Elucidation of the anti-NSCLC mechanism of flavonoid derivative JW4 by an integrative approach of network pharmacology and experimental verification.

P300 reduces TUBB4B expression to facilitate the biological process of migration and invasion of non-small cell lung cancer cells

Long non-coding RNAs are involved in the tumorigenesis of non-small cell lung cancer (NSCLC). Here we investigated whether LINC00476 affects the proliferation, invasion and migration of NSCLC cells via the SETDB1-activated Wnt/β-catenin pathway. The expression of LINC00476, SETDB1, Wnt1 and β-catenin were determined in NSCLC tumor tissues and the paired adjacent tissues, as well as in NSCLC cell lines and bronchial epithelioid cell lines. Cell proliferation, invasion and migration were determined using cell counting kit-8 assay and transwell assay. The relationship between LINC00476 and SETDB1 was elucidated using RNA pull-down, RNA immunoprecipitation and ubiquitination assays. LINC00476 was found to be significantly downregulated, while SETDB1, Wnt1 and β-catenin were upregulated in NSCLC tumor tissues and cell lines compared to the normal ones. Overexpression of LINC00476 promoted the proliferation, invasion and migration of NSCLC cells, and suppressed tumor growth in the mouse xenograft. Meanwhile, overexpression of LINC00476 induced the degradation of SETDB1 by promoting its ubiquitination. The simultaneous overexpression of LINC00476 and SETDB1 negated the inhibition of LINC00476 overexpression on the proliferation, invasion and migration of NSCLC cells. In conclusion, these findings indicate that LINC00476 acts as a tumor suppressor in NSCLC by downregulating SETDB1, which provides a novel target in the treatment of NSCLC.

BackgroundMicroRNAs (miRNAs) are short, non-coding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Although miR-196a has been implicated in several other cancers, its role in non-small cell lung cancer (NSCLC) is unknown. The aim of the present study was to examine the expression pattern of miR-196a in NSCLC and its clinical significance, as well as its biological role in tumor progression.MethodsExpression of miR-196a was analyzed in 34 NSCLC tissues and five NSCLC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of DNA methylation on miR-196a expression was investigated by 5-aza-2-deoxy-cytidine treatment and bisulfite sequencing. The effect of miR-196a on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Analysis of target protein expression was determined by western blotting. Luciferase reporter plasmids were constructed to confirm the action of miR-196a on downstream target genes, including HOXA5. Differences between the results were tested for significance using Student’s t-test (two-tailed).ResultsmiR-196a was highly expressed both in NSCLC samples and cell lines compared with their corresponding normal counterparts, and the expression of miR-196a may be affected by DNA demethylation. Higher expression of miR-196a in NSCLC tissues was associated with a higher clinical stage, and also correlated with NSCLC lymph-node metastasis. In vitro functional assays demonstrated that modulation of miR-196a expression affected NSCLC cell proliferation, migration and invasion. Our analysis showed that miR-196a suppressed the expression of HOXA5 both at the mRNA and protein levels, and luciferase assays confirmed that miR-196a directly bound to the 3’untranslated region of HOXA5. Knockdown of HOXA5 expression in A549 cells using RNAi was shown to promote NSCLC cell proliferation, migration and invasion. Finally, we observed an inverse correlation between HOXA5 and miR-196a expression in NSCLC tissues.ConclusionsOur findings indicate that miR-196a is significantly up-regulated in NSCLC tissues, and regulates NSCLC cell proliferation, migration and invasion, partially via the down-regulation of HOXA5. Thus, miR-196a may represent a potential therapeutic target for NSCLC intervention.

