Inhibition of OGG1 ameliorates pulmonary fibrosis via preventing M2 macrophage polarization and activating PINK1-mediated mitophagy.

BACKGROUND: 8-Oxoguanine DNA glycosylase (OGG1), a well-known DNA repair enzyme, has been demonstrated to promote lung fibrosis, while the specific regulatory mechanism of OGG1 during pulmonary fibrosis remains unclarified. METHODS: A bleomycin (BLM)-induced mouse pulmonary fibrosis model was established, and TH5487 (the small molecule OGG1 inhibitor) and Mitochondrial division inhibitor 1 (Mdivi-1) were used for administration. Histopathological injury of the lung tissues was assessed. The profibrotic factors and oxidative stress-related factors were examined using the commercial kits. Western blot was used to examine protein expression and immunofluorescence analysis was conducted to assess macrophages polarization and autophagy. The conditional medium from M2 macrophages was harvested and added to HFL-1 cells for culture to simulate the immune microenvironment around fibroblasts during pulmonary fibrosis. Subsequently, the loss- and gain-of function experiments were conducted to further confirm the molecular mechanism of OGG1/PINK1. RESULTS: In BLM-induced pulmonary fibrosis, OGG1 was upregulated while PINK1/Parkin was downregulated. Macrophages were activated and polarized to M2 phenotype. TH5487 administration effectively mitigated pulmonary fibrosis, M2 macrophage polarization, oxidative stress and mitochondrial dysfunction while promoted PINK1/Parkin-mediated mitophagy in lung tissues of BLM-induced mice, which was partly hindered by Mdivi-1. PINK1 overexpression restricted M2 macrophages-induced oxidative stress, mitochondrial dysfunction and mitophagy inactivation in lung fibroblast cells, and OGG1 knockdown could promote PINK1/Parkin expression and alleviate M2 macrophages-induced mitochondrial dysfunction in HFL-1 cells. CONCLUSION: OGG1 inhibition protects against pulmonary fibrosis, which is partly via activating PINK1/Parkin-mediated mitophagy and retarding M2 macrophage polarization, providing a therapeutic target for pulmonary fibrosis.

Xanthatin alleviates airway inflammation in asthmatic mice by regulating the STAT3/NF-κB signaling pathway.

Here, we aimed to investigate the role of Xanthatin in asthma and its underlying mechanism. BALB/c mice were treated with ovalbumin (OVA) to establis a mouse model of asthma. Our results showed that OVA injection significantly increased inflammatory cell infiltration and goblet cell hyperplasia in lung issues, while Xanthatin treatment and STAT3 inhibitor C188-9 administration relieved these symptoms. Moreover, OVA-induced OVA-specific immunoglobulin E level in serum and the number of total cell, macrophages, lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid (BALF) were markedly reduced by Xanthatin treatment and signal transducer and activator of transcription 3 (STAT3) inhibition. Additionally, Xanthatin treatment and STAT3 inhibition was also significantly decreased the levels of inflammatory cytokines in BALF in asthmatic mice. We further demonstrated that the STAT3/nuclear factor-kappaB (NF-κB) pathway was blocked by Xanthatin in asthmatic mice. Overall, we conclude that Xanthatin attenuates airway inflammation in asthmatic mice through blocking the STAT3/NFκB signaling pathway, indicating the potential of Xanthatin as a useful therapeutic agent for asthma.

miR-363-3p inhibits migration, invasion, and epithelial-mesenchymal transition by targeting NEDD9 and SOX4 in non-small-cell lung cancer.

miR-363-3p is downregulated in lung adenocarcinoma and can inhibit tumor growth. Here, we aimed to investigate the effect of miR-363-3p on non-small-cell lung cancer (NSCLC) metastasis. In our study, miR-363-3p overexpression inhibited cell migration and invasion via epithelial-mesenchymal transition inhibition, while miR-363-3p knockdown exhibited the opposite effects. Further studies demonstrated that miR-363-3p bound to 3'-untranslated regions of NEDD9 and SOX4, and negatively regulated their levels. Interestingly, NEDD9 or SOX4 knockdown rescued the metastasis-promoting effects of antagomiR-363-3p. The inhibitory effects of agomiR-363-3p were also blocked by NEDD9 or SOX4 overexpression. Moreover, lentivirus particles carrying pre-miR-363 (LV-pre-miR-363) significantly decreased, while LV-miR-363-3p inhibitor increased metastatic nodule numbers and the levels of NEDD9 and SOX4 in lungs. In conclusion, tumor suppressor miR-363-3p may be a potential target in NSCLC therapy.

