Transwell Experiments Research Articles

Extracellular vesicle loaded with miR-494-3p can suppress ovarian cancer cell

ObjectiveOvarian cancer (OC) accounts for the highest mortality rate among all gynecological malignancies. We intend to develop engineered exosomes to deliver the microRNA to inhibit the growth and progression of OC. MethodsExtracellular vesicles (EV) secreted by OC cells were isolated and loaded with miR-494-3p by electroporation to prepare the engineered miR-494-3p EV. The morphology and size of miR-494-3p EV were identified using the Transmission Electron Microscope and Nanoparticle Tracing Analysis. Western blot (WB) was conducted to detect the exosomal markers including TSG101 and CD63. The loading efficiency was assessed by qRT-PCR. Exosomes labeled with PKH26 were used to study the specific uptake by OC cells. OVCAR3 and SKOV3 cells were treated with EV, miR-NC EV, and miR-494-3p EV, respectively. EdU assay was conducted to evaluate proliferation. The level of miR-494-3p in OC tissues and cell lines was detected by qRT-PCR. Flow cytometry was used to detect apoptosis. Scratching assay was applied to evaluate migration. Transwell assay was used to evaluate migration and invasion. The levels of apoptosis-related, migration-related, and invasion-related proteins were assessed by WB. ResultsThe results of qRT-PCR showed that miR-494-3p was significantly lower expressed in OC tissues than in normal tissues. The same is true in OC cell lines. The results showed that miR-494-3p EV could be successfully absorbed by OVCAR3 and SKOV3 cells. The expression of miR-494-3p in miR-494-3p EV or miR-494-3p EV-treated OC cells was significantly increased. The results of the EdU experiment, scratch experiment, and Transwell experiment showed that miR-494-3p EV significantly inhibited the proliferation, migration, and invasion of OC cells. Flow cytometry results showed that miR-494-3p EV significantly increased OC cell apoptosis. These results were further verified by the WB experiment. ConclusionsIn summary, our study suggests that OC cell-derived miR-494-3p EV may play a role in the development of OC cells by inhibiting the proliferation, migration, and invasion of OC cells and promoting apoptosis. These findings may provide new therapeutic strategies for the clinical treatment of OC.

Elucidating the Influence of MPT-driven necrosis-linked LncRNAs on immunotherapy outcomes, sensitivity to chemotherapy, and mechanisms of cell death in clear cell renal carcinoma.

Clear cell renal carcinoma (ccRCC) stands as the prevailing subtype among kidney cancers, making it one of the most prevalent malignancies characterized by significant mortality rates. Notably,mitochondrial permeability transition drives necrosis (MPT-Driven Necrosis) emerges as a form of cell death triggered by alterations in the intracellular microenvironment. MPT-Driven Necrosis, recognized as a distinctive type of programmed cell death. Despite the association of MPT-Driven Necrosis programmed-cell-death-related lncRNAs (MPTDNLs) with ccRCC, their precise functions within the tumor microenvironment and prognostic implications remain poorly understood. Therefore, this study aimed to develop a novel prognostic model that enhances prognostic predictions for ccRCC. Employing both univariate Cox proportional hazards and Lasso regression methodologies, this investigation distinguished genes with differential expression that are intimately linked to prognosis.Furthermore, a comprehensive prognostic risk assessment model was established using multiple Cox proportional hazards regression. Additionally, a thorough evaluation was conducted to explore the associations between the characteristics of MPTDNLs and clinicopathological features, tumor microenvironment, and chemotherapy sensitivity, thereby providing insights into their interconnectedness.The model constructed based on the signatures of MPTDNLs was verified to exhibit excellent prediction performance by Cell Culture and Transient Transfection, Transwell and other experiments. By analyzing relevant studies, we identified risk scores derived from MPTDNLs as an independent prognostic determinant for ccRCC, and subsequently we developed a Nomogram prediction model that combines clinical features and associated risk assessment. Finally, the application of experimental techniques such as qRT-PCR helped to compare the expression of MPTDNLs in healthy tissues and tumor samples, as well as their role in the proliferation and migration of renal clear cell carcinoma cells. It was found that there was a significant correlation between CDK6-AS1 and ccRCC results, and CDK6-AS1 plays a key role in the proliferation and migration of ccRCC cells. Impressive predictive results were generated using marker constructs based on these MPTDNLs. In this research, we formulated a new prognostic framework for ccRCC, integrating mitochondrial permeability transition-induced necrosis. This model holds significant potential for enhancing prognostic predictions in ccRCC patients and establishing a foundation for optimizing therapeutic strategies.

