Targeting MET endocytosis or degradation to overcome HGF-induced gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma.

The overexpression of hepatic growth factor(HGF) is one of the important reasons for the development of gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma cells. Targeting the HGF receptor MET through endocytosis inhibition or degradation induction has been proposed as a potential strategy to overcome this resistance. However, the effectiveness of this approach remains needs to be evaluated. In this study, we observed that MET receptors undergo persistent endocytosis but rarely enter the degradation pathway in HGF-overexpressing cells. We showed that MET endocytosis can be inhibited by using gene silence method or MET inhibitors. CHC or DNM2 gene silence slightly increases the sensitivity of resistant cells to gefitinib without affecting MET activity, while GRB2 gene silence can simultaneously inhibit MET endocytosis and reduce MET activity, resulting in a significant reversal effect of gefitinib resistance. Similarly, MET inhibitors significantly reverse drug resistance, accompanied by simultaneous inhibition of MET endocytosis and activity, highlighting the importance of both endocytosis and activity in HGF-induced gefitinib resistance. Additionally, we demonstrated that promoting MET degradation through deubiquitinase (USP8 or USP32) gene silence is another effective method for reversing drug resistance. Overall, our findings suggest that targeting MET receptor endocytosis and degradation is an attractive strategy for overcoming HGF-induced gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma.

Sec3 knockdown inhibits TGF-ß induced epithelial-mesenchymal transition through the down-regulation of Akt phosphorylation in A549 cells.

The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-ß induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-ß stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-ß stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-ß induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-ß induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells.

miR-1-3p and miR-206 sensitizes HGF-induced gefitinib-resistant human lung cancer cells through inhibition of c-Met signalling and EMT.

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR-1-3p and miR-206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR-1-3p and miR-206 can overcome HGF-induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR-1-3p and miR-206 restored the sensitivities of lung cancer cells PC-9 and HCC-827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR-1-3p and miR-206 directly target HGF receptor c-Met in lung cancer. Knockdown of c-Met mimicked the effects of miR-1-3p and miR-206 transfections Meanwhile, c-Met overexpression attenuated the effects of miR-1-3p and miR-206 in HGF-induced gefitinib resistance of lung cancers. Furthermore, we showed that miR-1-3p and miR-206 inhibited c-Met downstream Akt and Erk pathway and blocked HGF-induced epithelial-mesenchymal transition (EMT). Finally, we demonstrated that miR-1-3p and miR-206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR-1-3p and miR-206 in overcoming HGF-induced gefitinib resistance in EGFR mutant lung cancer cell.

In vivo selection with lentiviral expression of Bcl2T69A/S70A/S87A mutant in hematopoietic stem cell-transplanted mice.

Current in vivo selections for hematopoietic stem cell (HSC)-based gene therapy are drug dependent and not without risk of cytotoxicity or tumorigenesis. We developed a new in vivo selection system with the non-phosphorylatable Bcl2 mutant Bcl2T69A/S70A/S87A (Bcl2AAA), which makes in vivo selection drug independent and without risk of cytotoxicity or tumorigenesis. We demonstrated in HSC-transplanted mice that Bcl2AAA facilitated efficient in vivo selection in the absence of any exogenously applied drugs under both myeloablative and non-myeloablative conditioning. In mice transplanted with retrovirally transduced sca-1-positive bone marrow cells, the marked cell level increased from 26.38% of input transduced cells to 92.61 ± 0.95% of peripheral blood cells for myeloablative transplantation or to 37.82 ± 6.35% for non-myeloablative transplantation 6 months after transplantation. Bcl2AAA did not induce tumorigenesis and does not influence hematopoiesis and the function of the reconstituted blood system. However, the high-level constitutive expression of Bcl2AAA mediated by retroviral vector induced exhaustion of the marked cells after tertiary transplantation. Fortunately, low-level constitutive expression of Bcl2AAA driven by an internal promoter in lentiviral vector could both maintain the marked cell level (24.13 ± 5.27%, 27.17 ± 5.51%, 24.33 ± 5.08%, and 22.07 ± 4.44% for primary, secondary, tertiary, and quaternary recipients) and avoid the exhaustion of the marked cells even in quaternary recipients. Importantly, the low-level constitutive expression of Bcl2AAA did not induce tumorigenesis. Thus, the in vivo selection employing the low-level constitutive expression of Bcl2AAA provides a general platform which is relevant for widespread applications of gene therapy.

Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway.

Lower Serum Levels of miR-29c-3p and miR-19b-3p as Biomarkers for Alzheimer's Disease.

MicroRNAs (miRNAs) are short noncoding RNA that participate in posttranscriptional gene regulation. However, little is understood about the roles of miRNAs in Alzheimer's disease (AD). In this study, we used next-generation sequencing on RNA extracted from the serum samples of 20 AD patients and 20 controls, yielding a total of 72 miRNAs with significantly changed expression levels. Among these candidates, we selected 9 miRNAs with most significant alteration in disease, and validated their expression levels using RT-qPCR analysis on serum samples from 45 AD patients and 40 control subjects. Thus, the serum levels of miR-146a-5p, 106b-3p, 195-5p, 20b-5p, and 497-5p were significantly higher, while those of miR-125b-3p, 29c-3p, 93-5p and 19b-3p were significantly lower in AD patients, compared with control subjects. Two miRNAs, miR-29c-3p and miR-19b-3p, were selected because both RNA deep-sequencing and q-PCR methods indicated lower serum levels of these miRNAs in AD patients. Computational analysis predicted that 3'-untranslated region of signal transduction and activator of transcription 3 (STAT3) mRNA is targeted both by miR-29c-3p and miR-19b-3p. Using SH-SY5Y human neuroblastoma cells, we showed that transfection with miR-29c-3p or miR-19b-3p inhibitor significantly increased STAT3 phosphorylation. Furthermore, Water maze test, which assesses the learning and memory deficits in rodents, showed that escape latency was significantly shorter in AD rats with overexpression of miR-29c-3p or miR-19b-3p than in control AD rats. These results suggest that miR-29c-3p or miR-19b-3p may contribute to the cognitive function. In conclusion, the serum levels of miR-29c-3p and miR-19b-3p are helpful biomarkers for AD.

Identification of carcinogenic potential-associated molecular mechanisms in CD133(+) A549 cells based on microRNA profiles.

This study aimed to identify carcinogenic potential-related molecular mechanisms in cancer stem cells (CSCs) in lung cancer. CD133(+) and CD133(-) subpopulations were sorted from A549 cells using magnetic-activated cell sorting. The abilities to form sphere and clone, proliferate, migrate, and invade were compared between CD133(+) and CD133(-) cells, as well as drug sensitivity. Thereafter, microRNA (miRNA) profiles were performed to identify differentially expressed miRNAs between CD133(+) and CD133(-) subpopulation. Following, bioinformatic methods were used to predict target genes for differentially expressed miRNAs and perform enrichment analysis. Furthermore, the mammalian target of rapamycin (mTOR) signaling pathways and CSC property-associated signaling pathways were explored and visualized in regulatory network among competitive endogenous RNA (ceRNA), miRNA, and target gene. CD133(+) subpopulation showed greater oncogenic potential than CD133(-) subpopulation. In all, 14 differentially expressed miRNAs were obtained and enriched in 119 pathways, including five upregulated (hsa-miR-23b-3p, -23a-3p, -15b-5p, -24-3p, and -4734) and nine downregulated (hsa-miR-1246, -30b-5p, -5096, -6510-5p, has-miR-7110-5p, -7641, -3197, -7108-5p, and -6791-5p). For mTOR signaling pathway, eight differential miRNAs (hsa-miR-23b-3p, -23a-3p, -15b-5p, -24-3p, -4734, -1246, -7641, and -3197) and 39 target genes (e.g., AKT1, AKT2, PIK3CB, PIK3CG, PIK3R1, PIK3CA, and PIK3CD) were involved, as well as some ceRNAs. Besides, for CSC property-related signaling pathways, six miRNAs (hsa-miR-1246, -15b-5p, -30b-5p, -3197, -4734, and -7110-5p) were dramatically enriched in Hedgehog, Notch, and Wnt signaling pathways via regulating 108 target genes (e.g., DVL1, DVL3, WNT3A, and WNT5A). The mTOR and CSC property-associated signaling pathways may be important oncogenic molecular mechanisms in CD133(+) A549 cells.