CASC15 contributes to proliferation and invasion through regulating miR-766-5p/ KLK12 axis in lung cancer.

Long non-coding RNAs (lncRNAs) are key mediators of cancer. The dysregulation of a lncRNA, CASC15, has been linked to several cancers, except lung cancer. Here, the aim of the study was to explore the role and mechanism of CASC15 in lung cancer regulation, with the focus on its interaction with a potential target, microRNA-766-5p (miR-766-5p) and an oncogene, kallikrein-related peptidase 12 (KLK12). Quantitative real-time PCR (qRT-PCR) was used to assess levels of CASC15, miR-766-5p and KLK12 in lung cancer tissues or cells. Western blot analysis was used to detect KLK12 protein expression. Ectopic expression of CASC15 was induced by a lentiviral system. CCK-8 and transwell assays were used to evaluate lung cancer cell proliferation and invasion, respectively. The interaction among CASC15, miR-766-5p and KLK12 was investigated by bioinformatical analysis and luciferase assay. In lung cancer tissue and cells, CASC15 was upregulated, while miR-766-5p was downregulated. Overexpression of CASC15 promoted lung cancer cell proliferation and invasion. A negative correlation was found between CASC15 and miR-766-5p levels. Overexpression of miR-766-6p reversed the cancer-promoting role of CASC15 in lung cancer cells, which was mediated by KLK12. The tumor-promoting role of CASC15 and tumor-suppressing role of miR-766-5p were also validated in vivo in tumor bearing mice, and KLK12 was also shown as an important mediator. CASC15 promotes lung cancer through the miR-766-5p/KLK12 axis, indicating that CASC15 is a potential therapeutic in lung cancer.

MicroRNA-206 promotes lipopolysaccharide-induced inflammation injury via regulation of IRAK1 in MRC-5 cells.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play crucial role in the airway inflammatory diseases. However, the involvement of miR-206 in airway inflammatory diseases is still uninvestigated. The study aimed to explore the effect of miR-206 on lipopolysaccharide (LPS)-induced inflammation injury in MRC-5 cells, and point out a potential relevance for chronic obstructive pulmonary disease (COPD). METHODS: LPS was utilized to expose MRC-5 cells, then cell viability, cell migration, apoptosis, apoptosis-associated factors, as well as the concentrations and protein levels of IL-6 and IL-8 were explored. After transfected with miR-206 mimic and inhibitor, above parameters were reassessed in LPS-injured cells. Expression level of IRAK1 was examined in miR-206 mimic/inhibitor transfected cells by using RT-qPCR. The effect of IRAK1 on LPS-induced inflammation injury was investigated in MRC-5 cells after transfection with pc-IRAK1 and sh-IRAK1. The effects of miR-206 and IRAK1 on MEK/ERK and JNK pathways were determined by western blot assay. RESULTS: LPS significantly triggered inflammation injury in MRC-5 cells by inhibiting cell viability, suppressing the healing of scratches, inducing cell apoptosis, down-regulating Bcl-2 expression and up-regulating Bax, cleaved-Caspase-3 and cleaved-Caspase-9 expression, and concurrently increasing the concentrations and the protein levels of IL-6 and IL-8. MiR-206 overexpression aggravated LPS-induced inflammation injury in MRC-5 cells. Up-regulation of IRAK1 was observed in miR-206 mimic-transfected cells. Moreover, IRAK1 overexpression promoted LPS-induced inflammation injury in MRC-5 cells. MiR-206 activated MEK/ERK and JNK pathways by regulating IRAK1. CONCLUSIONS: MiR-206 promotes LPS-induced inflammation injury through regulation of IRAK1 in MRC-5 cells.

