| Background and ObjectiveLung cancer is a malignant tumor which seriously threatens human life and health. It has become the main cause of death in the world. Lung cancer can be divided into small cell lung cancer(SCLC) and non small cell lung cancer(NSCLC) two categories. Surgery, chemotherapy, targeted therapy and radiotherapy are the main treatments for lung cancer. Although the level of diagnosis and treatment of lung cancer continues to progress, but the long-term survival of patients with lung cancer is not optimistic, the overall 5 year survival rate of only 10%- 20%. Local recurrence and distant metastasis are the main causes of death in patients with lung cancer. The occurrence and development of lung cancer is the result of multi gene change, which involves the activation of many oncogenes and the inactivation of tumor suppressor genes. At present, gene targeted therapy has become a new hot spot in the research of lung cancer. It has important clinical value and practical significance to find and recognize new therapeutic targets and to diagnose and treat lung cancer.Small RNA(miRNA, micro RNA) encoding length of 19-25 nucleotides, widely exists in plants and animals. The specific binding of m RNA with target m RNA can inhibit target m RNA translation or degradation of target. miRNA is a kind of abundant gene regulatory molecules in multicellular organisms, which affect the expression of many protein coding genes. Increasing evidence indicates that miRNA is involved in the regulation of many basic cellular behaviors, such as cell differentiation, proliferation, growth, migration, and apoptosis. The abnormal expression of miRNA plays an important role in tumor initiation, progression and recurrence, and plays a role in inhibiting cancer and promoting cancer. miR-92 b, miR-9, miR-224 and miR-183 and so on are cancer genes and promote the metastasis of lung cancer. The downregulation of miR-101, miR-133 a and miR-141 in lung cancer can significantly inhibit tumor metastasis. In addition, lung cancer is associated with the presence of miRNA in body fluids such as blood, serum, plasma and saliva.miRNA is a new tumor marker in lung cancer, which plays an important role in lung cancer.Integrin(ITGB) belongs to the cell surface molecules are a class of alpha and beta subunits by noncovalent bonds composed of transmembrane protein receptor mediated cell and between cells and extracellular matrix(extracellular matrix,ECM) adhesion. Because of the particularity of transmembrane structure, integrin has two-way signal transduction function. 18 kinds of alpha subunits and 8 beta subunits have been found, which can form 24 different kinds of two kinds of polymers, which are distributed in different tissues and organs of the body, and play their respective biological functions. At the same time, it is closely related to the occurrence and development of the disease, and its signal transduction pathway can also be involved in the occurrence and development of tumor. The mutation or abnormal expression of integrin is generally considered to be related to the occurrence and metastasis of tumor. Integrin beta 3 is a member of the beta subunit, which is expressed in different tissues of the whole body, and participates in various biological processes, and exerts different physiological functions. ITGB3 is a common receptor for a variety of proteins, such as fibronectin, laminin, matrix metalloproteinase-2, bone, and fibronectin. ITGB3 expression was observed in all kinds of malignant tumors. In leukemia, ITGB3 plays a key role in the development of Acute myeloid leukemia, making it a potential therapeutic target for AML. Studies have confirmed that ITGB3 is an important regulator of cell migration and invasion in colorectal cancer cells. In breast cancer, m RNA expression profiles showed that some of the blood vessel formation associated proteins(including ITGB3) were significantly up-regulated in metastatic tumor cells. In addition, the latest research shows that the expression of Let-7c in lung cancer tissue is down regulated by targeting ITGB3 to inhibit the migration and invasion of lung cancer cells.miR-338 family(miR-338, miR-338-3p, miR-338-5p) is located in the apoptosis related tyrosine kinase 7 intron. The expression of miR-338 in hepatocellular carcinoma, oral squamous cell carcinoma and esophageal squamous cell carcinoma has been confirmed. Overexpression of miR-338 can inhibit the proliferation and promote cell apoptosis of gastric cancer cells. Recovery of miR-338 levels in hepatocellular carcinoma can make cancer cells sensitive to Sola Fini therapy. Studies have confirmed that miR-338 can inhibit the invasion and migration of colorectal cancer cells by targeting Smoothened. But it is still not clear the mechanism of miR-338 in tumor metastasis.In this study, we tried to evaluate the expression of miR-338 in 5 lung cancer cell lines?1 human lung fibroblast cell line MRC-5and 115 cases lung cancer tissues and adjacent normal lung tissues by real-time fluorescent PCR(q RT-PCR) assay. The function of miR-338 in the proliferation, adhesion, invasion and metastasis of lung cancer cells was explored by constructing miR-338 over expression of lung cancer cell lines. And to detect the expression of ITGB3 in miR-338 over expression of lung cancer cell line, and preliminary to study the molecular mechanism of miR-338 inhibiting the metastasis of lung cancer.This paper contains the following three parts: the first part: The expression and significance of miR-338 in lung cancer cell lines and lung cancer tissues; the second part: Effects of up regulation of miR-338 expression on the biological behavior of lung cancer cell lines A549 and NCI-H292; the third part: miR-338 inhibits the metastasis of lung cancer by targeting integrin beta 3.Part 1: The expression and significance of miR-338 in lung cancer cell lines and lung cancer tissuesMethods:1.To culture 5 lung cancer cell lines(A549, NCI-H292, NCI-H460, NCI-H446, NCI-H1299) and 1 human lung fibroblast MRC-5 cell line.2. 