Identification of microRNA-93 as a functional dysregulated miRNA in triple-negative breast cancer.

MicroRNAs (miRNAs) are widely recognized as key players in cancer progression and drug resistance, but less is known about the role of miRNAs in triple-negative (estrogen receptor, progesterone receptor, and HER-2/neu) breast cancer (TNBC). The aim of the present study was to examine the expression profile of miRNAs and to explore their possible roles in TNBC. Differentially expressed miRNAs were identified by miRNA microarray and verified by quantitative real-time polymerase chain reaction. The expression of miR-93 was assessed by in situ hybridization in 119 cases of breast cancer. Cell proliferation potential was examined by MTT assay. Cell migration and invasion abilities were evaluated by a wound healing assay and transwell invasion or migration assay. Seven upregulated and ten downregulated miRNAs in TNBC were identified. The miR-93 expression level in TNBC tissues was significantly higher than that in non-triple-negative breast cancer tissues. The potentials of proliferation, invasion, and metastasis in breast cancer MCF-7 cells were promoted by ectopic transfection of miR-93. Our study found several distinct differentially expressed miRNAs in TNBC, as compared to non-triple-negative breast cancer. Among them, miR-93 may be considered as a biomarker associated with the biological and clinical characteristics of human TNBC.

Tumor-associated Macrophages Mediate Gefitinib Resistance in Lung Cancer through HGF/c-met Signaling Pathway.

BACKGROUND: The biological behavior of cells changes after they develop drug resistance, and the degree of resistance will be affected by the tumor microenvironment. In this study, we aimed to study the effects of M2 macrophages on gefitinib resistance. METHODS: We polarized THP-1 cells into M0 and M2 macrophages, and conducted various experiments to investigate the effects of M2 macrophages on gefitinib resistance in lung cancer. RESULTS: We found that M2 macrophages promote gefitinib resistance in HCC827 and PC9 cells. In addition, we used ELISA to measure the secretion level of HGF. HGF secretion levels were significantly increased in M2 macrophages. Exogenous HGF remarkably increased the proliferation and invasion in HCC827 and PC9 cells. However, the addition of anti-HGF antibodies abolished the proliferation and invasion of both HCC827 and PC9 cells promoted by M2 macrophages. Furthermore, M2 macrophages or exogenous HGF significantly increased the expression of p-met and p-ERK in HCC827 and PC9 cells, while anti-HGF antibodies diminished the expression of p-met and p-ERK by neutralizing HGF in M2 macrophages. CONCLUSION: Our results revealed that M2 macrophages promote gefitinib resistance by activating ERK and HGF/c-met signaling pathways in HCC827 and PC9 cells. Our findings provide a new therapeutic strategy for gefitinib resistance in lung cancer.

Silence of ezrin modifies migration and actin cytoskeleton rearrangements and enhances chemosensitivity of lung cancer cells in vitro.

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.

Lysosomal membrane permeabilization is involved in curcumin-induced apoptosis of A549 lung carcinoma cells.

