| Lung cancer is the leading cause of cancer related deaths in China and worldwide,80% to 85% of lung cancers are currently classified as non-small cell lung cancer (NSCLC). The mainstay treatments for NSCLC include surgery, chemotherapy, radiotherapy and targeted therapy. Notwithstanding the advance of therapeutics, the outcome of lung cancer is still gloomy. Less than 17% of all patients diagnosed with lung cancer survive 5 years, most patients die because of metastasis diseases. Investigating the underlying mechanisms of lung cancer initiation and development may identify more effective therapeutic targets, thereby designing more effective targeted drugs.The epidermal growth factor receptor (EGFR) is one of four members of the ErbB family of type-1-tyrosine kinases, which also includes ErbB2/Her2, Her3, and Her4. EGFR is frequently aberrantly activated in a wide range of solid tumors including NSCLC. EGFR has been recognized as an effective anti-cancer target since 1983, and EGFR-targeted therapies, including monoclonal antibodies (mAbs) and small-molecule tyrosine kinase inhibitors (TKIs), have been successfully applied in clinical, showing promising outcome in selected patients. However, innate or acquired resistance to these EGFR-targeted therapies remains a great challenge, which results in treatment failure, necessitating more effective targeted strategies.microRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that regulate gene expression post-transcriptionally. miRNAs suppress gene expression by binding to the complementary sequences in the 3’untranslated region (3’UTR). In mammals, miRNAs are predicted to regulate the expression of about 50% of all protein-coding genes. miRNAs are involved in almost every cellular processes including cell proliferation and differentiation, cell cycle, cell death and apoptosis, cell migration and invasion. Accumulating evidence suggest that miRNAs are involved in many human diseases, especially cancer. Aberrant miRNAs expression profiles are frequently observed in cancers; these miRNAs function as oncogene or tumor suppressor, which renders them a potential target for anti-cancer treatment. miRNAs-based therapy has been suggested to be a rational and potential approach to target EGFR in anti-EGFR therapies. To date, a number of miRNAs, such as miR-7, miR-23b/27b and miR-133a, have been demonstrated to target EGFR directly. However, more anti-EGFR miRNAs need to be explored since one mRNA can be the target of hundreds of miRNAs, and combination of multiple tumor suppressive miRNAs targeting an individual gene could improve therapeutic effect by reducing resistance.In the present study, we aimed to identify novel EGFR targeting miRNAs, and determine the biological function of identified miRNA in NSCLC cells. We identified a novel EGFR-targeting miRNA, miR-134, in NSCLC cells, and demonstrated that miR-134 functions as a tumor suppressor in NSCLC by targeting EGFR and ITGB1, implying that miR-134 may be a potential target for NSCLC treatment.Part 1. Identify miRNAs that target EGFR directly[Objectives]We aimed to select potential miRNAs that target EGFR directly using computational algorithms. Among the predicted conserved miRNAs with good mirSVR scores, we focused on those miRNAs that function as tumor suppressors but have not been identified to regulate EGFR. The candidate miRNAs were tested to select a miRNA that suppresses EGFR protein expression most severely.[Methods]1. Computational algorithms (microrna.org, TargetScan) were used to select candidate miRNAs that target EGFR directly.2. Western blot was used to detect the expression of EGFR protein in A549 and H1299 cells after transfection with miRNAs mimics. miR-134 that inhibits EGFR expression most severely was selected for further experimental validation.3. Western blot was used to detect the expression of EGFR protein in four additional NSCLC cell lines (H460, H520, H1975 and PC9) after transfection with miR-134 mimics; qRT-PCR was used to detect the expression of EGFR mRNA.4. Dual luciferase reporter assays were performed to test that whether the 3’UTR of EGFR mRNA is the direct target of miR-134.5. Western blot was used to detect the alteration of EGFR signaling after transfection with miR-134 mimics.[Results]1. Three candidate miRNAs (miR-134, miR-200a and miR-373) were selected, with miR-7 as positive control.2. As presented by Western blot, in A549 and H1299 cells, EGFR protein expression levels were suppressed significantly 48 hours and 72 hours after transfection with miR-7 mimics. Among three tested miRNAs, miR-134 exerted the most severe inhibitory effect on EGFR expression in both cells 48 hours and 72 hours after transfection. Therefore, we chose miR-134 for further investigation.3. As shown by Western blot, miR-134 inhibited EGFR expression in H520 and H1975, but not in H460 and PC9. qRT-PCR demonstrated that transfected cells showed significantly high levels of miR-134 with inhibited EGFR mRNA expression in A549, H1299, H520 and H1975, but not in H460 and PC9.4. Dual luciferase reporter assays suggested that miR-134 regulated EGFR expression by targeting 3’UTR of EGFR mRNA directly.5. As shown by Western blot, miR-134 down-regulated p-EGFR in NSCLC cell lines (A549, H1299, H520 and H1975), and suppressed specific EGFR-associated signaling in a cellular context dependent way.