Numerous microRNAs (miRs) are dysregulated in non‑small cell lung cancer (NSCLC), serving pivotal roles in its formation and progression. miR‑625 is dysregulated in several types of human cancer, but its involvement in the formation and development of NSCLC remains poorly understood. In the present study, we aimed to investigate miR‑625 expression in NSCLC and its role in regulating NSCLC cell behavior. miR‑625 expression in NSCLC tissues and cell lines was detected using reverse transcription‑quantitative polymerase chain reaction. The effects of miR‑625 overexpression on NSCLC cell proliferation, apoptosis, migration and invasion in vitro were assessed using an MTT assay, flow cytometry, and cell migration and invasion assays, respectively. The effects of miR‑625 upregulation on NSCLC growth were evaluated in an in vivo xenograft model. The molecular mechanisms underlying the tumor‑suppressing roles of miR‑625 in NSCLC were explored in detail. miR‑625 expression was determined to be downregulated in NSCLC tissues and cell lines. This decreased expression was associated with advanced clinical features and poor overall survival of patients with NSCLC. Exogenous miR‑625 expression suppressed NSCLC cell proliferation, migration and invasion, and induced apoptosis in vitro. miR‑625 upregulation hindered NSCLC tumor growth in vivo. Homeobox B5 (HOXB5) was proposed to be the direct target gene of miR‑625 in NSCLC cells. The tumor‑suppressing effects of HOXB5 silencing were similar to those of miR‑625 overexpression in NSCLC cells. In rescue experiments, HOXB5 overexpression partially reversed the inhibitory effects of miR‑625 in NSCLC cells. miR‑625 upregulation directly targeted HOXB5 to deactivate the Wnt/β‑catenin signaling pathway in NSCLC cells in vitro and in vivo. miR‑625 was determined to be associated with HOXB5 suppression and Wnt/β‑catenin pathway deactivation, which in turn inhibited the aggressive behavior of NSCLC cells in vitro and in vivo.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-associated mortality worldwide. Non-small cell lung cancer (NSCLC) is the predominant type of lung cancer, and accounts for ~85% of all lung cancer cases. An increasing number of studies suggest that microRNAs (miRs) may be involved in the regulation of NSCLC carcinogenesis and progression. However, the expression and function of miRNA-219 in NSCLC, and its underlying mechanisms of action, remain unknown. In the present study, miR-219 expression in NSCLC tissues and cell lines was determined using reverse transcription-quantitative polymerase chain reaction. Following transfection with miR-219 mimics, the effects of miR-219 overexpression on NSCLC cell proliferation, migration and invasion were examined. Furthermore, the miR-219 target in NSCLC was investigated. miR-219 was observed to be downregulated in NSCLC tissues and NSCLC cell lines. In addition, miR-219 was demonstrated to function as a tumor suppressor in NSCLC, through inhibiting cell proliferation, migration and invasion invitro. Furthermore, high mobility group AT-hook 2 (HMGA2) was identified to be a direct target of miR-219 in NSCLC, and downregulation of HMGA2 suppressed NSCLC cell proliferation, migration and invasion invitro. HMGA2 expression was upregulated in NSCLC tissues, and was inversely correlated with miR-219 expression. In conclusion, miR-219 functions as a tumor suppressor and may be important in inhibiting the growth and metastasis of NSCLC cells via directly targeting HMGA2. Therefore, miR-219 may present a potential novel therapeutic target for NSCLC.

It has been certified that GABPB1-AS1 is aberrantly expressed and plays as a vital role in some kinds of cancers. However, its expression pattern and functions in non-small cell lung cancer (NSCLC) are still largely unknown. This study aims to assess GABPB1-AS1 expression and biological roles in NSCLC. The expression of GABPB1-AS1 was detected in NSCLC specimens and adjacent normal specimens. CCK8 and Transwell assays were performed to evaluate the effects of GABPB1-AS1 on NSCLC cell proliferation, migration and invasion. Bioinformatics tools and luciferase reporter assays were applied to predict and verify GABPB1-AS1's direct targets. The results revealed that GABPB1-AS1 is sharply reduced in NSCLC specimens and cell lines. CCK8 assays indicated that overexpression of GABPB1-AS1 dramatically reduced NSCLC cell growth, and Transwell assays proved that NSCLC cell migration and invasion were distinctly inhibited by GABPB1-AS1. Exploration of the mechanism uncovered that miRNA-566 (miR-566)/F-box protein 47 (FBXO47) is directly targeted by GABPB1-AS1 in NSCLC. The study demonstrated that GABPB1-AS1 inhibited NSCLC cell proliferation, migration and invasion by targeting miR-566/FBXO47.