The positive feedback loop of lncRNA DANCR/miR-138/Sox4 facilitates malignancy in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most frequent cancers worldwide. The abnormal expression of long non-coding RNAs (lncRNAs) has been reported to be closely associated with the progression of human cancers, including NSCLC. Here, we demonstrated that differentiation antagonizing noncoding RNA (DANCR) was overexpressed in NSCLC tissues. Upregulation of DANCR expression was significantly associated with larger tumor size, advanced TNM stage and lymph node metastasis, and also predicted poor prognoses of patients with NSCLC. Functional experiments showed that DANCR enhanced NSCLC growth and metastasis both in vitro and in vivo. Further investigation revealed that DANCR could compete with the Sox4 mRNA to bind with miR-138, thus affecting Sox4 expression. In addition, we found that Sox4 bound to the promoter regions of DANCR gene to activate DANCR expression, suggesting a positive feedback loop of DANCR/miR-138/Sox4 in NSCLC. Taken together, these results provide a comprehensive analysis of the roles of DANCR as a competing endogenous RNA (ceRNA) in NSCLC progression.

Reduced interleukin-38 in non-small cell lung cancer is associated with tumour progression.

Lung cancer continues to be the leading cause of cancer-related deaths worldwide due to its high incidence, malignant behaviour and lack of major advancements in treatment strategy. The occurrence and development of lung cancer is closely related to inflammation. Thus, we conducted the present study to investigate the effects of IL-38 (interleukin-38), a newly identified anti-inflammatory factor, on non-small cell lung cancer (NSCLC), which accounts for about 85% of all lung cancers. We first evaluated the IL-38 expression in 384 pairs of NSCLC samples and their adjacent normal mucosa by real-time PCR, ELISA (enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-38 on patient survival rates, cancer progression and their sensitivity to chemotherapy drugs was assessed. IL-38 was barely expressed in the NSCLC tissues but highly expressed in the adjacent normal tissues. The downregulation of IL-38 was significantly correlated with the results of the American Joint Committee on Cancer stage and degree of differentiation, and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with NSCLC. Overexpression of IL-38 in NSCLC cells suppressed cell migration, invasion, proliferation and colony formation through suppressing ß-catenin. IL-38 inhibited NSCLC formation in a mice model and sensitized the cancer cells to chemotherapy drugs. Our results show that IL-38 plays an inhibitory role in NSCLC development and functions as a novel prognostic indicator and a potential therapeutic target.

MALAT1/miR-101-3p/MCL1 axis mediates cisplatin resistance in lung cancer.

In this study, we investigated the mechanism by which lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) mediates cisplatin resistance in lung cancer. Lung cancer patients with high MALAT1 levels were associated with cisplatin resistance and low overall survival. Moreover, cisplatin-resistant A549/DDP cells showed higher MALAT1 expression than cisplatin-sensitive lung cancer cells (A549, H460, H1299 and SPC-A1). Dual luciferase reporter and RNA immunoprecipitation assays showed direct binding of miR-101-3p to MALAT1. MALAT1 knockdown in lung cancer cells resulted in miR-101-3p upregulation and increased cisplatin sensitivity. In addition, miR-101-3p decreased myeloid cell leukemia 1 (MCL1) expression by binding to the 3'-untranslated region (3'-UTR) of its mRNA. These results demonstrate that MALAT1/miR-101-3p/MCL1 signaling underlies cisplatin resistance in lung cancer.

Silencing of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human lung cancer cells.

Activating enhancer-binding protein (AP)-4 is a member of the basic helix-loop-helix transcription factors, and is involved in tumor biology. However, the role of AP-4 in human lung cancer remains to be fully elucidated. In the present study, the expression of AP-4 in human lung cancer tissues and cells was investigated by reverse transcription-quantitative polymerase chain reaction, and it was observed that the level of AP-4 was increased in tumor tissues and cells compared with their normal counterparts. AP-4 expression was knocked down by transfection with a specific small interfering RNA (siRNA) in lung cancer cells, and this indicated that siRNA-mediated silencing of AP-4 inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase and induced apoptosis by modulating the expression of p21 and cyclin D1. The results of the present study suggest that AP-4 may be an oncoprotein that has a significant role in lung cancer, and that siRNA-mediated silencing of AP-4 may have therapeutic potential as a strategy for the treatment of lung cancer.