Suppression of hepatocellular carcinoma progression by long noncoding RNA apolipoprotein C1 pseudogene via the regulation of the microRNA-106b-PTEN axis.

Numerous researches have reported that long noncoding RNAs (lncRNAs) participate in tumor development and progression. LncRNA apolipoprotein C-I pseudogene 1 (APOC1P1), a pseudogene located in 19q13.2 between apolipoprotein C-I and apolipoprotein C-IV, is involved in a variety of diseases. However, the role of lncRNA APOC1P1 in hepatocellular carcinoma (HCC) remains unknown. Quantitative polymerase chain reaction (qPCR) was performed to examine the expression of APOC1P1, miR-106b, and PTEN (phosphatase and TENsin homolog deleted on chromosome 10) in HCC tissues, adjacent normal tissues, and specific cell lines (LO2, Bel-7407, HCCLM3, MHCC-97H, Hep G2, and Huh-7). Upregulation of APOC1P1 and downregulation of miR-106b were conducted via application of vector transfection and microRNA (miRNA) inhibitor. Bioinformatics analysis and luciferase reporter assay were used to verify the binding sites of APOC1P1, miR-106b, and PTEN. Cell proliferation and invasion were determined with Cell Counting Kit-8 (CCK-8) and Transwell experiments. Subcellular location analysis was used to determine the distribution of APOC1P1 in cells, and Western blotting was used to detect the expression of PTEN. It was found that the expressions of APOC1P1 and PTEN were downregulated, while that of miR-106b was upregulated in HCC tissues and cells. Subcellular location analysis showed that APOC1P1 was localized in cytoplasm and competitively bound to miR-106b. APOC1P1 overexpression and miR-106b inhibition suppressed HCC cell proliferation and invasion. qPCR indicated the negative correlation between APOC1P1 expression and miR-106b expression in HCC tissues and a positive correlation between APOC1P1 and PTEN. Our findings suggested that the lncRNA APOC1P1 inhibits HCC progression by competitively binding to miR-106b, leading to elevated PTEN expression, inhibiting cell proliferation and invasion in HCC cells. These results provide new insights into the diagnosis and therapy of HCC.

Paeonol impacts ovarian cancer cell proliferation, migration, invasion and apoptosis via modulating the transforming growth factor beta/smad3 signaling pathway.

Paeonol (2-hydroxy-4-methoxyphenylacetophenone) is a natural phenolic component isolated from the root bark of peony with multiple pharmacological activities and has been proven to have anti-cancer effects. The objective of this study is to investigate the influence mechanism of paeonol on the proliferatory and apoptotic activities of ovarian cancer (OC) cells by modulating the transforming growth factor beta (TGF-β)/Smad3 pathway. The SKOV3 cells were pretreated with various concentrations of paeonol (0, 25, 50, 100, 200, 400 μg/mL) for 48 hours to determine the optimal experimental concentration of paeonol. Following this, the TGF-β overexpression vector was constructed and transfected into the SKOV3 cells. The assessment of cell proliferation, invasion, and migration was conducted through MTT, colony formation, flow cytometry, transwell, and wound-healing experiments. The detection of TGF-β/Smad3 pathway-related proteins and apoptosis-related proteins (B-cell lymphoma (Bcl-2) Bcl-2-associated X protein (Bax)) was performed using Western blot analysis. Paeonol exhibited a significant inhibitory effect on SKOV3 cell viability when administered at concentrations ranging from 50-400 μg/mL, with an IC50 value of 200 μg/mL. Within the concentration range of 50 to 200 μg/mL, paeonol exhibited a dose-dependent effect on the progression of SKOV3 cells, including a reduction in the anti-apoptotic protein Bcl-2, an increase in the pro-apoptotic protein Bax (P<0.05), inhibition of cell migration and invasion (P<0.05), and promotion of cell apoptosis (P<0.05), particularly at a concentration of 200 μg/mL. These effects were found to be more pronounced. The aforementioned effects of paeonol can be ascribed to its inhibition of the TGFβ/Smad3 pathway, according to a mechanistic viewpoint. It is noteworthy that the inhibitory impact of paeonol on SKOV3 cell progression is counteracted by the elevation of TGF-β levels following overexpression. We conclude that paeonol exerts regulatory effects on the TGF-β/Smad3 pathway, leading to the inhibition of proliferation, migration, and invasion of OC cells, thereby attenuating malignant behavior of cancer cells.

