| Background and ObjectiveLung cancer is a malignant tumor with the highest morbidity and mortality in the world, which is serious threat to human health and life security. Lung cancer is generally divided into squamous cell carcinoma, adenocarcinoma, large cell carcinoma and small cell carcinoma according to the histological classification, the first three are collectively referred to non small cell lung cancer (non small cell lung cancer, NSCLC) in clinic, accounting for about80%of all lung cancer cases.Early lung cancer patients always have no obvious symptoms, most are late lung cancer stage when confirmed. In recent years, although predominantly surgical comprehensive treatment has achieved great progress, but the5-year survival rate is still less than15%.The only way to improve the NSCLC long-term survival is early diagnosis and treatment, and to achieve early diagnosis, clear NSCLC molecular mechanism of occurrence and development have a vital role.MicroRNAs(miRNAs) are a class of small noncoding RNAs (19-24nucleotide), which regulate the expression of target genes through binding to the target genes3’-untranslated region (31UTR) resulting in translational repression or mRNA degradation. A large number of studies have shown that abnormal expression of micrornas with closely related to the development of tumor.It not only can be used as a tumor suppressor gene through down-regulation of oncogene activated, but also can be used as a cancer gene through down-regulation of tumor suppressor gene activated.Therefore, as a kind of new molecular target, micrornas have raised more attention in tumor and other biomedical research field.At present, as RNA sequencing (RNA-seq) and miRNA chip hybridization method was widely used, many malignant tumor miRNA expression profiles have been established, Some of these miRNAs become the new target of tumor therapy and become the prognosis of tumor markers.Of course, more and more research about miRNAs in human non-small cell lung cancer(NSCC) have been reported, with the deepening of the study, we have known the mechanism of a large number of miRNAs in non-small cell lung cancer(NSCC). Recent studies showed that miR-675expression was up-regulated in human glioma tissue, gastric cancer, liver cancer and other malignant tumors, And through the regulation of Cadherin13, RUNX1(Runt Domain Transcription Factorl), Twistl (twist basic helix-loop-helix transcription factor1), different target genes involved with tumor development and progression.Anther study found down regulated the expression of adrenal cortical carcinoma, suggesting that miR-675expression in different tumor and its mechanism are different.Expression of miR-675in non small cell lung cancer and impact on biological characteristics and the related mechanism has not been reported in the literature. This project intends to use molecular biology, cell biology, and immunohistochemistry method to analyze the expression level of miR-675-5p in non small cell lung cancer and its clinical pathological significance.On this basis, analyze whether affect malignant biological characteristics of the cells of non small cell lung cancer and the mechanism of carcinogenesis when the expression level of miR-675-5p was changed.Methods:1. Detected the expression of miR-675-5p in NSCC (cancer tissues and adjacent tissues of cancer) and cell lines by real-time quantitative PCR(qRT-PCR), especially focused on the analysis of miR-675-5p and clinical pathological characteristics of the patients with NSCC;2. We Constructed of LV-miR-675-precursor and LV-miR-675-5p-inhibition lentiviral vector and packaging preparation into virus (random sequence as a negative control),Infected to A549and HTB-182cells by lentivirus,analyzed the effect of biology(cell proliferation, invasion and migration and tumor growth in nude mice)after up-regulated and down-regulated the expression of miR-675-5p in A549cell lines and HTB-182cell lines.3. Bioinformatics was used to predict the target gene of miR-675-5p.The luciferase reporter assay was used to assess the target genes of miR-675-5p in A549cell lines and HTB-182cell lines which was used to determine whether the miR-675-5p directly regulates the target gene GPR553’-UTR. The protein level of target gene GPR55in the A549and HTB-182cell lines which was transfected with LV-miR-675-precursor and LV-miR-675-5p-inhibition were observed by Western blot. The expression of GPR55in tumor tissue which comformed with subcutaneous injection of LV-miR-675-precursor was observed by immunohistochemical method. 4. The expression level of GPR55protein in non small cell lung cancer and normal lung tissue chip was observed by immunohistochemical method and analyze its relationship with lymph node metastasis was analyzed.5. We constructed siRNA interference plasmid which transfect into non small cell lung cancer cell line A549-LV-miR-675-5p-inhibition with high expression of GPR55, investigated the effect of the effect of biology(cell proliferation, invasion and migration and tumor growth in nude mice)in non small cell lung cancer cell line A549-LV-miR-675-5p-inhibition after GPR55was knocked down.Further analyze the function of miR-675-5p mediated by GPR55.Results:1.The expression levels of miR-675-5p in NSCC tissues were significantly reduced compared to that in adjacent non-cancerous tissues, meanwhile the expression levels of miR-675-5p in six human NSCC cell lines were also lower than that in normal lung cell line HBE. The expression of miR-675-5p in patients with non small cell lung cancer had a negative correlation with lymph node metastasis and TNM stage.2. Down-regulation of miR-675-5p promoted cell growth, proliferation, clone formation, invasion and migration, and promoted the tumorigenicity graft growth of nude mice in vivo;whereas up-regulation of miR-675-5p had a contrary effect.3. The luciferase reporter assay showed that GPR55was a direct target gene of miR-675-5p. Up or down regulation of miR-675-5p can lead to the down-regulation or up-regulation of GPR55.4. Compared with normal lung tissue, the expression of GPR55protein was significantly higher in patients with non-small cell lung cancer tissues,and had a proportional relationship with lymph node metastasis of tumor It suggested the target gene GPR55miR-675-5p be involved in NSCLC carcinogenesis.5. Down regulation of GPR55in A549-LV-miR-675-5p-inhibition cells significantly inhibited cell proliferation, invasion, migration, and can inhibited tumor growth in nude mice. It further confirmed that miR-675-5p was involved in the occurrence and development of NSCLC through regulation of the target gene expression of GPR55.Conclusion:1. The expression levels of miR-675-5p in NSCC tissues and cells were down-regulated. The expression of miR-675-5p in patients with non small cell lung cancer had a negative correlation with lymph node metastasis and TNM stage.2. miR-675-5p inhinbited cell growth, proliferation, clone formation, invasion and migration, and inhinbited the tumorigenicity graft growth of nude mice in vivo.3. GPR55was a direct target gene of miR-675-5p. The regulation of miR-675-5p to GPR55was a post-transcriptional regulation. The the expression of GPR55was knocked down, proliferation and invasion of A549LV-miR-675-5p-inhibition cell in vitro were inhibited,and tumor growth of nude mice in vivo were inhibited.4. Compared with normal lung tissue, the expression of GPR55protein was significantly higher in patients with non-small cell lung cancer tissues,and had a proportional relationship with lymph node metastasis of tumor. |
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