We previously reported that curcumin inhibited lung cancer A549 cells growth and promoted cell apoptosis in vitro. In this study, we further examined the apoptosis-related parameters, including lysosomal damage and cathepsin activation, in A549 cells exposed to curcumin. We found that curcumin caused lysosomal membrane permeabilization (LMP) and cytosolic relocation of cathepsin B (cath B) and cathepsin D (cath D). However, only Z-FA-fmk (a cath B inhibitor) but not pepstatin A (a cath D inhibitor) inhibited curcumin-induced cell apoptosis, mitochondrial membrane potential loss, and cytochrome c release. The antioxidant N-acetylcysteine and glutathione attenuated LMP, suggesting that lysosomal destabilization was dependent on the elevation of reactive oxygen species and which precedes mitochondrial dysfunction. These findings indicated a novel pathway for curcumin regulation of ROS-lysosomal-mitochondrial pathway and provided the key mechanism of regulation of LMP in cell apoptosis, which may be exploited for cancer treatment.

[Expression of ezrin in human non-small cell lung cancer and its relationship with metastasis and prognosis].

OBJECTIVE: To explore the expression of ezrin protein in human non-small cell lung cancer (NSCLC) tissues and lung cancer cell lines, and the association between the expression of ezrin protein and the expression of E-cadherin and CD44V6 proteins. METHODS: The expression of ezrin protein and mRNA in lung cancer cell lines was detected by RT-PCR and Western blotting. Ezrin, E-cadherin and CD44V6 were detected by immunohistochemical SP staining in tumor tissues from 150 lung cancer cases and in adjacent normal lung tissues from 30 patients. Furthermore, the expression of ezrin in 30 freshly-taken NSCLC tissues was also detected by Western blot. RESULTS: The expression of ezrin protein and mRNA was up-regulated in highly metastatic human lung cancer. The positive rate of ezrin, E-cadherin and CD44V6 expression in the lung cancer was 61.3%, 54.0% and 58.7%, respectively. The up-regulation of ezrin expression was significantly correlated with lymph node metastasis, but not correlated with age, sex, tumor size, histological type, clinical TNM system and pathological grade. Western blot analysis showed that the level of ezrin in the NSCLC tissues was significantly higher than that in the normal tissues (t = 5.013, P < 0.01). Survival analysis showed that the 5-year survival rate of patients with negative ezrin expression was 29.3%, significantly higher than that of patients with positive ezrin expression (15.2%, χ(2) = 4.128, P = 0.042). Multivariate Cox regression analysis showed that ezrin expression (RR = 3.012, P = 0.047) and lymph node metastasis (RR = 4.827, P = 0.035) were significantly independent prognostic factors for patients with lung cancer. Furthermore, a negative correlation was observed between the expressions of ezrin and E-cadherin in lung cancer, and a positive correlation between the expressions of ezrin and CD44V6 in lung cancer. CONCLUSIONS: Ezrin, E-cadherin and CD44V6 play an important role in the regulation of growth and meastasis of lung cancer. Combined detection of ezrin, E-cadherin and CD44V6 expression is helpful in evaluating the metastasis and prognosis of non-small cell lung cancer.

Anlotinib Inhibits Cell Proliferation, Migration and Invasion via Suppression of c-Met Pathway and Activation of ERK1/2 Pathway in H446 Cells.

BACKGROUND: Small Cell Lung Cancer (SCLC) represents the most aggressive pulmonary neoplasm and is often diagnosed at late stage with limited survival, despite combined chemotherapies. The purpose of this study was to investigate the effect of anlotinib on SCLC and the potential molecular mechanisms. METHODS: Cell viability was assessed by CCK-8 assay to determine the adequate concentration of anlotinib. Then, effects of anlotinib on cell apoptosis, cell cycle distribution, migration and invasion were analyzed by flow cytometry, PI staining, wound healing assay and transwell assay, respectively. The protein expression of c-met and ERK1/2 pathways in H446 cells were assessed by western blot analysis. RESULTS: In this study, we found that anlotinib significantly reduced the cell viability of H446 cells, induced G2/M cell cycle arrest and decreased invasion and migration of H446 cells. Futhermore, we also found that anlotinib could suppress c-met signal transduction and activate the ERK1/2 pathway in H446 cells. More importantly, c-met was involved in the effects of anlotinib on migration and invasion in H446 cells. CONCLUSION: Taken together, our results demonstrated that anlotinib was a potential anticancer agent that inhibited cell proliferation, migration and invasion via suppression of the c-met pathway and activation of the ERK1/2 pathway in H446 cells.