PSMA7 promotes the malignant proliferation of esophageal cancer

BackgroundIt is important to explore novel molecules that play a key role in esophageal cancer (ESCA) progression. MethodsTwo ESCA tissue expression profile microarrays (GSE92396 and GSE17351) data from GEO were downloaded, and differentially expressed genes (DEGs) were analyzed using GEO2R. The DEGs common to both microarrays were analyzed for protein-protein interactions, KEGG and GO. The altered expression of proteasome 20S subunit α 7 (PSMA7) in ESCA tissues was analyzed using information from publicly available databases (GEO, TCGA, TNMplot). PSMA7 was overexpressed or knocked down in Eca109 and KYSE150 cells using transfection, and the effects on cell proliferation, migration, invasion and apoptosis were examined using CCK-8, Transwell, and flow cytometry experiments. Results284 common DEGs were identified, and 10 core proteins, HSP90AA1, AURKA, CDC6, PCNA, MCM5, KAT2B, GRB2, MYBL2, PSMA7, and CKAP5, involved in ESCA progression were identified. PSMA7 mRNA level was significantly increased in ESCA tissues. PSMA7 overexpression significantly promoted the proliferation, migration and invasion of Eca109 and KYSE150 cells, and significantly promoted apoptosis. In contrast, PSMA7 knockdown inhibited their proliferation and motility, and significantly suppressed apoptosis. ConclusionThis study analyzed multiple proteins that may play a key role in ESCA progression, and identified the pro-cancer role of PSMA7.

CTSK and PLAU as Prognostic Biomarker and Related to Immune Infiltration in Pancreatic Cancer: Evidence from Bioinformatics Analysis and qPCR

Pancreatic adenocarcinoma (PAAD) is a malignancy with the highest mortality rate worldwide. There is a pressing need for novel biomarkers that can facilitate early detection and serve as targets for therapeutic interventions beyond the commonly utilized CA199 marker. This study utilized microarray datasets (GSE15471, GSE62165, and GSE28735) from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs) and construct a protein-protein interaction network using STRING and Cytoscape. Hub genes were selected using BiNGO. Expression profiles and clinical data from the Cancer Genome Atlas (TCGA) were then used to compare the expression levels of CTSK and PLAU in pancreatic cancer and healthy pancreatic tissues via the Wilcoxon rank-sum test, with further validation using qPCR. Functional enrichment analysis was conducted to explore potential signaling pathways and biological functions. Prognostic values were assessed by the Kaplan-Meier and Cox regression analyses, and an overall survival (OS) nomogram was created to predict 1-, 2-, and 3-year survival after cancer diagnosis. The infiltration of immune cells was evaluated by single-sample gene set enrichment analysis. The methylation status of both genes was analyzed using the UALCAN and MethSurv databases. The results demonstrated that CTSK and PLAU were overexpressed in pancreatic cancer and that the hypomethylation status of both genes was associated with a poor prognosis. The overexpression of both genes was positively correlated with various immune cells, and functional enrichment analysis revealed that they were associated with immune cell infiltration. Besides, the effects of PLAU on the migration and invasion of pancreatic cancer cells were also verified by scratch and transwell experiments. Consequently, CTSK and PLAU have potential as prognostic biomarkers for pancreatic cancer.