Study on expression of lncRNA RGMB-AS1 and repulsive guidance molecule b in non-small cell lung cancer.

BACKGROUND: The relationships between lncRNAs and tumors have currently become one of the focuses on cancer studies. However, there are a few studies about lncRNAs in non-small cell lung cancer (NSCLC) at present. METHODS: Microarray analysis was designed to study the expression patterns of lncRNAs in three pairs of NSCLC tissues. The expression of lncRNA RGMB-AS1 and repulsive guidance molecule b (RGMB) were detected in 72 paired NSCLC tissues and adjacent normal tissues by qRT-PCR assay. The relations of lncRNA RGMB-AS1 and RGMB expression with clinicopathological factors of NSCLC patients were explored. A549 and SPC-A-1 cells were transfected with siRNA of lncRNA RGMB-AS1 and negative control. RGMB expression level was detected by qRT-PCR assay and western blot analysis. RESULTS: The results of microarray found that 571 lncRNAs were differentially expressed in NSCLC tissues (Fold change cut-off: 5.0, P < 0.05), including 304 upregulated and 267 downregulated lncRNAs. The results of qRT-PCR showed that lncRNA RGMB-AS1 expression was significantly higher in NSCLC tissues than in adjacent normal tissues (P < 0.05), while RGMB mRNA showed an opposite trend (P < 0.05). Correlation analysis indicated that the expression of lncRNA RGMB-AS1and RGMB mRNA were inversely correlated (R(2) = 0.590, P < 0.05). While lncRNA RGMB-AS1 and RGMB expression levels in NSCLC tissues were associated with the occurrence of differentiation status, lymph node metastases and TNM stage (P < 0.05). Transfection with siRNA of lncRNA RGMB-AS1, subsequent results showed that RGMB mRNA and protein expression were upregulated (P < 0.05) in A549 and SPC-A-1 cells compared to the control groups. CONCLUSION: We identified lncRNA RGMB-AS1 was upregulated and RGMB was downregulated in NSCLC patients. Both were related to differentiation status, lymph node metastases and TNM stage. Studies also indicated that lncRNA RGMB-AS1and RGMB were inversely correlated. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7911587521528276.

MiR-144 inhibits proliferation and induces apoptosis and autophagy in lung cancer cells by targeting TIGAR.

BACKGROUND: MiRNAs are noncoding RNAs of 20-24 nucleotides that function as post-transcriptional negative regulators of gene expression. MiRNA genes are usually transcribed by RNA polymerase II in the nucleus. Their initial products are pre-miRNAs which have cap sequences and polyA tails. The p53-induced glycolysis and apoptosis regulator (TIGAR) was discovered through microarray analysis of gene expression following activation of p53. However, little is known about the effect of miR-144 on cell proliferation and apoptosis and how it interacts with TIGAR. METHODS: We performed real-time PCR, western blotting, CCK8, colony formation, tumor growth, flow cytometry, Caspase3/7 activity, Hoechst 33342 staining, MDC staining of autophagic cells and luciferase reporter assays to detect the influence of miR-144 to lung cancer cells. RESULTS: miR-144 targeted TIGAR, inhibited proliferation, enhanced apoptosis, and increased autophagy in A549 and H460 cells. CONCLUSIONS: Our study improves our understanding of the mechanisms underlying lung cancer pathogenesis and may promote the development of novel targeted therapies.

Silencing BMP-2 expression inhibits A549 and H460 cell proliferation and migration.