BackgroundNumerous studies have shown that long non-coding RNAs (lncRNAs) play key roles during multiple cancer processes, such as cell proliferation, apoptosis, migration and invasion. The previous studies found that NKILA interacted with and suppressed the nuclear translocation of NF-KappaB, which influenced metastasis and prognosis in breast cancer. However the clinical significance and biological role of NKILA in non-small cell lung cancer (NSCLC) remains unknown.MethodsWe examined expression levels of NKILA in 106 pairs of NSCLC tissues and cell lines. The expression level of NKILA after TGF-β1 stimulation also was examined by qRT-PCR and validated by Chromatin immunoprecipitation (ChIP). Gain-of-function and loss-of-function assays were performed to examine the effect of NKILA on proliferation, migration and invasion of NSCLC cells. RNA immunoprecipitation (RIP), western blot and rescue experiments were carried out to reveal the interrelation between NKILA, NF-κB and EMT signal pathway.ResultsThe expression of NKILA was down-regulated in NSCLC cancer tissues compared with matched adjacent noncancerous tissues, and lower NKILA expression in tumor tissues were significantly correlated with lymph node metastasis and advanced TNM stage. We found that the expression of NKILA was mainly regulated by classical TGF-β signal pathway in NSCLC cells rather than NF-κB pathway reported in breast cancer. Gain and loss of function assays found that NKILA inhibited migration, invasion and viability of NSCLC cells. Mechanistic study showed that NKILA attenuated Snail expression via inhibiting the phosphorylation of IκBα and NF-κB activation, subsequently suppressed the expression of markers of epithelial-mesenchymal transition process.ConclusionsThe present study found that the expression of NKILA was downregulated in tumor tissues of NSCLC, which improved the metastasis of NSCLC patients. In vitro studies further clarified that the expression of NKILA was regulated through classical TGF-β signal pathway, which subsequently inhibited migration and invasion of NSCLC cells through interfering NF-κB/Snail signal pathway in NSCLC cells.

Non‐small cell lung cancer (NSCLC) is the most common type of lung cancer. We aimed to investigate the role of LINC00184 in NSCLC. Migration, proliferation and invasion of NSCLC cells were analysed using the wound healing assay, cell counting kit‐8 assay and transwell assay, respectively. Apoptosis and cell cycle were assessed using flow cytometry. Online bioinformatics tools were utilized to predict downstream microRNAs (miRNA) or genes related to LINC00184 expression. The RNA pull‐down experiment and luciferase reporter assay were performed to verify the predictions thereof. LINC00184, miR‐524‐5p, and high mobility group 2 protein (HMGB2) expression levels in NSCLC tissues and cell lines were detected using quantitative real‐time polymerase chain reaction. An NSCLC mouse model was constructed for in vivo experiments. LINC00184 overexpression was observed in NSCLC tissues and cell lines and was found to be correlated with poor prognosis. LINC00184 knockdown inhibited cell proliferation, migration and invasion, induced cell cycle arrest and accelerated apoptosis in NSCLC cell lines. LINC00184 suppressed tumour growth and proliferation in NSCLC mouse models and directly targeted the miR‐524‐5p/HMGB2 axis. Moreover, the expression levels of LINC00184 and HMGB2 were negatively correlated with miR‐524‐5p expression, whereas LINC00184 expression was positively correlated with HMGB2 expression. LINC00184 affected the cell cycle, proliferation, apoptosis, migration and invasion in NSCLC via regulation of the miR‐524‐5p/HMGB2 axis.

BackgroundBrain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). However, the exact parts played by miR-330-3p in BM of NSCLC remain unknown. Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Here, we evaluated the expression and biological effects of miR-330-3p in NSCLC cells and explored the underlying mechanism of miR-330-3p in promoting cell migration and invasion in NSCLC.MethodsStable over-expression and knockdown of miR-330-3p in NSCLC cells was constructed with lentivirus. Expression levels of miR-330-3p in NSCLC cells were quantified by quantitive real-time PCR (qRT-PCR). The effects of miR-330-3p on NSCLC cells were investigated using assays of cell viability, migration, invasion, cell cycle, apoptosis, western blotting, immunohistochemical, and immunofluorescence staining. A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis. The potential target of miR-330-3p in NSCLC cells was explored using the luciferase reporter assay, qRT-PCR, and western blotting. The miR-330-3p targets were identified using bioinformatics analysis and verified by luciferase reporter assay. The correlation between GRIA3 and DNA methyltransferase (DNMT) 1 and DNMT3A was tested by RT-PCR, western blotting, and co-immunoprecipitation (IP).ResultsmiR-330-3p was significantly up-regulated in NSCLC cell lines. MTT assay, transwell migration, and invasion assays showed that miR-330-3p promoted the growth, migration, and invasion of NSCLC cells in vitro and induced tumor growth and metastasis in vivo. Luciferase reporter assays showed that GRIA3 was a target of miR-330-3p. qRT-PCR and western blotting exhibited that miR-330-3p promoted the growth, invasion, and migration of NSCLC cells by activating mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinases (ERK) signaling pathway. Furthermore, miR-330-3p up-regulated the total DNA methylation in NSCLC cells, and co-IP-demonstrated GRIA3 was directly related with DNMT1 and DNMT3A.ConclusionsmiR-330-3p promoted the progression of NSCLC and might be a potential target for the further research of NSCLC brain metastasis.