MicroRNA-152 specifically targets kinesin family member 14 to suppress the advancement of bladder cancer cells via PI3K/AKT pathway

BackgroundKinesin family member 14 (KIF14) overexpression has been linked to tumor progression and metastasis in different malignancies, but its precise molecular mechanism in bladder cancer (BLCA) remains unclear. MethodsThe expression of KIF14 in BLCA and its relationship with clinical outcomes were assessed. Functional investigations on KIF14 were conducted using CCK-8, Transwell experiment, colony formation, scratch motility assays, and flow cytometry. We examined the downstream route of KIF14 and identified its upstream regulatory factor through luciferase reporter experiments and bioinformatics tools. ResultsOur findings demonstrated that increased KIF14 expression was associated with poor survival prognosis in BLCA patients. Deletion of KIF14 affected cell cycle progression, induced apoptosis, and inhibited cell growth, migration, and invasion. GSEA analysis revealed a strong association between KIF14 expression and the PI3K/AKT signaling pathway. Further research showed that KIF14 deletion decreased the levels of p-PI3K, p-AKT, FOXM1, and CCNB1. We also found that has-miR-152-3p (miR-152) suppressed BLCA cell growth by post-transcriptionally regulating KIF14 expression. ConclusionsOur findings suggest that targeting KIF14 could alter the PI3K/AKT and FOXM1-CCNB1 axis, leading to growth inhibition, cell cycle arrest, and induction of apoptosis in BLCA cells. Additionally, miR-152 directly regulates KIF14 expression at the post-transcriptional level. Overall, KIF14 represents a promising therapeutic target for BLCA clinical therapy.

A novel long noncoding RNA (lncRNA), LINC02657(LASTR), is a prognostic biomarker associated with immune infiltrates of lung adenocarcinoma based on unsupervised cluster analysis.

Non-small cell lung cancer (NSCLC) has long been the deadliest malignancy worldwide, with adenocarcinoma (AD) being the most common pathological subtype. Here we focused on the value of LASTR in LUAD. Using expression analysis, enrichment analysis, immune cell infraction analysis, we found that the expression level of LASTR was significantly increased in LUAD tissue. Meanwhile, LASTR was significantly associated with differential infiltration of various immune cells. Kaplan-Meier survival analysis showed that LUAD related with a poor prognosis in terms of OS, PFI, and DSS compared with high-expression LASTR. The enrichment analysis showed that LASTR is related to the pathays like PI3K-AKT signaling pathway. Thus, the present findings could be helpful in a better understand of LASTR in LUAD. RT-PCR was used to verify the high expression of LASTR in LUAD tissues, and the apoptosis of LUAD cell lines was promoted by CCK8 and Transwell experiments to verify the ability of LASTR to promote the migration and invasion of lung cancer cells in vitro.

Effects of miR-143 on the migration and invasion of osteosarcoma cells by regulating MMP-13 expression

To explore the effect of miR-143 regulating matrix metalloproteinase(MMP)-13 expression on migration and invasion of osteosarcoma cells. The mouse osteosarcoma cell line 143B cells were cultured in 96-well plates, and blank group, negative group, positive group, and intervention group were set up. Then, the blank group did no treatment 50 μg miR-143 mimic was added to positive group, negative group added equal mimic NC (control sequence of miR-143 mimic), the intervention group was added 50 μg miR-143 mimic and 10 μg MMP-13 protein, all groups continued to culture for 3 to 6 hours, and finally the serum was aspirated to treat for half an hour. The protein expressions of miR-143 and MMP-13 in each group were measured by fluorescence quantitative PCR experiment and Western blot experiment, respectively, and the invasion and migration abilities of cells were measured by Transwell and scratch experiments. The expression of MMP-13 protein in the positive group and the intervention group was significantly lower than that in the blank group, and the positive group was lower than the intervention group (P<0.05);The mean numbers of invasive cells in blank group, negative group, positive group and intervention group were (1 000.01±44.77), (959.25±46.32), (245.04±4.33), (634.06±33.78) cells/field, respectively;the scratch healing rate of the positive group and the intervention group was significantly lower than that of the blank group, and the positive group was lower than the intervention group (P<0.05). MMP-13 is a target of miR-143, which can reduce the migration and invasion ability of osteosarcoma cells by inhibiting the expression of MMP-13.

