Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.     

Identification of key long noncoding RNAs and their biological functions in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) are vital regulators in tumorigenesis and metastasis. However, the pathological role of lncRNAs in hepatocellular carcinoma (HCC) is still unclear. In this study, we filtered out three lncRNAs from The Cancer Genome Atlas (TCGA) data that were screened for basic expression and clinical research. We selected lncRNA-NEAT1 for further study to explore its function in HCC progression and its regulatory mechanism. We identified three differentially expressed lncRNAs (DElncRNAs) in tumor and adjacent normal tissues from the TCGA library using data mining methods: lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11. Their basic expression levels were detected by qPCR. Then, we selected lncRNA-NEAT1 as a potentially important lncRNA to verity its biological function and mechanism in HCC cell lines. lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11 were overexpressed in liver cancer tissues and cell lines. We found that silencing NEAT1 in vitro can inhibit the proliferation of HuH-7 and Li-7 cells, inhibit cell migration, and induce apoptosis as well as significantly increase the level of miR-16-5p. We also confirmed that miR-16-5p has a significant correlation with Bcl-2. When NEAT1 is silenced, the expression of Bcl-2 decreases. Inhibiting miR-16-5p can restore Bcl-2 to its original level. We conclude that miR-16-5p1/lncRNA NEAT1 plays a crucial role in regulating the delivery of Bcl-2 in HCC. Overall, the miR-16-5p/lncRNA-NEAT1/Bcl-2 signaling axis may be a promising target for HCC treatment.     

Silencing of long non-coding RNA CCHE1 inhibits the ovarian cancer SKOV3 cell invasion and migration and inactivates the p38-MAPK signaling pathway

Ovarian cancer (OC) is a major cause of cancer-related deaths among gynaecological malignancies. Emerging studies suggest that the long non-coding RNA (lncRNA) may be the potential biomarker for the diagnosis and prognosis of the cancer. The current study was carried out to investigate the role of lncRNA CCHE1 silencing in OC cell invasion and migration. Expression of lncRNA CCHE1 in normal ovarian cell Hose and OC cell lines HO 8910, A2780 and SKOV3 was detected. LncRNA were transfected with siRNA, and then the proliferation of cells was detected by using MTT assay. Cell invasion and migration was measured by using Transwell assay and scratch test, respectively. The protein levels of E-cadherin, N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK in cells after siRNA transfection were detected by using Western blot analysis. Consequently, lncRNA CCHE1 expression was highly expressed in OC cell lines, especially in SKOV3 cells. siRNA1, siRNA2 and siRNA3 all decreased. lncRNA CCHE1 expression in SKOV3 cells and siRNA2 showed the best silencing efficacy. Silencing of lncRNA CCHE1 decreased proliferation, invasion and migration, and reduced the protein levels of N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK, while reducing the protein level of E-cadherin in SKOV3 cells. Collectively, our study proved that the silencing of lncRNA CCHE1 could inhibit SKOV3 cell invasion and migration via inactivating the p38-MAPK signaling pathway.     

LncRNA LINC01772 promotes metastasis and EMT process in cervical cancer by sponging miR-3611 to relieve ZEB1

Cervical cancer (CC), has been identified as one of the most frequent malignant tumors all over the world, with high mortality in females. A growing number of investigations have confirmed that long noncoding RNAs (lncRNAs) play a crucial role in the progression of multiple cancers. Nonetheless, the biological function and regulatory mechanism of LINC01772 in CC haven’t been explored so far. In this study, LINC01772 expression was found to be upregulated in tissues and cells of CC. Knocking down LINC01772 suppressed CC cell proliferation, migration and epithelial-mesenchymal transition (EMT) process. Through molecular mechanism assays, LINC01772 was verified to be bound with miR-3611 and LINC01772 acted as a sponge for miR-3611. Zinc finger E-box binding homeobox 1 (ZEB1) was a downstream target gene of miR-3611. ZEB1 was negatively regulated by miR-3611 but positively regulated by LINC01772. Rescue assays confirmed that miR-3611 inhibitor or ZEB1 overexpression offset the inhibitive effect of LINC01772 depletion on cell proliferation, migration and EMT process in CC. In a word, our study validated that LINC01772 promoted cell metastasis and EMT process in CC by sponging miR-3611 to upregulate ZEB1 expression, indicating that LINC01772 could serve as a new therapeutic target for patients with CC.     