Lung cancer is the most common cause of cancer- associated mortality for men and women worldwide. An increasing number of studies have reported that the abnormal expression of microRNAs contributes to the pathogenesis of the majority of human cancer types, including non-small cell lung cancer (NSCLC). The present study aimed to measure microRNA-650 (miR-650) expression in NSCLC and evaluate its function in NSCLC cells. Reverse transcription-quantitative polymerase chain reaction was used to determine miR-650 expression in NSCLC tissue samples and cell lines. Assays for cell proliferation, migration and invasion were performed to investigate the roles of miR-650 on NSCLC progression. Furthermore, the mechanisms underlying the effects of miR-650 on NSCLC cell growth and metastasis were determined. In the current study, miR-650 was demonstrated to be highly expressed in NSCLC tissue samples and cell lines. Inhibition of expression of miR-650 suppressed NSCLC cell proliferation, migration and invasion in vitro. Additionally, large tumor suppressor kinase 2 (LATS2) was identified as a direct target gene of miR-650 in NSCLC. LATS2 was revealed to be significantly downregulated in NSCLC tissues and was negatively correlated with miR-650 expression. Notably, LATS2 re-expression decreased NSCLC cell proliferation, migration and invasion; similar to the effects induced by miR-650 underexpression. In conclusion, the results of the current study suggest that miR-650 may serve as an oncogene by direct targeting LATS2 in NSCLC formation and progression.

Background: Non-small cell lung cancer (NSCLC) is a highly malignant tumor. Accumulating evidence suggested that prostate cancer non-coding RNA 1 (PRNCR1) participated in the pathogenesis of NSCLC, whereas the elaborate mechanism remains unclear. Hence, the role of PRNCR1 in the progression of NSCLC was investigated.Methods: Levels of PRNCR1, microRNA-126-5p (miR-126-5p), and metadherin (MTDH) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured using Cell Counting Kit-8 (CCK-8). Flow cytometry was conducted to determine cell apoptosis. Besides, transwell assay was performed to detect cell migration and invasion in NSCLC cells. The expression levels of E-cadherin, N-cadherin, Vimentin, and MTDH were detected via Western blot. Dual-luciferase reporter, RNA immunoprecipitation, and RNA pull down assays were employed to verify the relationship between miR-126-5p and PRNCR1 or MTDH.Results: PRNCR1 and MTDH levels were highly expressed, while miR-126-5p expression was lowly expressed in NSCLC tissues and cell lines. Knockdown of PRNCR1 promoted cell apoptosis, impeded proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in NSCLC cells, and these effects were abrogated by its target gene of miR-126-5p inhibitor. Moreover, MTDH as the target of PRNCR1, its overexpression reversed the impacts of miR-126-5p mimic on cell behaviors and EMT in vitro. Finally, PRNCR1 and miR-126-5p regulated MTDH expression.Conclusion: PRNCR1 modified cell behaviors and EMT via miR-126-5p/MTDH axis in NSCLC cells, providing a novel thinking for clinical treatment of NSCLC.

Non-small cell lung cancer (NSCLC) is a malignant tumor associated with poor prognosis. The clinical value of long non-coding RNAs (lncRNAs) in the pathomechanism of various types of human malignancy has attracted increasing attention. The present study aimed to investigate the expression of LINC01272 in NSCLC and to determine its prognostic value and biological role. Tumor and adjacent non-tumor tissues from 108 patients with NSCLC and NSCLC cell lines were used in this study. The expression levels of LINC01272 and microRNA (miR)-1303 in tissues of patients and NSCLC cell lines were evaluated by reverse transcription quantitative PCR. The relationship between LINC01272 and the overall survival of patients with NSCLC was analyzed by Kaplan-Meier survival curve and log-rank test. Cox regression analysis confirmed the prognostic value of LINC01272 in patients with NSCLC. Cell Counting Kit-8 assay was used to evaluate the proliferation of NSCLC cells. The migration and invasion of NSCLC cells were determined using Transwell assays. The interaction between LINC01272 and miR-1303 in NSCLC was confirmed by dual-luciferase reporter assay. LINC01272 downregulation in NSCLC tissues was associated with worse overall survival in patients based on bioinformatics analysis. Furthermore, LINC01272 expression, which was decreased in NSCLC tumor tissues and NSCLC cells, was considered as an independent prognostic biomarker in NSCLC. In addition, LINC01272 overexpression inhibited NSCLC cell proliferation, migration and invasion. miR-1303 expression, which was increased in tumor tissues, was sponged by LINC01272 and negatively correlated with LINC01272 expression. miR-1303 expression reversed the inhibitory effects of LINC01272 on NSCLC cell function. In summary, the findings from this study suggested that LINC01272 expression, which was decreased in NSCLC tumor tissues and NSCLC cells, may be used as an independent prognostic biomarker for patients with NSCLC and that its overexpression may suppress NSCLC cell proliferation, migration and invasion by inhibiting miR-1303.