Epidemiologic Characteristics of and Prognostic Factors for COVID-19 Among Hospitalized Patients: Updated Implications From Hubei Province, China.

Introduction: The roles of some indicators in the prognosis of patients with coronavirus disease-19 (COVID-19) remain unclear and controversial. This study aimed to explore the epidemiologic characteristics of and prognostic factors for COVID-19 to provide updated recommendations for its prevention, diagnosis, and treatment. Methods: For this retrospective study, demographic, epidemiologic, and clinical data were extracted from the medical records of patients admitted to the Maternal and Child Hospital of Hubei Province (Optical Valley) with COVID-19 between February 19, 2020, and March 19, 2020. The primary outcome was the prognosis that was determined at discharge as mentioned in the medical records. Descriptive statistics, univariate analyses, and stepwise logistic regression analysis were used for data analysis. Results: Of the 1,765 patients included, 93.1% were cured and the mortality was 1.8%. Univariate analyses identified 63 factors significantly associated with COVID-19 prognosis. Logistic regression analysis revealed that a poorer prognosis was associated with undergoing resuscitation, complex disease manifestations, consultation with outside specialists, elevated basophil or lymphocyte counts, an albumin (ALB)/globulin (A/G) ratio > 2.4, and elevated levels of serum aspartate aminotransferase (AST) or creatinine. Patients had a better prognosis if the following conditions were met: dry cough reported as an initial symptom, fatigue as a clinical manifestation, and a diagnosis based on laboratory testing. Conclusion: To prevent clinical deterioration, clinicians should provide special care to patients who underwent resuscitation, with a critical disease, or requiring consultation with outside specialists. Extra attention should be paid to patients with high basophil or lymphocyte counts, a high A/G ratio, and elevated AST or creatinine levels.

miR-1/133a and miR-206/133b clusters overcome HGF induced gefitinib resistance in non-small cell lung cancers with EGFR sensitive mutations.

It has been reported that clustered miRNAs can be transcribed coordinately and exhibit similar functions by regulating the same targets. miR-1/133a and miR-206/133b are well-characterized miRNA clusters. However, the effect of these clusters on EGFR-TKI resistance is not clear. In this study, we demonstrated that lentivirus-mediated HGF overexpression was able to induce gefitinib resistance in non-small cell lung cancers with EGFR sensitive mutations. miR-1/133a and miR-206/133b clusters could overcome HGF induced gefitinib resistance. Furthermore, the clusters were more effective than individual miRNA. Transcriptome RNA sequencing and bioinformatics analysis revealed that multiple pathways, including 'EGFR tyrosine kinase inhibitor resistance' pathway, were involved in anti-resistance mechanisms of miR-1/133a and miR-206/133b clusters. Western blotting results confirmed the inhibitory effect of miRNA clusters on MET expression and downstream pathway activation. In conclusion, miR-1/133a and miR-206/133b clusters are able to exhibit the synergetic effect on overcoming HGF-induced gefitinib resistance in NSCLC and the mechanisms are through targeting multiple genes related to gefitinib resistance.

Mogroside V Inhibits Hyperglycemia-induced Lung Cancer Cells Metastasis through Reversing EMT and Damaging Cytoskeleton.