Exosomal miR-218 regulates the development of endometritis in dairy cows by targeting TGIF2/TGF-β pathway

Endometritis affects the reproductive capacity of dairy cows and leads to serious economic losses in dairy farming. Clarification of the pathogenesis of endometritis is necessary to improve the reproductive efficiency of dairy cows. Exosomes and their miRNAs have been proven to play an important role in inflammatory regulation. Exosomal miR-218 is a differentially expressed miRNA found in endometrial epithelial cells (EECs) under endometrial inflammation. Therefore, we investigated the expression of miR-218 in the uterine tissue of dairy cows, lipopolysaccharide (LPS) treated EECs, exosomal vesicles, and regulation of exosomal miR-218 by targeting TGIF-2 inducible factor homology frame 2 (TGIF2)/transforming growth factor-beta (TGF-β). The expression of miR-218 was suppressed in inflammatory uterine tissues and LPS treated EECs. The expression of TGIF2 and TGF-β in inflammatory uterine tissues and LPS treated EECs was significantly higher than those in healthy uterine tissues and EECs (p < 0.01). Interestingly, miR-218 derived from donor cells was found to regulate the expression of the target gene TGIF2 in recipient cells through the fusion of exosomes. Concurrently, the expression of its target gene TGIF2 was also suppressed by miR-218 in donor cells resulting in fewer TGIF2 being transported into recipient cells with exosomal fusion. This may be a novel mechanism of miRNAs-mediated regulation and provides a new reference for analyzing the pathogenesis of endometritis in dairy cows.     

Elp3 modulates neural crest and colorectal cancer migration requiring functional integrity of HAT and SAM domains

Cell migration is a finely tuned biological process that often involves epithelial-mesenchymal transition (EMT). EMT is typically characterized by the upregulation of mesenchymal markers such as Snail1. This process has been shown to be of critical importance to normal developmental processes, including neural crest migration and invasion. Interestingly, similar mechanisms are utilized in disease processes, such as tumor metastasis and migration. Notably, EMT and EMT-like processes confer tumor cells with the ability to migrate, invade, and adopt stem cell-like properties that largely account for immunosuppression and tumor recurrence. Therefore, identifying sensitive EMT markers may contribute to cancer prognosis and diagnosis in many facets. Previously, we showed that Elp3 plays an essential role during neural crest migration by stabilizing Snail1. In the current study, we further elucidate the molecular mechanism underlying colorectal cancer migration. We found that mElp3 exerted an identical function to xElp3 in modulating neural crest migration, and both HAT and SAM domains are imperative during this migratory process. Interestingly, overexpression of mElp3 in SW480 cells promoted cell migration and invasion, and we further showed that Elp3 stabilized Snail1 requiring integrity of both SAM and HAT domains. Our findings warrant further exploration of the specific target of Elp3 in cancer cells.     

<i>LINC00609</i> inhibits A549 cells progression through the regulation of <i>miR-128-3p/RND3</i> axis

Background: Long-chain non-coding RNA (lncRNA) LINC00609 is a potential tumor suppressor, but the mechanism of action in non-small cell lung cancer (NSCLC) is yet to be understood.Objectives: The effects of LINC00609 on A549 cell proliferation, apoptosis, and cell cycle arrest were investigated. Methods: The LINC00609 levels in NSCLC and normal tissues were analyzed by bioinformatics. Expressions of LINC00609, miR-128-3p, and Rho family GTPase 3 (RND3) in NSCLC cells (A549) were determined by qRT-PCR. Bioinformatics analysis predicted target genes and dual-luciferase reporter assays to ensure that LINC00609 targeted miR-128-3p and miR-128-3p targeted RND3. The proliferation of cells was determined using EDU and CCK-8. Flow cytometry was used to evaluate cell apoptosis rate and cell cycle. The western blotting assay identified proteins related to proliferation and apoptosis. Results: In NSCLC tissues, LINC00609 was expressed in low levels, while its high expression was associated with a higher survival rate. LINC00609 affected cell proliferation, apoptosis, cell cycle arrest, and expression of related proteins. Dual-luciferase reporter assay showed that LINC00609 binds specifically to miR-128-3p, and miR-128-3p binds to RND3. MiR-128-3p overexpression could neutralize the effects of LINC00609. A siRNA targeting RND3 could reverse the effect of the miR-128-3p inhibitor. Silencing RND3 resulted in a decrease in apoptosis rate and the number of cells in the S-phase and an increase in the number of cells in the G1-phase. Furthermore, phosphorylation levels of the AKT protein and mTOR protein, and Bcl2 expression, increased; however, the expression of RND3, Bax, and caspase3 decreased. Conclusions: LINC00609 regulated miR-128-3p/RND3 axis to modulate A549 cell proliferation, apoptosis, and cell cycle arrest. In the case of NSCLC, LINC00609 could be a potential target for therapy.     

