| [Background and Objective]Lung cancer is the most common type of malignant tumor and the leading cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for ～80% of primary lung cancer, and approximately two-thirds of NSCLC patients are diagnosed in advanced stages, which contribute to the high mortality levels. Because the majority of patients present with invasive and metastatic disease, understanding the basis of lung cancer progression is vital. Chemotherapy mostly based on cDDP is useful treatment for patients with NSCLC, but NSCLC usually is insensitive to chemotherapy in a clinical setting, which causes the treatment failure. Many factors, including oncogenes and tumor suppressors have been reported to be involved in lung tumorigenesis or progression, but the underlying mechanisms remain to be fully determined.MicroRNAs (miRNAs) are small non-coding RNAs that usually repress gene expression through partially complementary binding to the target mRNA. The discovery of miRNAs has revealed a new regulatory layer of gene expression that affects the development and progression of various diseases, especially including cancer. Numerous studies have demonstrated that miRNAs can function as oncogenes or tumor suppressor as results of their fundamental roles in diverse cellular processes, such as cell differentiation, proliferation, apoptosis, invasion and metastasis. The aberrant expression of specific miRNAs has been found to be associated with development and clinical outcomes of various cancers, but mechanisms of deregulated miRNAs and their roles in tumorigenesis are still largely unknown.In the present study, we confirmed the decrease of miR-101 expression in lung cancer and indicated that miR-101 sensitized lung cancer cells to chemotherapy and inhibited invasion via directly targeting RUNX1 in lung cancer cells. Reciprocally, we identified RUNX1 physically binds to the miR-101 promoter and negatively transcriptionally regulates miR-101 expression. These findings suggest the miR-101/RUNX1 feedback regulatory loop in lung cancer as valuable biomarker and potential therapeutic target.[Methods]1. To confirm the miR-101 expression pattern in lung cancer, we initially evaluated the expression levels of miR-101 in matched non-small cell lung cancer (NSCLC) and adjacent non-tumor lung tissues and lung cancer cells (A549, H460, H1299, H1975,95C and 95D) and a normal diploid human cell line of lung fibroblasts MRC5 used qRT-PCR.2. Using the public database-TargetScan (http://www.targetscan.org) to predicte the potential targets of miR-101;3. Using qRT-PCR and western blot to evaluated the expression levels of RUNX1 in matched non-small cell lung cancer (NSCLC) and adjacent non-tumor lung tissues and lung cancer cells (A549, H460, H1299, H1975,95C and 95D) and a normal diploid human cell line of lung fibroblasts MRC5;4. To address whether miR-101 and RUNXl have potential in-parallel relationship, we analyzed miR-101 and RUNX1 expression using Pearson correlation coefficient.5. We transfected H1299 and 95D cells with miR-101 mimics or scramble control, the expression level of miRNA-101 and RUNX1 was detected by qRT-PCR, separately, and the expression of RUNX1 protein was massured by western blot;6. To determine whether miR-101 suppresses RUNX1 through a process triggered by the interaction between miR-101 and RUNX1 3’-UTR region, we constructed a reporter plasmid containing firefly luciferase fused with RUNX1 3’-UTR sequence of miR-101 binding site and cotransfected the reporter plasmid with miR-101 mimics or scramble control in 95D or H1299 cells for 48 h.7. Overexpressed miR-101 via transfection of miR-101 mimics sensitized H1299 and 95D cells to cDDP detected by MTS assay. RUNX1 knockdown via transfection of RUNX1 specific siRNAs enhanced the sensitivity of H1299 and 95D cells to cDDP. Overexpressed RUNX1 via transfection of RUNX1 expressing plasmid pcDNA3.1-RUNX1 reversed miR-101 mediated chemotherapeutic sensitization in lung cancer cells.8. The transwell assay were performed on differnet cell groups, including scramble control, miR-101 mimics transfected; si-control, si-RUNX1 transfected, miR-101 mimics plus pcDNA3.1/control and miR-101 mimics plus pcDNA3.1/RUNX1;9. Detected miR-101 expression level in cells with RUNX1 knockdown using qRT-PCR, and analyzed the response elements of a cohort of transcription factors within a 2 kb region upstream of the miR-101 precursor start site using the online software "The JASPAR database";10. ChIP-qPCR assays were performed to confirm the direct association of RUNX1 with the miR-101 promoter;11. To observe whether the 2 kb region indeed has promoter activity, the 2 kb DNA was cloned into the pGL4 reporter plasmid and the luciferase reporter assay was performed.