115 cases of lung cancer tissues and corresponding adjacent normal lung tissues were collected.3. RNA was extracted from all cell lines and 115 cases of lung cancer tissues and corresponding adjacent normal lung tissues, and the expression of miR-338 was detected by q RT-PCR.4. To analyze the correlation between miR-338 expression and gender, age, smoking history, tumor size, tumor emboli, lymph node metastasis, tumor recurrence and TNM stage in lung cancer.5. Using SPSS 13.0 to analyze the datas, which are in the form of the average value ± the standard deviation(mean ± SD). Using paired samples t test to estimate the difference between the two groups. Using single factor analysis of variance(ANOVA) to analyze the relationship between miR-338 expression and the clinical pathological factors, and using the log rank test to draw the survival curve according to the Kaplan-Meier method. If p<0.05, then the difference between the two groups was statistically significant.Results:1. Compared with the human lung fibroblast MRC-5 cell, the expression of miR-338 in all lung cancer cell lines was significantly reduced(A549 1.0vs0.15± 0.01?NCI-H292 1.0vs0.35±0.12?NCI-H460 1.0vs0.3±0.02?NCI-H446 1.0vs0.25± 0.07?NCI-H1299 1.0vs0.275±0.01,p<0.05).2. Compared with the normal lung tissue adjacent to cancer, the expression of miR-338 in 115 cases of lung cancer was significantly reduced(0vs-2.99±3.69, p<0.001). The decrease of miR-338 expression in lung cancer tissues was not related to gender, age, smoking history, tumor size and lymph node metastasis(p>0.05). The decrease of miR-338 expression in lung cancer tissues was related to tumor emboli, tumor recurrence, and TNM stage(P=0.005, 0.004, 0.025). The 5 year overall survival rate of the low miR-338 expression group was significantly lower than that of the high miR-338 expression group(median survival time 44 months vs 53 months, P=0.001).Part 2: Effects of up regulation of miR-338 expression on the biological behavior of lung cancer cell lines A549 and NCI-H292Methods:1. To construct and culture A549 and NCI-H292 cell lines with recombinant miR-338 lentivirus infection, and to detect the expression of miR-338 in the cells by q RT-PCR.2. CCK-8 method was used to detect the proliferation and growth of A549, NCI-H292 cells with recombinant miR-338 lentivirus infection and control cells.3. To detect the adhesion ability of A549, NCI-H292 cells with recombinant miR-338 lentivirus infection and control cells.4. Transwell assay detection was used to detect the migration and invasion of A549 and NCI-H292 cells with recombinant miR-338 lentivirus infection and control cells.5.Nude mice transplanted tumor model experimental was used to detect the effects of NCI-H292 cells with recombinant miR-338 lentivirus infection and control cells in nude mice lung transplantation tumor.6.Using SPSS 13.0 to analyze the datas, which are in the form of the average value ± the standard deviation(mean ± SD).Using paired sample t test to estimate the difference between the two groups. If p<0.05, then the difference between the two groups was statistically significant.Results:1. Compared with the control cells, the miR-338 expression was significantly increased in A549, NCI-H292 cells with recombinant miR-338 lentivirus infection(A549 646.37±35.45vs23.05±3.56?NCI-H292 330.86±11.05vs10.54±2.35,P<0.05). The lung cancer cell lines with high expression of miR-338 were successfully constructed.2. Up regulation of miR-338 expression in vitro can inhibit the proliferation(p<0.05), adhesion(A549 average cell count 1233±249vs310±22, p=0.035)(NCI-H292 average cell count 180±16vs72±10,p=0.028), migration(A549 averagecell count 347±41vs190±8,p=0.029)(NCI-H292 average cell count 287±33vs127± 21,p=0.045) and invasion(A549 average cell count 197±12vs68±14,p=0.008)(NCI-H292 average cell count 106±10vs41±3,p=0.006) of the lung cancer cells A549, NCI-H292.3.Nude mice xenograft tumor model experiment showed that miR-338 high expression can effectively inhibit the growth of lung transplantation tumor in nude mice(average tumor clone number 20±7vs4±2, p=0.004).Part 3:miR-338 inhibits the metastasis of lung cancer by targeting integrin beta 3Methods:1. The bioinformatics analysis suggested that ITGB3 was a target gene of miR-338.2. The ITGB3 expression was detected by Western blotting in the lung cancer cell lines A549 and NCI-H292 with miR-338 over expression and control cells.3. To verify the miR-338 target gene with Dual luciferase reporter assay.4. Using SPSS 13.0 to analyze the datas, which are in the form of the average value ± the standard deviation(mean ± SD).Using paired sample t test to estimate the difference between the two groups. If p<0.05, then the difference between the two groups was statistically significant.Results:1. Compared with the control cells, the expression of ITGB3 protein was significantly reduced in the lung cancer cell lines A549,NCI-H292(A549 1.0vs0.35±0.04, p<0.001)(NCI-H292 1.0vs0.69±0.07, p=0.01).2. Compared with the control group, dual luciferase activity assay revealed that the the luciferase activity was reduced in miR-338 over expression group(A549 1.0vs0.75±0.08,p=0.03)(NCI-H292 1.0vs0.62±0.06,p=0.008).Conclusions:1. In the lung cancer cell lines and lung cancer tissues, the expression of miR-338 was significantly lower than that of normal cells and tissues. The expression level of miR-338 in lung cancer patients was not related to gender, age, smoking history, tumor size and lymph node metastasis. The decrease of miR-338 expression in lung cancer tissues was related to tumor emboli, tumor recurrence, and TNM stage. The 5 year overall survival rate of the low miR-338 expression group was significantly lower than that of the high miR-338 expression group.2. Up regulation of miR-338 expression in lung cancer cells in vitro can effectively inhibit the proliferation and adhesion of lung cancer cells, and reduce the migration and invasion of lung cancer cells.3. miR-338 plays a negative regulation post transcription by targeting the integrin beta 3 3'UTR m RNA region. miR-338 plays a role in inhibiting cancer.Integrin beta 3 is a new miR-338 lung cancer target gene,and is a potential new target for lung cancer gene therapy. |
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