| Backgrounds:Lung cancer, as a common malignant tumor, becomes to be the leading cause of cancer death. While, there is few available treatment options. Recently, morbidity of lung cancer presents high and lends to occur ahead of time. In china, lung cancer is also the major reason of death from maniglant tumor. Large of work about lung cancer has been carried out, but the mechanisms of lung cancer threatening our health were still unclear. We argue that environment especially smoking and gene were pivotal factors. In addition, it’s reported that 60 gene mutations were related to lung cancer in all probility. It presents a new insight of lung cancer. But we even don’t know how they work.Fortunately, the development of molecular biology provided a method to solve these problems. MicroRNA, as an important biomarker, regulated the expression of various genes known as epigenetics. MiRNAs are endogenous non-coding segments of RNA, 18-25 nucleotides in length, which negatively regulate gene expression at the post-transcriptional level and have a role in networking and fine-tuning gene expression in the cell.and it was thought to be a key factor in lung cancer owing to its tumor-supression activity and carcinogenicity.Dysregulation and aberrant expression of microRNA-100 have been reported to be invovled in tumorigenesis and tumor expression of several cancer types, suggesting that miR-100 might serve as a diagnostic and prognostic marker for human malignancy. We previously found that miR-100-3p overexpressed in the BEAS-2B cell line and rarely expressed in the NSCLC cell line. From this, we hypothesized that whether low expression of miR-100-3p will promote the development of lung cancer.In this article, we aim to explore the real mechanism how miR-100-3p regulate the carcinoma of lung via cell biology and histology methods. Most of all, we will discuss the proliferation, cell cycle and migration of lung cancer cell on the premise of knockdown the expression of miR-100-3p.Research contents:1. Measuring the level of miR-100-3p expression in BEAS-2B, A549, L18 cell lines,20 NSCLC patients’samples and 16 healthy controls by qRT-PCR.2. Established the stable expression of miR-100-3p inhibitor and inhibitor control in A549 cell using lentivirus vector, and established the stable expression of miR-100-3p mimics and negative control in L18 cell using lentivirus vector.3. Study the NSCLC cell biology function when established the stable expression of miR-100-3p in A549 and L18 cells. Followed by:(1) MTT and colony formation assay detected A549 and L18 cells proliferation and draw the growth curve.(2) Flow cytometry for detection of cell cycle change A549 and L18 cells.(3) Adhesion assay for detection of the adhesion change A549 and L18 cells.(4) Soft agar semisolid colony forming assay is to observe the capacity of anchor-independent growth.(5) Transwell/Matrigel observed A549 and L18 cells migration and invasion abilities.4. Analysis the possible molecular mechanisms through Western blotting.Research results:1. miR-100-3p expression was significantly lower especially in the A549 and L18 NSCLC cell line, compared with the bronchial epithelial cell(BEAS-2B), (p<0.01); the expression of miR-100-3p in 20 cases of patients with NSCLC was significantly Lower than the corresponding 16 healthy controls (p<0.001).2. The expression of miR-100-3p in A549 cell was effectively inhibited after transfection miR-100-3p inhibitor, and overexpression of miR-100-3p in L18 cell with using lentivirus vector.3. The effects of miR-100-3p on the NSCLC cells growth:MTT and colony formation assay results found that the proliferation capability of A549 cells was increased after inhibiting the expression of miR-100-3p, but this capability of L18 cells was inhibited after the overexpression of miR-100-3p.4. The effects of miR-100-3p on the NSCLC cell cycle:Cell cycle test shows inhibition of miR-100-3p resulted in a higher proportion of cells (A549) in S phase, but overexpression of miR-100-3p resulted in a lower proportion of cells (L18) in S phase.5. The effects of miR-100-3p on the NSCLC cell anchor-independent growth:Soft agar clone forming assay results displayed that the ability of anchor-independent growth of A549 cells increased when the expression of miR-100-3p was down-regulated, forming a larger colony. But this capability of L18 cells was inhibited after the overexpression of miR-100-3p.6. The effects of miR-100-3p on the NSCLC cell adhesion:Adhesion assay results show that the adhesion of cells enhanced which inhibiting the expression of miR-100-3p in A549 cells. But the cell adhesion ability of L18 cells was reduced when the expression of miR-100 was increased.7. The effects of miR-100-3p on the NSCLC cell migration and invasion:The migration and invasion assay results show that the migration and invasion ability of A549 cells was enhanced when the expression of miR-100 was inhibited, but the migration and invasion ability of L18 cells was reduced when the expression of miR-100 was increased.8. The miR-100-3p inhibitor induction of EMT in A549 cells:Cell morphology assay showed that epithelial cells change cytoskeleton, lose polarity and the space between the cell connection, epithelial cells translate into mesenchymal cells. Western Blot assay displayed that after inhibiting the expression of miR-100-3p in A549 cells, the expression levels of E-cadherin was significantly decreased and the expression of vimentin were markedly increased.9. Analysis the possible molecular mechanisms:(1) We found that inhibiting the expression of miR-100-3p in A549 cells, the expression of epithelial marker was downregulation and the expression of mesenchymal marker was upregulation by Western Blotting. But overexpression of miR-100-3p in A549 cells, the expression of epithelial marker was upregulation and the expression of mesenchymal marker was downregulation by Western Blotting.(2) We found that changes the expression of miR-100 may regulate β-catenin-dependent Wnt pathway in A549 cells and L18 cells.Conclusion:1. Human lung cancer (A549/L18) cells have been infected with lentiviral vector expressing microRNA-100-3p successfully.2. Inhibition of miR-100-3p increases proliferation, migration, and invasion of NSCLC cells in vitro. MiR-100-3p plays the antioncogene role in NSCLC cells.3. miR-100-3p enhances malignant behavior of non-small cell lung cancer cells via EMT.MiR-100-3p may play a pivotal role in activating the Wnt-β-catenin signaling through targeting FZD8. |
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