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) is a member of the TGF-ß superfamily that is closely correlated with many malignancies, particularly lung cancer. However, the effects of silenced BMP-2 on lung cancer cell proliferation and migration are not clear. METHODS: Using quantitative real-time RT-PCR, BMP-2 mRNA expression was detected in 61 non-small cell lung cancer (NSCLC) samples. Survival curves were generated using follow-up data. Relationships between clinical or pathological characteristics and prognosis were analyzed. Cell viability assays and transwell migration assays were used to evaluate the effects of BMP-2 silencing on cell proliferation and migration of A549 and H460 cells. RESULTS: BMP-2 mRNA expression was higher in NSCLC tissues compared to matched adjacent normal tissues (P<0.01). High BMP-2 expression levels were significantly associated with the occurrence of lymph node metastases and tumor stage (P<0.05). There were significant differences in survival curves between groups with metastatic lymph nodes and non-metastatic lymph nodes, as well as between groups with low BMP-2 expression and groups with high BMP-2 expression. In addition, we observed decreased proliferation and migration rates of the NSCLC-derived cell lines A549 and H460 that were transfected with siBMP-2 (P<0.05). CONCLUSION: BMP-2 mRNA is overexpressed in NSCLC samples and is a risk factor for survival in patients with NSCLC. BMP-2 silencing can significantly inhibit A549 and H460 cell proliferation and migration. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4263254471298866.

Up-regulation of microRNA-138 induce radiosensitization in lung cancer cells.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to identify the association between miR-138 and Sentrin/SUMO-specific protease 1 (SENP1) as a radiosensitization-related gene and characterize the biological function by which SENP1 was regulated by miR-138 to influence radiosensitization in lung cancer cells. In this study, we showed that miRNA-138 is reduced in both lung cancer clinical specimens and cell lines and is effective to inhibit SENP1 expression. Moreover, high levels of miR-138 are associated with lower levels of lung cancer cell proliferation and colony formation. Then, we investigated the underlying mechanisms responsible for the increase in the radiosensitivity of lung cancer cells when SENP1 is inhibited by miR-138. We further show that the increased radiosensitivity may be the result of an increased γ-H2AX expression, an increased rate of apoptosis, and changes in the cell cycle. In conclusion, our data demonstrate that the miR-138/SENP1 cascade is relative to radiosensitization in lung cancer cells and is a potential radiotherapy target.

Expression analysis of serum microRNAs in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology with considerable morbidity and mortality. Seeking informative diagnostic markers with greater clinical significance is essential for the early diagnosis of IPF. microRNAs (miRNAs or miRs) have emerged as novel serum diagnostic biomarkers for various diseases. In this study, we performed microarray analysis of the miRNA expression profile in the serum of patients with IPF compared to that of control subjects. We then performed a preliminary analysis of biological functions for the most differentially expressed miRNAs. Some of the microarray results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results from this study provide evidence to link the biological role of miRNAs to IPF, and suggest that miRNAs may undertake a variety of functions. Additionally, we found that the altered expression levels of miR-21, miR-155 and miR-101-3p were associated with forced vital capacity (FVC) and radiological features in IPF. Our data may serve as a basis for further investigation, preferably in large prospective studies, before miRNA can be used as a non-invasive screening tool for IPF in routine clinical practice.

Downregulation of microRNA-182 inhibits cell growth and invasion by targeting programmed cell death 4 in human lung adenocarcinoma cells.

Lung cancer is a major cause of cancer death worldwide. Programmed cell death 4 (PDCD4), an important tumor suppressor, influences transcription and translation of multiple genes and modulates different signal transduction pathways. However, the upstream regulation of this gene is largely unknown. In our study, we found that microRNA-182 (miR-182) was upregulated, whereas PDCD4 was downregulated in lung cancer cell lines. We performed methyl thiazolyl tetrazolium and colony formation assays to study the influence of miR-182 on proliferation of the lung cancer cell lines A549 and SPC-A-1. We also carried out Transwell and wound healing assays to investigate the effect of miR-182 on invasion and migration of A549 and SPC-A-1. Finally, using the luciferase reporter assay and restore assay, we demonstrated that PDCD4 is a direct target of miR-182. These results suggest that in lung adenocarcinoma cells, miR-182 plays an oncogenic role as a direct negative regulator of PDCD4.

MiR-495 regulates proliferation and migration in NSCLC by targeting MTA3.