Investigation of the biological activity of hydroxyapatite on vascular endothelial cells

ObjectiveThis study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. MethodsWe employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. ResultsAt extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. ConclusionHydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

Nattokinase enhances the sensitivity of cancer cells to oxaliplatin through mitochondrial pathway and induction of apoptosis

Combination therapy consisting of anticancer drugs has been used to decrease the adverse effects and increase the effectiveness of the drugs. The present study aimed to examine the synergistic effect and potential mechanisms of oxaliplatin and nattokinase (NK) combined treatment for BGC-823, HepG-2, HeLa, and NCI-H460 cells. NK was isolated and purified from black soybean natto by dialysis and gel filtration chromatography, which had a nattokinase activity of 600,391 U/g. MTT experiment, clone formation assay, Hoechst 33,342 staining, DCFH-DA staining, and JC-1 staining showed that NK had no cytotoxicity and oxaliplatin could inhibit cell proliferation. Oxaliplatin combined with NK could enhance its anticancer effect and lead to mitochondrial dysfunction by causing the increase of intracellular ROS, which eventually induced apoptosis. Compared to oxaliplatin alone, nattokinase in combination with oxaliplatin can effectively inhibit DNA synthesis and increase cell uptake, ultimately inhibiting cell proliferation. Transwell and angiogenesis experiments proved that the combination of NK and oxaliplatin could more effectively inhibit cell migration, invasion, and in vitro angiogenesis, thereby preventing the spread and metastasis of tumors. RT-qPCR showed that the mechanism of inhibition of cell proliferation and thus induction of apoptosis by the combination of NK and oxaliplatin may be achieved through the upregulation of BAD, BAX, and Caspase-3 genes. Molecular docking results show that NK could act as the carrier of oxaliplatin. In conclusion, the above results suggested that NK could play a synergistic anticancer role with oxaliplatin by inducing apoptosis through the mitochondrial pathway, enhance the sensitivity of cancer cells to oxaliplatin, and provide a new strategy for cancer treatment.

Exosomal miR-331-3p derived from chemoresistant osteosarcoma cells induces chemoresistance through autophagy

BackgroundOsteosarcoma is a common malignant bone tumor, and chemotherapy can effectively improve the prognosis. MicroRNA-331 (MiR-331) is associated with poor cancer outcomes. However, the role of miR-331 in osteosarcoma remains to be explored.MethodsDrug-resistant osteosarcoma cells were cultured, and their exosomes were purified. The secretion and uptake of exosomes by drug-resistant osteosarcoma and osteosarcoma cells were confirmed using a fluorescence tracking assay and Transwell experiments. The effects of drug-resistant exosomes on cell proliferation were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. siRNA-Drosha and neutral sphingomyelinase inhibitor GW4869 were used to determine the transfer of miRNAs. qRT-PCR and western blotting were used to detect the role of autophagy in the regulation of drug-resistant cell-derived exosomal miR-331-3p.ResultsExosomal miR-331-3p levels in drug-resistant cells were higher than in exosomes from osteosarcoma cells. The exosomes secreted by the drug-resistant osteosarcoma cells could be absorbed by osteosarcoma cells, leading to acquired drug resistance in previously non-resistance cells. Inhibition of miRNAs resulted in reduced transmission of drug resistance transmission by exosomes. Exosomes from drug-resistant osteosarcoma cells transfected with siRNA-Drosha or treated by GW4869 could not enhance the proliferation of MG63 and HOS cells. Finally, miR-331-3p in the exosomes secreted by drug-resistant osteosarcoma cells could induce autophagy of osteosarcoma cells, allowing them to acquire drug resistance. The inhibition of miR-331-3p decreased drug resistance of osteosarcoma cells.ConclusionExosomes secreted from chemoresistant osteosarcoma cells promote drug resistance through miR-331-3p and autophagy. Inhibition of miR-331-3p could be used to alleviate drug resistance in osteosarcoma.