HMGB1 promotes the proliferation and invasion of oral squamous cell carcinoma via activating epithelial-mesenchymal transformation

This study aimed to investigate the role of high mobility group box-1 (HMGB1) expression in oral squamous cell carcinoma; HMGB1 promoted the proliferation and invasion of oral squamous cell carcinoma via activating epithelial-mesenchymal transformation (EMT). In this study, RNA transfection was used to silence the expression of HMGB1 in oral squamous cell carcinoma cells. CCK-8, cell clone formation and trans-well assays were used to detect the proliferation and invasion of cells before and after HMGB1 silencing. qRT-PCR and Western blot were used to detect changes in EMT marker protein expression before and after transfection. HMGB1 was significantly higher in OSCC tissues than in adjacent tissues, and of the cell lines examined, HMGB1 was highest in SCC-9 cells. Additionally, HMGB1 silencing decreased SCC-9 cell proliferation and viability. Down-regulation of HMBG1 expression inhibited not only the proliferation but also the invasion of SCC-9 cells. The expression of N-cadherin, Snail, and Slug, but not E-cadherin, were promoted after silencing HMGB1. The expression of HMGB1 in OSCC tissue and cell lines was higher, and HMGB1 silencing decreased SCC-9 cell proliferation and invasion, suggesting that HMGB1 has positive effects on OSCC development. Down-regulation of HMBG1 expression regulates EMT markers, suggesting that HMBG1 promotes OSCC cell proliferation and invasion is likely to be associated with EMT activation.     

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.     

Chrysophanol inhibits the progression of gastric cancer by activating nod-like receptor protein-3

Aim: Gastric cancer (GC) is one of the most common malignant tumors. Chrysophanol has been reported to possess antitumor effects on a variety of cancers; however, its role in GC remains unclear. This study aimed to investigate the effects of chrysophanol on the proliferation, pyroptosis, migration, and invasion of GC cells. Methods: Human GC cell lines MKN 28 and AGS cells were treated with different concentrations of chrysophanol, then cell proliferation, migration, invasion and pyroptosis were determined by CCK-8, colony-forming assay, wound healing assay, Transwell assay, and flow cytometry. Cell migration and invasion were reassessed in these transfected cells following the transfection of nod-like receptor protein-3 (NLRP3) siRNA in MKN 28 and AGS cells. To examine the downstream signaling pathway of the NLRP3 signaling pathway, NLRP3, caspase-1, gasdermin-D, interleukin (IL)-1β, and IL-18 were detected by quantitative real-time-polymerase chain reaction or western blotting. Results: Chrysophanol inhibited the proliferation of GC cells, caused pyroptosis, inhibited cell migration and invasion, and increased the expression of NLRP3 inflammasomes in GC cells. Knockdown of NLRP3 inhibited the effects of chrysophanol on proliferation, pyroptosis, migration, and invasion of GC cells. Chrysophanol plays an anticancer role by enhancing NLRP3. Conclusions: Chrysophanol exerts anti-neoplastic effects in vitro in GC cells by modulating NLRP3, thus highlighting its therapeutic potential in GC.     

Effect of microRNA-143-3p-mediated CTNND1 on the biological function of lung cancer cells

Lung cancer poses a serious threat to human life with high incidence and miRNA is an important biomarker in tumors. This study aimed to explore the effect of miR-143-3p on the biological function of lung cancer cells and the underlying mechanism. Eighty-seven samples of lung cancer tissues and 81 samples of tumor-adjacent tissues from patients undergoing radical lung cancer surgery in our hospital were collected. The lung cancer cells and lung fibroblast cells (HFL-1) were purchased, and then miR-143-3p-mimics, miR-NC, si-CTNND1, and NC were transfected into A549 and PC-9 cells to establish cell models. MiR-143-3p and CTNND1 expression levels were measured by the qRTPCR, Bax, Bcl-2, and CTNND1 expression levels by the Western Blot (WB), and cell proliferation, invasion, and apoptosis by the MTT assay, Transwell assay, and flow cytometry. Dual luciferase report assay was used to determine the relationship between miR-143-3p and CTNND1. In this study, miR-143-3p was lowly expressed in lung cancer and CTNND1 was highly expressed in lung cancer. The overexpression of miR-143-3p inhibited cell proliferation and invasion, promoted cell apoptosis, significantly increased Bax protein expression, and decreased Bcl-2 protein expression. The inhibition of CTNND1 led to opposite biological characteristic in cells. The dual luciferase reporter assay demonstrated that miR-143-3p was a target region of CTNND1. Such results suggest that miR-143-3p can inhibit the proliferation and invasion of lung cancer cells by regulating the expression of CTNND1 and promote the apoptosis of lung cancer cells, sott is expected to be a potential target for lung cancer.     

