effect of overexpressed miR-519 d on invasion of lung cancer cellsMethods1 A549 and H1299 lung cancer cells were divided into three groups,respectively.The groups were as follows: miR-519 d agomir groups,miR-519 d scramble groups,blank groups.2 Transwell assay was used to investigate the effect of miR-519 d expression on the invasion ability of each group cells.3 Statistical analysis,experimental data obtained in the chapter were analyzed using statistical software SPSS21.0,the mean and standard deviation(Mean + SD)of experimental data were used to process statistical analysis with inspection level ?=0.05.Results1 Transwell assay showed that the number of invaded cells in miR-519 d agomir group was significantly lower than that in miR-519 d scramble groups and blank groups(P<0.05).Chapter 4 Effect of overexpressed miR-519 d on growth of xenografts in nude miceMethods1 30 female BALB/C nude mice were purchased and reared.2 According to the different conditions of subcutaneous injection,the nude mice were divided into 6 groups(5/each group).The groups were as follows: the group of A549 cells which were transfected with miR-519 d agomir,the group of A549 cells which were transfected with miR-519 d scramble and the blank group which transfected nothing;the group of H1299 cells which were transfected with miR-519 d agomir,the group of H1299 cells which were transfected with miR-519 d scramble and the blank group which were transfected nothing 3 q RT-PCR was used to detect the expression of miR-519 d 4 Xenograft tumor experiment was used to detect the proliferation ability of lung cancer cells in vivo 5 After 4 weeks of rearing,the subcutaneous transplantation tumors were dissected out from the nude mice and the Ki67 assay was used to detect the cell proliferation.Results1 In the A549 and H1299 lung cancer cells,the expression of miR-519 d was significantly upregulated in cells of miR-519 d agomir groups compared with the miR-519 d scramble groups and blank groups.2 The bioluminescence signal of miR-519 d agomir groups was significantly weaker than both the blank groups and the miR-519 d scramble groups(P<0.05).3 The number of Ki67-positive cells in the miR-519 d agomir groups was significantly lower than the control groups(P<0.05).Chapter 5 Preliminary exploration on the mechanisms of miR-519 d in lung adenocarcinomaMethods1 Bioinformation software was used to predict whether there exists complementary binding sites between miR-519 d and e IF4 H 3'UTR.2 Constructed recombined wild and mutant double luciferase reporter vector of e IF4 H 3'UTR.3 Dual luciferase reporter assay was used to test whether miR-519 d could target to e IF4 H.4 the expression of e IF4 H protein level was detected by Western Blot assay.5 Statistical analysis,experimental data obtained in the chapter were analyzed using statistical software SPSS21.0,the mean and standard deviation(Mean + SD)of experimental data were used to process statistical analysis with inspection level ?=0.05.Results1 There existed complementary binding sites between miR-519 d and e IF4 H 3'UTR.2 Wt-pmir GLO-e IF4 H and Mt-pmir GLO-e IF4 H double luciferase reporter vectors were successfully constructed.3 The protein level of e IF4 H was significantly downregulated in cells of miR-519 d agomir groups(P<0.05).4 Double luciferase reporter assay demonstrated that e IF4 H was one of the targets of miR-519 d in lung adenocarcinoma cellsChapter 6 Comparison of biological effects of downregulated e IF4 H expression and upregulated miR-519 d expression in A549 and H1299 cellsMethods1 si-e IF4 H was composed to silence the expression of e IF4 H in cells 2 A549 and H1299 cells were transfected with miR-519 d agomir to upregulate the expression of miR-519 d,and were transfected with si-e IF4 H to downregulate the expression of e IF4 H.The A549 and H1299 cells were respectively divided into 4 groups,miR-519 d agomir groups,si-e IF4 H groups,miR-519 d scramble groups,and blank groups.3 The expression of e IF4 H protein was detected by Western Blot assay 4 Cell proliferation and invasion were detected by CCK-8 assay,colony formation assay and Transwell assay respectively.5 Statistical analysis,experimental data obtained in the chapter were analyzed using statistical software SPSS21.0,the mean and standard deviation(Mean + SD)of experimental data were used to process statistical analysis with inspection level ?=0.05.Results1 Compared with miR-519 d scramble groups and blank groups,e IF4 H protein expression in si-e IF4 H groups was at a low level similar to that observed in the miR-519 d agomir groups(P>0.05).2 The results of CCK-8 aasay and colony formation assay showed that compared with miR-519 d scramble groups and blank groups,cell proliferation in si-e IF4 H groups was at a low level similar to that observed in the miR-519 d agomir groups(P>0.05).3 The results of Transwell assay showed that compared with miR-519 d scramble groups and blank groups,cell invasion in si-e IF4 H groups was at a low level similar to that observed in the miR-519 d agomir groups(P>0.05).Conclusions1 The expression of miR-519 d in lung adenocarcinoma tissues was down regulated,e IF4 H expression was significantly increased,there is an obviously negative correlation between the expression of miR-519 d and e IF4 H,and the two sides were related to lymph node metastasis.2 e IF4 H is one of the downstream targets of miR-519 d in lung adenocarcinoma.Overexpressed miR-519 d could inhibit cell proliferation and invasion at least partly through downregulating the expression of e IF4 H. |
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