[Conclusions]1. Over-expression of miR-134 down-regulates EGFR mRNA and protein expression levels in NSCLC cell lines (A549, H1299, H520 and H1975).2. miR-134 regulates EGFR expression by targeting 3’UTR of EGFR mRNA directly.3. In NSCLC cell lines (A549, H1299, H520 and H1975), over-expression of miR-134 down-regulated p-EGFR, and suppressed specific EGFR-associated signaling in a cellular context dependent way, suggesting that miR-134 may function as a tumor suppressor in NSCLC.Part 2. miR-134 inhibits NSCLC cells proliferation and migration as well as invasion in vitro[Objectives]Based on the conclusion of part 1 that miR-134 may function as a tumor suppressor, we further investigated whether miR-134 could inhibit NSCLC cells proliferation and migration as well as invasion in vitro, and explored the underlying mechanisms.[Methods]1. MTT assays were used to detect the impact of miR-134 on NSCLC (A549, H1299, H520 and H1975) cells proliferation. Cell death and apoptosis analysis was performed to detect the impact of miR-134 on cell death, and cell cycle analysis was used to determine the impact of miR-134 on cell cycle.2. Transwell migration and invasion assays were used to detect the impact of miR-134 on NSCLC (A549 and H1299) cells migration and invasion.3. RNAi and functional rescue experiments were performed to test that whether EGFR down-regulation is responsible for the tumor suppressive function of miR-134, which means whether EGFR is a functional target of miR-134.4. Computational algorithms (microrna.org, TargetScan) in combination with literature review were used to identify other functional targets of miR-134.5. RNAi and functional rescue experiments were performed to test whether ITGB1 is a functional target of miR-134 in NSCLC.[Results]1. In NSCLC cell lines (A549, H1299, H520, H1975), transfection of miR-134 significantly inhibited cells proliferation by inducing cell apoptosis and/or cell cycle arrest in G0/G1 phase.2. As shown by Transwell assays, miR-134 significantly inhibited migration as well as invasion in A549 and H1299 cells.3. EGFR knockdown in A549 and H1299 cells significantly suppressed cell proliferation and migration as well as invasion. Functional rescue experiments confirmed that only the cell proliferation suppressive function of miR-134 is mediated by down-regulating EGFR.4. After integrating both targets prediction as well as literature review, three pertinent oncogenes, KRAS, FOXM1 and ITGB1, were selected for further mRNA validation. qRT-PCR suggested that ITGB1 mRNA expression levels were suppressed more severely than KRAS and FOXM1 by miR-134 in A549 and H1299 cells. Therefore, ITGB1 was selected for Western blot validation, which further confirmed the down-regulation of ITGB1 protein by miR-134.5. RNAi experiments confirmed that knocking down of ITGBI could significantly inhibit cell proliferation and migration as well as invasion, mimicing the phenotype of miR-134 over-expression. Functional rescue experiments in A549 and H1299 cells stably expressing ITGB1 (A549-ITGB1 and H1299-ITGB1) further elucidated that ITGB1 is the functional target of miR-134. In A549-ITGB1 and H1299-ITGB1 cells, the tumor suppressive effects of miR-134, including inhibition of cell proliferation and migration as well as invasion, were partly abolished.[Conclusions]1. miR-134 inhibits NSCLC cells proliferation and migration as well as invasion, suggesting that miR-134 functions as a tumor suppressor in NSCLC.2. Down-regulation of EGFR by miR-134 partly contributes to the proliferation suppression of miR-134 in NSCLC, but does not involve in migration and invasion suppressive role of miR-134.3. ITGB1 is another important functional target of miR-134, which involves in the tumor suppressive effects of miR-134 in NSCLC.4. miR-134 functions as a tumor suppressor in NSCLC by targeting EGFR and ITGB1.Part 3. miRNA-134 suppresses NSCLC growth and metastasis in vivo[Objectives]Based on the conclusion of part 2 that miR-134 functions as a tumor suppressor in NSCLC by targeting EGFR and ITGBI in vitro, we continued to test the tumor suppressive role of miR-134 in vivo.[Methods]1.4-5 weeks old female nude mice were used to establish A549 lung cancer xenograft model. When the diameter of tumor reached about 5-6 mm, these nude mice were randomly divided into two groups. miR-134 agomir or negative control (NC) agomir was then directly injected into the implanted tumor at the dose of 5nmol per mouse every 3 days for five times. Tumor volume was monitored every 3 days, and tumor growth curves were painted.2.48 hours after the last injection, animals were sacrificed and tumor tissues were resected. qRT-PCR was performed to detect the expression of miR-134, Western blot was used to detect the protein expression of EGFR and ITGB1.3. Tumor tissues were fixed in formalin and imbedded in paraffin. Sections of 5μm were cut from the embedded tissues and mounted on polylysine-coated slides. IHC staining was used to detect the expression of EGFR, ITGB1, Ki-67 (marker of cell proliferation), Cleaved PARP (marker of apoptosis), E-cadherin and Vimentin (EMT markers).[Results]1. miR-134 agomir suppressed tumor growth of A549 xenograft in nude mice.2. qRT-PCR confirmed elevated expression of miR-134 in A549 xenografts injected with miR-134 agomir. Western blotting and IHC showed decreased expression of EGFR and ITGB1.3. IHC showed that, in A549 xenografts injected with miR-134 agomir, the expression of Ki-67 was decreased, whereas the expression of Cleaved PARP was increased, meanwhile, the expression of epithelial marker E-cadherin was increased, and the expression of mesenchymal marker vimentin was decreased.[Conclusions]1. miR-134 could inhibit A549 tumor growth in vivo, which renders it a potential target for NSCLC treatment.2. miR-134 could down-regulate EGFR and ITGB1 simultaneously, which results in decreased proliferation, increased apoptosis, and reversed EMT, ultimately leading to suppressed tumor growth and metastasis in vivo. |
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