Our previous studies have showed that metastasis-associated protein 3 (MTA 3) is overexpressed in non-small cell lung cancer (NSCLC) tissue, and increased MTA3 mRNA levels is a risk factor of lymph node metastasis. Using bioinformatics analyses, we found that MTA3 was a potential target of miR-495. However, the pathophysiological role of miR-495 and its relevance to the growth and development of NSCLC have yet to be investigated. The purpose of this study was to elucidate the molecular mechanisms by which miR-495 acts as a tumor suppressor in NSCLC. qRT-PCR data showed significant downregulation of miR-495 in 56 NSCLC tissue samples and 5 lung cancer cell lines, compared with their adjacent normal tissue; furthermore, western blotting analysis revealed MTA3 protein was overexpressed in the tumor samples compared with the matched adjacent normal tissue. MiR-495 was shown to not only inhibit the proliferation of lung cancer cells (A549 and Calu-3) but also to inhibit cell migration in vitro. Using western blotting and luciferase assays, MTA3 was identified as a target of miR-495. These findings suggest the importance of miR-495 targeting of MTA3 in the regulation of lung cancer growth and migration.

Analysis of MAT3 gene expression in NSCLC.

BACKGROUND: Many studies have suggested different roles of Metastasis-associated protein 3 (MAT3) in different types of human cancers. However, expression of MAT3 in primary lung cancer and its relationship with clinicopathological factors have not been examined and the biological roles of MTA3 in lung cancer cells are still unclear. METHODS: The expression of MAT3 mRNA and protein were detected with quantitative real-time RT-PCR and immunohistochemical methods in 118 NSCLC samples and corresponding non-neoplastic samples. Survival curves were made with follow-up data. The relations of the prognosis with clinical and pathological characteristics were analyzed. RESULTS: The expression level of MAT3 mRNA and the positive rate of MAT3 protein were significantly higher in NSCLC samples than that in non-neoplastic samples, and in NSCLC samples with lymph node metastasis than that in NSCLC samples without lymph node metastasis (P < 0.01). MAT3 mRNA expression level was a risk factor of lymph node metastasis in patients with NSCLC (P = 0.006). There were significant differences in survival curves between lymph node metastatic group and non-metastatic group (P = 0.000), among groups of MAT3 positive and negative (P = 0.000), among groups of TNM stage I, II and III (P = 0.000) and among groups of tumor status T1, T2 and T3T4 (P = 0.000); but no statistical significance between male patients and female patients (P = 0.516), between ≥ 60 years old patients and <60 years old patients (P = 0.133), between histology types adenocarcinoma and squamous cell carcinoma (P = 0.865) and between well differentiation and moderate-poor differentiation (P = 0.134). The level of MAT3 mRNA (P = 0.000) and protein (P = 0.000) were risk factors of survival. CONCLUSION: Our study showed that MAT3 over-expression in NSCLC tissue, and MAT3 mRNA level is a risk factor of lymph node metastasis. The level of MAT3 mRNA and protein were risk factors of survival in patients with NSCLC. It suggested that this antigen could be used as a simple and efficient parameter with which to identify high-risk patients. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5585901065503943.

Identification of miR-1293 potential target gene: TIMP-1.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a glycosylated protein with multiple activities in the regulation of biological processes, such as cell growth and apoptosis as well as tumor invasion and metastasis. Bioinformatics analysis using TargetScan and miRanda suggested tissue inhibitors of TIMP-1 are among the targets of miR-1293. To confirm this, we cloned both wild-type and mutant TIMP-1 3'UTR fragments by overlap extension PCR, constructed the recombinant plasmids pGL3-TIMP-1-wt, -mut, and pcDNA 3.1(+)/TIMP-1-CDS and, respectively, co-transfected them into 293T cells with the miR-1293 inhibitor, mimics or the miR inhibitor-NC using a BTX ECM 2001 square-wave electroporator. We used a luciferase assay to investigate binding of miR-1293 to the 3'UTR of TIMP-1. Effects on the levels of the TIMP-1 protein were analyzed by Western blot experiments. The luciferase reporter assay showed a statistically significant (P < 0.05) upregulation of activity. Western blot analysis showed a significant increase of expression of the TIMP-1 gene co-transfected with the miR-1293 inhibitor, and demonstrated direct binding of miR-1293 to the 3'UTR of TIMP-1. In this study, we identified TIMP-1 as a novel direct target for miR-1293, which provides the basis for further study of the multifunctional mechanisms of miR-1293 and TIMP-1 in the regulation of a variety of diseases.