MicroRNA-221-3p promotes post-burn HUVEC proliferation, migration, and angiogenesis by regulating CDKN1B.

Previous studies have shown that miR-221-3p plays an important role in vascular remodeling, but it is unclear whether it contributes to angiogenesis after burn injury. The purpose of this study was to investigate the effect of miR-221-3p on angiogenesis in HUVECs after burn injury and to reveal its underlying molecular mechanism. The burn HUVECs model was established by heat treatment. Plasmid or oligonucleotide transfection altered the expression of miR-221-3p and CDKN1B in HUVECs. MTT, colony formation, Transwell, flow cytometry, and tube formation experiments were applied to assess the proliferation, migration, apoptosis, cell cycle, and tube formation capacity of HUVECs. miR-221-3p, CDKN1B, Ki-67, and PCNA expression was assessed by RT-qPCR or Western blot. The dual-luciferase reporter assay verified the targeting relationship between miR-221-3p and CDKN1B. miR-221-3p was lowly expressed and CDKN1B was highly expressed in burn HUVECs. Overexpression of miR-221-3p promoted the proliferation, migration, and tube formation ability of burn HUVECs and inhibited apoptosis and the proportion of cells in the G0/G1 phase, whereas overexpression of CDKN1B had the opposite effect. Knockdown of miR-221-3p further inhibited the angiogenic capacity of burn HUVECs, but this effect was reversed by knockdown of CDKN1B. Mechanistically, miR-221-3p targeted CDKN1B. miR-221-3p improves the angiogenesis of burn HUVECs by targeting CDKN1B expression, and the miR-221-3p/CDKN1B axis may serve as a potential molecular target for future burn therapy.

FAXDC2 inhibits the proliferation and invasion of human liver cancer HepG2cells.

The reprogramming of lipid metabolism serves an important role in occurrence and development of liver cancer. Fatty acid hydroxylase domain containing 2 (FAXDC2) is a hydroxylase involved in the synthesis of cholesterol and sphingomyelin and downregulated in various types of cancer. There are no reports on the relationship between FAXDC2 and liver carcinogenesis. The present study used multiple portals and publicly available tools to explore its correlation with liver cancer. The results showed that the expression of FAXDC2 decreased in liver cancer and the methylation level near the promoter increased. Patients with liver cancer and with low expression of FAXDC2 had a poor prognosis. Gain of function and loss of function strategies were performed to evaluate its roles in liver cancer cells. CCK-8 assay showed that overexpression of FAXDC2 inhibited the viability of liver cancer cells (HepG2). Flow cytometry analysis indicated that HepG2 cells with overexpressing FAXDC2 showed an S phase arrest, associated with cyclin-dependent kinase 2 decreased. Transwell experiments showed that increasing FAXDC2 inhibited HepG2 cell invasion ability, accompanied by the upregulation of E-cadherin. Notably, knockdown of FAXDC2 had no significant effect on cell cycle and invasion functions. Based on the cBioPortal platform, FAXDC2 was predicted to closely correlate to the ERK signal in tumorigenesis. Western blotting results showed that overexpression of FAXDC2 decreased the phosphorylation level of ERK in liver cancer cells. The present study first identified FAXDC2 as a liver cancer suppressor, which might inhibit the proliferation and invasion of liver cancer cells through the mechanism associated with ERK signaling. The present study provided a possible new target for the diagnosis and treatment of liver cancer.

OSM May Serve as a Biomarker of Poor Prognosis in Clear Cell Renal Cell Carcinoma and Promote Tumor Cell Invasion and Migration.