BACKGROUND: Diabetes Mellitus (DM) accelerates progress of lung cancer. Hyperglycemia, a critical feature of DM, promotes lung cancer metastasis. Mogroside V is a triterpenoid glycoside from Siraitia grosvenorii. Interestingly, mogroside V not only plays an anti-diabetic role, but also has anti-tumor effects. OBJECTIVE: In this study, we investigated the metastatic efficiency of mogroside V in lung cancer cells cultured in hyperglycemia. METHODS: Two lung cancer cell lines-A549 and H1299 were cultured in normoglycemia (5.5mM glucose) and hyperglycemia (25mM glucose). Cellular proliferation was tested by MTT, invasion was examined by transwell assay, migration was measured by wound healing assay, cytoskeleton was stained by Phalloidin-TRITC and the expressions of EMT markers and Rho-GTPase family protein were detected by western blot. RESULTS: Hyperglycemia promoted the invasion and migration of A549 and H1299 cells compared with normoglycemia. Mogroside V inhibited the hyperglycemia-induced invasion and migration. Hyperglycemia promoted epithelial-mesenchymal transition (EMT), while mogroside V could reverse this process through up-regulating E-Cadherin expression and down-regulating N-Cadherin, Vimentin, Snail expressions. Furthermore, mogroside V fractured microfilaments and reduced Rho A, Rac1, Cdc42 and p-PAK1 expressions under hyperglycemic conditions. CONCLUSION: These results suggest that mogroside V inhibits hyperglycemia-induced lung cancer cells migration and invasion through reversing EMT and damaging cytoskeleton.

Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro.

A combination of extrinsic hematopoietic growth regulators, such as stem cell factor (SCF), interleukin (IL)-3 and IL-6, can induce division of quiescent hematopoietic stem cells (HSCs), but it usually impairs HSCs' self-renewal ability. However, intrinsic negative cell cycle regulators, such as p18INK4C (p18), p27Kip1 (p27) and MAD1, can regulate the self-renewal of HSCs. It is unknown whether the removal of some extrinsic regulators and the knockdown of intrinsic negative cell cycle regulators via RNA interference (RNAi) induce ex vivo expansion of the HSCs. To address this question, a lentiviral vector-based RNAi tool was developed to produce two copies of small RNA that target multiple genes to knockdown the intrinsic negative cell cycle regulators p18, p27 and MAD1. Colony-forming cells, long-term culture-initiating cells (LTC-IC) and engraftment assays were used to evaluate the effects of extrinsic and intrinsic regulators. Results showed that the medium with only SCF, but without IL-3 and IL-6, could maintain the sca-1+c-kit+ bone marrow cells with high LTC-IC frequency and low cell division. However, when the sca-1+c-kit+ bone marrow cells were cultured in a medium with only SCF and simultaneously knocked down the expression of p18, p27 and MAD1 via the lentiviral vector-based RNAi, the cells exhibited both high LTC-IC frequency and high cell division, though engraftment failed. Thus, the simultaneous knockdown of p18, p27 and MAD1 with a medium of only SCF can induce LTC-IC expansion despite the loss of engraftment ability.

Identification of lymph node metastasis-related microRNAs in lung adenocarcinoma and analysis of the underlying mechanisms using a bioinformatics approach.

This study aimed to screen lymphatic metastasis-related microRNAs (miRNAs) in lung adenocarcinoma and explore their underlying mechanisms using bioinformatics. The miRNA expression in primary lung adenocarcinoma, matched adjacent non-tumorigenic and lymph node metastasis tissues of patients were profiled via microarray. The screened metastasis-related miRNAs were then validated using quantitative real-time PCR in a second cohort of lung adenocarcinoma patients with lymphatic metastasis. Significance was determined using a paired t-test. Target genes of the metastasis-related miRNAs were predicted using TargetScan, and transcription factors (TFs) were predicted based on the TRANSFAC and ENCODE databases. Furthermore, the related long non-coding RNAs (lncRNAs) were screened with starBase v2.0. The miRNA-TF-mRNA and lncRNA-miRNA-mRNA networks were constructed to determine the key interactions associated with lung adenocarcinoma metastasis. According to the miRNA microarray results, there were 10 miRNAs that were differentially expressed in metastatic tissues compared with primary tumor and adjacent non-tumorigenic tissues. Among them were increased levels of miR-146a-5p, miR-342-3p, and miR-150-5p, which were validated in the second cohort. Based on the miRNA-TF-mRNA network, vascular endothelial growth factor A and transcription factors (TFs) including TP53, SMAD4, and EP300 were recognized as critical targets of the three miRNAs. Interactions involving SNHG16-miR-146a-5p-SMAD4 and RP6-24A23.7-miR-342-3p/miR-150-5p-EP300 were highlighted according to the lncRNA-miRNA-mRNA network. miR-146a-5p, miR-342-3p, and miR-150-5p are lymphatic metastasis-related miRNAs in lung adenocarcinoma. Bioinformatics analyses demonstrated that SNHG16 might inhibit the interaction between miR-146a-5p and SMAD4, while RP6-24A23.7 might weaken miR-342-3p-EP300 and miR-150-5p-EP300 interactions in metastasis.

