| Background and ObjectivesThe morbidity and mortality of lung cancer is now in the first place of all malignant tumor. Statistical studies have found that in recent 50 years, higher incidence of lung cancer has increased in 16% to 69% of countries and regions than in the past. Non-small cell lung cancer is one of the main types of lung cancer, the incidence of lung cancer is particularly obvious rise in recent years. The tumor recurrence and metastasis are the main reasons of the bad prognosis of non-small cell lung cancer and lymphatic metastasis occupy important position. In some cases lymph node metastasis happened earlier than blood transfer or even the only way of metastasis in non-small cell lung cancer. The molecular mechanism and signal transduction pathways study of lymph node metastases in non-small cell lung cancer has important theoretical and clinical significance. Although lymph node metastasis and cell signal transduction trials of non-small cell lung cancer were carried out by scholars at home and abroad, and the tumor lymphatic generation had been confirmed one of the important link of lymph node metastasis, the molecular mechanism of lymph node metastasis and lymphatic generated in lung adenocarcinoma has not been fully elucidated.MicroRNA(miRNAs)are a class of endogenous non-coding RNA with about 18 to 25 nucleotide. They can degradate or inhibit mRNA translation process by combine with the 3’ untranslated region(UTR) of the target genes. New study confirmed that the miRNA can also act as the original oncogene and tumor suppressor genes and played an important role in gene regulation, cell apoptosis, cell differentiation and tumor related DNA damage repair. It had discovered that a variety of microRNAs has the efficacy of inhibiting tumor growth. miR-520 a is from miR- 520 family which is located in human chromosome 19. The present study found that miR- 520 a play an important role in tumor suppression in colon cancer, breast cancer, esophageal squamous carcinoma, myeloid leukemia and other tumors, but the function of miR-520a-3p on the occurrence and metastasis in non-small cell lung cancer is still not clear.This study was to research the role of miR-520a-3p in the occurrence and metastasis of non-small cell lung cancer. Non-small cell lung cancer clinical specimens detection and analysis found that there is a certain correlation between miR-520a-3p and MAP3K2. We discussed the specific molecular mechanisms of miR-520a-3p in non-small cell lung cancer occurrence, development and metastasis by in vitro and in vivo tests.We studied the mechanism between miR-520a-3p and by intervening in mi R-520a-3p expression level, using bioinformatics tools and fluorescein reported enzyme assay. Eventually it was clear that miR-520a-3p played a specific role in the process of non-small cell lung cancer. This study provided a new direction for the diagnosis and treatment of non-small-cell lung cancer.Methods1. Non-small cell lung cancer patient’s tumor, peritumoral and lymph nodes the sample were clinical collected, and the relationship of miR-520a-3p and the development, invasion and metastasis of non-small cell lung cancer according to the miR-520a-3p relative expression levels preliminary analyzed in different tissues and tumor TNM staging, metastasis of lymph node number, location, etc..Further, new lymphatic vessels in the tumor tissues were detected by the expression level of lymphatic vessels markers LYVE-1 and the relationship between LYVE-1 and miR-520a-3p were used to analyze the possible mechanisms that miR-520a-3p regulate non-small cell lung cancer.2. A high and low expression of miR-520a-3p cell lines in non small cell lung cancer cell line A549 were constructed as study by using the miR-520a-3p high specific inhibitors and miR-520a-3p analogue expression method. Then MTT cell proliferation experiment, flow cytometry technology testing, transwell cell climbing hole experiment and scratches experiment were choose to determine the proliferation, invasion, migration and apoptosis and other biology behavior of miR-520a-3p on non-small cell lung cancer cell.3. A nude mouse transplantation tumor model was built to make the tumor occurrence, development process and the tumor size, weight and transfer case comparison between tumors with different expression level of miR-520a-3p. Western blot test was choose to detect the expression levels of tumor lymphatic endothelial markers LYVE-1 groups in samples of transplantation to further research the ability of miR-520a-3p in the invasion and lymphatic metastasis in non-small cell lung cancer.4. To further verified the relationship between miR-520a-3p and MAP3K2, caspase 3, BCL-2, and look for other key molecular in the process of signal pathway that miR-520a-3p regulate MAP3K2. And explore the mechanism of miR-520a-3p inhibite lymph node metastasis in non-small cell lung cancer.Result1. We adopted qRT-PCR to detect the mi R-520a-3p expression level in the tumor