Quantitative and qualitative methods using fluorescence microscopy for the study of modified low density lipoproteins uptake

Atherosclerosis and heart disease are the main cause of death in United States. The development of atherosclerosis includes lipid deposition and foam cell formation in the artery wall. Scavenger Receptors A‐I and II (SRA‐I/II) have an important role of in foam cell formation and atherogenesis. Most of the SRA‐I/II studies had been performed using Iodine‐125‐radiolabeled modified low‐density lipoprotein. This report attempts to validate the use of fluorescence microscopy techniques as an alternative to obtain qualitative and quantitative information of the uptake of fluorescence‐labeled acetylated low‐density lipoprotein (AcLDL) in adherent CHO cells expressing SRA‐I/II. After verifying the protein expression of SRA‐I and II, uptake was quantified using a Laser Scan Cytometer, and images of cells containing fluorescent AcLDL were obtained. A significant increase in fluorescence was found in the cells transfected with SRA‐I/II vs. those with empty vector. When SRA‐I/II competitive ligands were used, the uptake of AcLDL was significantly decreased. In conclusion, the use of fluorescence microscopy techniques in obtaining qualitative and quantitative information of the uptake of fluorescence‐labeled AcLDL by adherent cells, such as CHO cells, is an alternative to the traditional use of radiolabeled iodine. SCANNING 31: 167–173, 2009. © 2009 Wiley Periodicals, Inc.     

MicroRNA-181a is elevated by 10-hydroxycamptothecin and represses lung carcinoma progression by downregulating FOXP1

Tumor progression is usually characterized by proliferation, migration, and angiogenesis, which is essential for supplying both nutrients and oxygen to the tumor cells. Therefore, targeting angiogenesis has been considered a promising therapeutic strategy for cancer prevention and treatment. In the present study, we demonstrated that in addition to suppressing lung cancer cell proliferation and migration in vitro, 10-hydroxycamptothecin (10-HCPT) is also capable of inhibiting angiogenesis in vivo with a miR-181a-dependent manner. Mechanistically, by upregulating miR-181a, which in turn downregulating FOXP1, 10-HCPT can inhibit the PI3K/Akt/ERK signaling pathwaymediated angiogenesis. Furthermore, reduced levels of miR-181a have been found in both lung cancer cell lines and xenograft with concurrently elevated levels of FOXP1, VEGF, bFGF, and HDGF. Consistent with the findings from the in vitro experiments, miR-181a impairs neovascularization in our xenograft model. In summary, our findings have not only established the anti-oncogenic role of miR-181a in lung cancer angiogenesis but also suggest that 10-HCPT could be a potential therapeutic reagent for lung cancer treatment.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

miR-181b promotes the oncogenesis of renal cell carcinoma by targeting TIMP3

Renal cell carcinoma (RCC) has a poor prognosis due to limited diagnosis and treatment. Thus, it is necessary to find novel prognostic biomarkers and therapeutic targets. The aberrant expression of microRNAs plays an important role in RCC oncogenesis. Tissue inhibitors of metalloproteinase 3 (TIMP3) acts as a downstream target of miR-181b. The aim of this study was to understand the role and molecular mechanism of miR-181b in RCC oncogenesis. The results showed that miR-181b expression was significantly higher in RCC tumour tissues, especially in those with significant invasion or metastasis. miR-181b overexpression promoted proliferation and migration of the RCC cell line 786-O, while miR-181b knockdown had the opposite effect. In addition, miR-181b was inversely correlated with TIMP3 expression in RCC tumour tissues. miR-181b overexpression reduced TIMP3 expression in RCC cell line 786-O or OS-RC-2, while miR-181b knockdown had the inverse effect. Mechanistically, a luciferase reporter assay confirmed the binding sites of miR-181b on the 3’-UTR of TIMP3, confirming the targeting effect of miR-181b on TIMP3. Overall, miR-181b promotes the development and progression of RCC by targeting TIMP3 expression, indicating the potential use of miR-181b in the diagnosis and treatment of RCC.