Exploration of inhibitory mechanisms of curcumin in lung cancer metastasis using a miRNA- transcription factor-target gene network.

The present study was aimed to unravel the inhibitory mechanisms of curcumin for lung cancer metastasis via constructing a miRNA-transcription factor (TF)-target gene network. Differentially expressed miRNAs between human high-metastatic non-small cell lung cancer 95D cells treated with and without curcumin were identified using a TaqMan human miRNA array followed by real-time PCR, out of which, the top 6 miRNAs (miR-302b-3p, miR-335-5p, miR-338-3p, miR-34c-5p, miR-29c-3p and miR-34a-35p) with more verified target genes and TFs than other miRNAs as confirmed by a literature review were selected for further analysis. The miRecords database was utilized to predict the target genes of these 6 miRNAs, TFs of which were identified based on the TRANSFAC database. The findings of the above procedure were used to construct a miRNA-TF-target gene network, among which miR-34a-5p, miR-34c-5p and miR-302b-3p seemed to regulate CCND1, WNT1 and MYC to be involved in Wnt signaling pathway through the LEF1 transcription factor. Therefore, we suggest miR-34a-5p/miR-34c-5p/miR-302b-3p -LEF1-CCND1/WNT1/MYC axis may be a crucial mechanism in inhibition of lung cancer metastasis by curcumin.

Integrated microRNA and gene expression profiling reveals the crucial miRNAs in curcumin anti-lung cancer cell invasion.

BACKGROUND: Curcumin (diferuloylmethane) has chemopreventive and therapeutic properties against many types of tumors, both in vitro and in vivo. Previous reports have shown that curcumin exhibits anti-invasive activities, but the mechanisms remain largely unclear. METHODS: In this study, both microRNA (miRNA) and messenger RNA (mRNA) expression profiles were used to characterize the anti-metastasis mechanisms of curcumin in human non-small cell lung cancer A549 cell line. RESULTS: Microarray analysis revealed that 36 miRNAs were differentially expressed between the curcumin-treated and control groups. miR-330-5p exhibited maximum upregulation, while miR-25-5p exhibited maximum downregulation in the curcumin treatment group. mRNA expression profiles and functional analysis indicated that 226 differentially expressed mRNAs belonged to different functional categories. Significant pathway analysis showed that mitogen-activated protein kinase, transforming growth factor-ß, and Wnt signaling pathways were significantly downregulated. At the same time, axon guidance, glioma, and ErbB tyrosine kinase receptor signaling pathways were significantly upregulated. We constructed a miRNA gene network that contributed to the curcumin inhibition of metastasis in lung cancer cells. let-7a-3p, miR-1262, miR-499a-5p, miR-1276, miR-331-5p, and miR-330-5p were identified as key microRNA regulators in the network. Finally, using miR-330-5p as an example, we confirmed the role of miR-330-5p in mediating the anti-migration effect of curcumin, suggesting the importance of miRNAs in the regulation of curcumin biological activity. CONCLUSION: Our findings provide new insights into the anti-metastasis mechanism of curcumin in lung cancer.