and peritumoral tissues of NSCLC patients, found that the mi R-520a-3p expression level in tumor tissue was significantly lower than in the peritumoral tissues, and the miR-520a-3p expression level was highly correlated with the incidence of lymph node metastases and the prognosis of NSCLC patients. Patients with a low expression level of mi R-520a-3p had higher probability of lymph node metastases and poor prognosis.2. The Influence of miR-520a-3p on non-small cell lung cancer A549 cell proliferation, apoptosis, invasion and migration ability were determined by MTT, flow cytometry, scratches, transwell experiments. The experiment proves that miR-520a-3p can significantly inhibit the proliferation, invasion and migration ability, and to promote cell apoptosis of non small cell lung carcinoma. This suggests that miR-520a-3p may inhibit tumor cell proliferation, invasion and migration, thus inhibiting tumor growth and metastasis as a tumor suppressor factor.3. High and low expression of non-small cell lung cancer cell line A549 and normal A549 cells were injected into right side of the hip of nude mice to make nude mice subcutaneous transplantation tumor model. During the construction process, we found that the growth of A549 cells with high expression of miR-520a-3p were relatively slow, the final tumor volume and weight are small, and the tumor with low expression of miR-520a-3p grew faster than A549 cell with high expression of miR-520a-3p, the final tumor volume and weight are relatively larger. Therefore, miR-520a-3p may act as a tumor suppress factor to inhibit the growth of non-small cell lung cancer. Further studies showed that the xenograft tumor tissue with a lower expression level of miR-520a-3p expressed a high level of lymphatic endothelial cell-specific marker LYVE-1 in tumor and peritumoral tissues, whereas tumor with higher expression of miR-520a-3p expressed a lower level of LYVE-1 in tumor tissues and peritumoral tissues. Therefore, miR-520a-3p may regulate the lymphatic metastasis by inhibiting tumor lymphangiogenesis.4. Experiments in vivo and in vitro were given for the investigation and analysis of the possible mechanisms for the regulation of miR-520a-3p to the proliferation and metastasis of non-small cell lung cancer. Firstly, bioinformatics analysis based on miR-520a-3p sequence showed that MAP3K2 may be the target molecules of miR-520a-3p. The wild-type HEK-293 cells and 3’-noncoding region MAP3K2 mutant HEK-293 cells were choose and miR-520a-3p inhibitor treatment was used on both of them and expression levels of MAP3K2 were detected. The results suggest that MAP3K2 protein levels OF the wild-type HEK-293 cells were significantly reduced, while MAP3K2 protein level in 3’untranslated region MAP3K2 mutant HEK-293 cells had no significant changes. Further by detecting the MAP3K2 protein expression levels in A549 cells with different miR-520a-3p expression levels found that MAP3K2 expression level was negative correlated with miR-520a-3p expression levels.i.e. A549 cells with lower miR-520a-3p level can highly expressed MAP3K2 protein and the A549 cells with low expression of miR-520a-3p after miR-520a-3p inhibitor treatment had a higher MAP3K2 protein expression level. Western blot showed that MAP3K2, caspase 3, bcl-2 expression levels changed with the average expression of miR-520a-3p levels In vivo, in which MAP3K2, caspase 3 and miR-520a-3p had a negative correlation, while bcl-2 was positively correlated with miR-520a-3. Therefore concluded: miR-520a-3p can regulate non-small cell lung cancer growth, proliferation, invasion and metastasis through influence the expression of MAP3K2, caspase-, bcl-2.Conclusions1. miR-520a-3p expression in tumor tissue was lower than in peritumoral tissues in patients with NSCLC; The tumor cells and tissues with higher degree of malignancy had the lower the expression of miR-520a-3p level; The specimens with the occurrence of lymph node metastasis had lower miR-520a-3p expression levels; The miR-520a-3p negatively correlated to LYVE-1 expression.2. Non-small cell lung cancer A549 cells had a low expression level of miR-520a-3p; The low expression level of miR-520a-3p was negatively correlated with the proliferation, invasion and migration ability of A549 cell and positively correlated with apoptosis of A549 cells.3. The right hip subcutaneous heterotopic xenograft model in nude mice was successfully constructed; The miR-520a-3p expression level and tumorigenesis, development negatively correlated; The miR-520a-3p expression level was negatively correlated with the LYVE-1 expression level and tumor lymphangiogenesis.4. The miR-520a-3p expression level was negatively correlated with MAP3K2, caspase 3 and correlated with bcl-2; In another word, miR-520a-3p may regulate growth, proliferation, invasion and metastasis of non-small cell lung cancer by influencing the expression of MAP3K2 3, caspase 3 and bcl-2. |
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