Increasing number of studies have indicated aberrant microRNA (miRNA) expression could affect normal biological progress in non-small cell lung cancer (NSCLC) cells. This study was performed to evaluate the biologic functions of microRNA-224 (miR-224) in NSCLC. Real-time PCR was performed to evaluate the expression of miR-224 and Homeobox D10 (HOXD10) in NSCLC cell lines and tissues. Transwell assays were performed to investigate the function of miR-224 on NSCLC cell migration and invasion. Moreover, western blotting and luciferase assays were used to investigate HOXD10 as miR-224 downstream targets. miR-224 is increased in NSCLC metastatic tissues and cell lines. Increased miR-224 expression promoted NSCLC cell migration and invasion, while low miR-224 expression suppressed NSCLC cell migration and invasion. Furthermore, HOXD10 was targeted directly by miR-224 in NSCLC cells. Moreover, we found that HOXD10 was a functional target and influenced tumour-inductive functions of miR-224 on progression of NSCLC. These findings suggest that miR-224 may be used in the treatment of NSCLC. Targeting this novel strategy, miR-224/HOXD10 axis may be helpful as promising biomarker and therapeutic method to control NSCLC cell metastasis.

Non-small cell lung cancer (NSCLC) is the most common malignant tumor worldwide. This work focuses on investigating the role of circ_0000353 in NSCLC and its potential mechanism of action. The expression levels of circ_0000353 and miR-411-5p in NSCLC and their matched normal lung tissues were detected by real-time PCR (RT-PCR). The correlation between the circ_0000353 expression and the clinicopathological parameters of NSCLC patients was also analyzed. CCK-8, BrdU and colony formation assays were adopted to detect the role of circ_0000353 in the proliferation of NSCLC cells. The metastasis of NSCLC cells was measured by Transwell assay. The dual-luciferase reporter gene assay was used to confirm the targeting relationship between circ_0000353 and miR-411-5p. The expression level of FOXO1 was detected by western blot. Circ_0000353 was significantly down-regulated in NSCLC tissues and cell lines, and the decreased expression was significantly linked to the increased clinical stage, larger tumor volume, and metastasis. The circ_0000353 over-expression restrained the proliferation, migration, and invasion of NSCLC cells in vitro. Additionally, up-regulation of miR-411-5p was observed in NSCLC tissues and cell lines, and luciferase assay and RT-PCR assay showed that circ_0000353 over-expression could target miR-411-5p and suppress its expression. Further studies confirmed that circ_0000353 and miR-411-5p modulated the FOXO1 expression. Circ_0000353 repressed the proliferation, migration, and invasion of NSCLC cells via inhibition of miR-411-5p and up-regulation of FOXO1.

MicroRNAs (miRs) regulate the proliferation and metastasis of numerous cancer cell types. It was previously reported that miR-143 levels were downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and that the migration and invasion of NSCLC cells was inhibited upon suppression of cell proliferation and colony formation by the upregulation of miR-143. Epidermal growth factor receptor (EGFR), which is a vital factor in the promotion of cancer cell proliferation and has been investigated as a potential focus in cancer therapy, has been reported to be a possible target of miR-143. The present study aimed to investigate the role of miR-143 in NSCLC using NSCLC cell lines and primary cells from NSCLC patients. NSCLC cells were co-transfected with EGFR and miR-143, and the mRNA and protein expression of EGFR were analyzed. Furthermore, the activity of the transfected cancer cells with regard to colony formation, migration, invasion and apoptosis were evaluated. The levels of miR-143 were decreased in the NSCLC cell lines and primary cells from patients with NSCLC compared with the controls. Following transfection with miR-143, the ability of NSCLC cells to proliferate, form colonies, migrate and invade was inhibited. Similarly, knockdown of EGFR led to the suppression of NSCLC cell proliferation. The mRNA and protein expression levels of EGFR were significantly reduced following miR-143 overexpression, and the level of miR-143 was inversely correlated with that of EGFR in NSCLC cells. The results of the present study demonstrated that miR-143 was able to suppress NSCLC cell proliferation and invasion by inhibiting the effects of EGFR, suggesting that EGFR may be considered a potential target for NSCLC therapy.