[Results]1. MiR-101 was primarily expressed in normal tissues with a 76.47% reduction in tumors; RUNX1 with a critically conserved binding site in the 3-UTR region of mRNA; RUNX1 mRNA levels in lung cancer tissues were much higher than in paired non-tumor tissues; miR-101 levels were highly decreased, whereas RUNX1 mRNA and protein was markedly elevated in lung cancer cells, when compared with MRC5 cells; miR-101expression was dramatically decreased in NSCLC tissues, especially from patients with metastasis; In contrast, RUNX1 mRNA levels were elevated in NSCLC tissues; miR-101 downregulation was accompanied by RUNX1 upregulation, whereas higher RUNX1 levels were seen in patients carrying lower miR-101 levels in fresh-frozen patient tissues, cell lines and formalin-fixed paraffin-embedded tissues.2. Transfection with miR-101 mimics successfully upregulated miR-101 levels with a significant decrease of RUNX1 mRNA and protetin, overexpressed miR-101 caused a remarkable decrease of luciferase activity driven by 3’-UTR in 95D or H1299 cells. Additionally, the lucifarase reporter assay performed in HEK293T cells showed miR-101 reduced the luciferase activity of the vector driven by 3’-UTR.3. Overexpressed miR-101 via transfection of miR-101 mimics significantly enhanced the sensitivity of 1299 and 95D cells to cDDP with marked decrease of IC50 values. In agreement, RUNX1 knockdown markedly sensitized H1299 and 95D cells to cDDP. To investigate whether miR-101 exerts its chemotherapy sensitizer role via suppressing RUNX1 expression, here H1299 cells and 95D were co-transfected with miR-101 mimics and RUNX-1 expressing plasmid pcDNA3.1-RUNX1without RUNX1-3’-UTR that significantly elevated RUNX1 protein level upon miR-101 overexpressed. Importantly, we found that ectopic RUNX1 expression significantly abolished miR-101 mediated chemotherapy sensitization.4. We found enforced miR-101 expression remarkably inhibited the invasion potential of H1299 and 95D cells determined by transwell assays. Coincidentally, RUNX1 knockdown significantly decreased the invasion potential of 95D and H1299 cells. Furthermore, we demonstrated that ectopic expressed RUNX1 obviously impaired miR-101 induced invasion inhibition in H1299 and 95D cells, suggesting RUNX1 overexpression is responsible for the effect of miR-101 downregulation on invasion in lung cancer cells.5. MiR-101 expression level in cells with RUNX1 knockdown and found miR-101 expression increased after RUNX1 knockdown in 95D and H1299 lung cancer cells and found three putative RUNX1 binding sites within 2 kb region upstream of the miR-101 precursor start site. RUNX1 most significantly bound to site A and site C. expectedly, RUNX1 knockdown decreased RUNX1 binding to miR-101 promoter in 95D and H1299 cells. Luciferase activity driven by the potential promoter of miR-101 was much higher in MRC5 cells than that in 95D or H1299 cells. Additionally, RUNX1 knockdown enhanced the luciferase activity in 95D and H1299 cells, but overexpressed RUNX1 attenuated the luciferase activity in MRC5 cells[Conclusions]1. The expression of miR-101 was decreased in lung cancer tissue, especially in the matestatic lung cancer.2. RUNX1 is a direct target of miR-101 in human lung cancer cells, and the transcription factor RUNX1 feedback transcriptionally regulates miR-101 in lung cancer cells.3. The miR-101/RUNXl feedback loop can attenuated the invasion ability and enhanced the sensitivity of chemotherapy drug of human lung carcinoma. |
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