Currently, the role of oncostatin M (OSM) in clear cell renal cell carcinoma (ccRCC) has not been investigated. This study will explore the impact of OSM on ccRCC expression, prognosis, and cell function. In this study, we used The Cancer Genome Atlas (TCGA) database to evaluate OSM expression characteristics, pathogenic factor distribution, and prognostic aspects in ccRCC. We also combined this analysis with qRT-PCR to verify OSM mRNA expression levels at the tissue level. Then, the effects of OSM on the proliferation, invasion, and migration abilities of ccRCC cells were explored through CCK8, Transwell, Western blotting, and immunofluorescence experiments. Finally, the oncogenic mechanisms associated with OSM in ccRCC were explored through signaling pathway enrichment and single-cell analysis. The results demonstrated that OSM was significantly more expressed in ccRCC than in normal tissues. According to the survival analysis, OSM in ccRCC was considerably worse in the group with high expression than in the group with low expression. Also, the univariate and multivariate Cox analyses of clinical characteristics show that OSM in ccRCC may be able to predict a poor prognosis on its own as a biomarker. In vitro cellular experiments demonstrated that high OSM expression had no discernible impact on ccRCC cell proliferation compared to the control group, but it did promote tumor cell invasion and migration. Signaling pathways and single-cell analysis revealed that OSM might promote ccRCC invasion and migration through M2 macrophages. In conclusion, OSM may serve as an independent poor prognostic biomarker in ccRCC and promote tumor cell invasion and migration. This discovery is expected to provide a new therapeutic target for patients with recurrent and metastatic ccRCC.

The long non-coding RNA NEAT1 promotes the progression of human ovarian cancer through targeting miR-214-3p and regulating angiogenesis

BackgroundAngiogenesis and metastasis contributes substantially to the poor outcome of patients with ovarian cancer. We aimed to explore the role and mechanisms of the long non-coding RNA NEAT1 (nuclear enriched abundant transcript 1) in regulating angiogenesis and metastasis of human ovarian cancer. NEAT1 expression in human ovarian cancer tissues and cell lines including SKOV-3 and A2780 was investigated through in situ hybridization. Gene knockdown and overexpressing were achieved through lentivirus infection, transfection of plasmids or microRNA mimics. Cell viability was measured with the cell counting kit-8 assay, while apoptosis was determined by flow cytometry. Cell migration and invasion were evaluated by transwell experiments, and protein expression was determined by western blot assays or immunohistochemistry. Duo-luciferase reporter assay was employed to confirm the interaction between NEAT1 and target microRNA. In vivo tumor growth was evaluated in nude mice with xenografted SKOV-3/A2780 cells, and blood vessel formation in tumor was examined by histological staining.ResultsNEAT1 was highly expressed in ovarian cancer tissues of patients and cell lines. MiR-214-3p was identified as a sponging target of NEAT1, and they antagonizedeach other in a reciprocal manner. NEAT1-overexpressing SKOV-3 and A2780 cells had significantly increased proliferation, reduced apoptosis, and augmented abilities of migration and invasion, while cells with NEAT1-knockdown displayed markedly attenuated traits of malignancies. Additionally, the levels of NEAT1 appeared to be positively correlated with the expression levels of angiogenesis-related molecules, including Semaphorin 4D (Sema4D), Sema4D receptor Plexin B1, T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1), and Rho-like GTPases Rac1/2/3. In the xenograft mouse model, more NEAT1 expression resulted in faster in vivo tumor growth, more blood vessel formation in tumor tissues, as well as higher expression levels of angiogenesis-related molecules and CD31.ConclusionsNEAT1 promotes angiogenesis and metastasis in human ovarian cancer. NEAT1 and miR-214-3p are promising targets for developing therapeutics to treat human ovarian cancer.

Inhibition of UFM1 expression suppresses cancer progression and is linked to the dismal prognosis and immune infiltration in oral squamous cell carcinoma.

Ubiquitin fold modifier 1 (UFM1) overexpression is associated with cancer cell proliferation, migration and invasion. However, the roles and pathways of UFM1 in oral squamous cell carcinoma (OSCC) has remained undefined. The expression of UFM1 and the relationship between UFM1 expression and prognosis were investigated using data of OSCC patients from The Cancer Genome Atlas (TCGA) database. The UFM1 co-expressed genes, and the association between the UFM1 expression and immune cells and ubiquitination were explored. The effects of UFM1 expression on the growth and migration of OSCC cells were investigated by siRNA interference, Cell Counting Kit-8 (CCK-8), Transwell, Western blotting, and wound healing experiments. UFM1 was highly expressed in OSCC. UFM1 overexpression was associated with short overall survival, disease-specific survival, and progression-free interval, and was an adverse factor for prognosis in OSCC. UFM1-related nomograms were significantly associated with poor prognosis in OSCC patients. Decreased UFM1 expression could inhibit the proliferation, migration, and invasion of OSCC cells. UFM1 was associated with the immune cells (such as the Th17 cells, T helper cells, and cytotoxic cells) and ubiquitination. Elevated UFM1 expression was associated with poor prognosis, ubiquitination and immune infiltration in OSCC, and inhibition of UFM1 expression delayed OSCC progression, showing that UFM1 could be a biomarker for prognosis and treating OSCC patients.

Graphene quantum dots promote migration and differentiation of periodontal ligament stem cells.

Graphene and its derivatives have attracted much attention as nanomaterials in bone tissue engineering because of their remarkable ability to induce cell osteogenic differentiation. However, graphene quantum dots (GQDs), as graphene derivatives, little is known about their osteodifferentiation- and osteoinduction-promoting capabilities, especially in the restoration of bone defect caused by periodontitis. Therefore, there is a growing need to investigate the effect of GQDs on periodontal ligament stem cells (PDLSCs). Here, we postulated that GQDs are a promising biocompatible nanomaterial that facilitated the migration and differentiation of PDLSCs, and use laboratory methods like CCK-8, transwell experiments, qRT-PCR, Alizarin red staining and immunofluorescence staining to evaluate. Our experiments confirmed that GQDs did not inhibit cell viability, with most cells remaining viable even at GQDs concentrations of up to 30μgmL-1. Moreover, GQDs were found to significantly enhance PDLSC migration, with the peak effect observed at concentrations of 5 and 10μgmL-1. Furthermore, GQDs accelerated osteoblastic differentiation in PDLSCs and induced the mineralization of calcium nodules. Additionally, GQDs were shown to promote fibroblast differentiation in PDLSCs compared to the control group. Thus, GQDs not only possessed low cytotoxicity and good biocompatibility, but also displayed the beneficial capability to migration and differentiation of PDLSCs, which indicated GQDs might be a potential nanomaterial for bone regeneration.

TREM1 is involved in the mechanism between asthma and lung cancer by regulating the Toll‑like receptor signaling pathway.

Lung cancer and asthma are both global health problems with significant economic consequences. Recent studies have demonstrated that asthma may be a risk factor for lung cancer. The present study aimed to explore the pathogenesis between these two diseases through a comprehensive analysis. Differentially expressed genes (DEGs) screened in the asthma-related GSE165934 dataset were analyzed to find relevant inflammatory pathways. Overlapping genes regulated by inflammatory pathways and lung cancer-DEGs from The Cancer Genome Atlas (TCGA) were obtained and subjected to survival and gene-wide mutation analyses, and nomogram construction to determine the hub gene. The hub gene was further analyzed through expression validation, immunoassays and functional experiments to investigate its role and mechanism in lung cancer. Functional enrichment analysis showed that 1,275 DEGs from GSE165934 were closely associated with the Toll-like receptor signaling pathway, and 8 overlapping genes were identified from 12 genes regulated by the Toll-like receptor signaling pathway and 3,134 TCGA-DEGs. After a series of bioinformatics analyses, it was found that triggering receptor expressed on myeloid cells 1 (TREM1) was the hub gene involved in the mechanism of asthma and lung cancer. TREM1 was also found to be a suppressor gene in lung cancer correlated with immune cells, immune checkpoint-related genes and tumor mutational burden score. Additionally, the results of Cell Counting Kit-8 and Transwell experiments demonstrated that overexpression of TREM1 could significantly inhibit the invasion, proliferation and migration of lung cancer cells. Reverse transcription-quantitative PCR and western blotting demonstrated that the overexpression of TREM1 could also significantly reduce the level of Toll-like receptor signaling pathway proteins. The present findings suggest that TREM1 is associated with the mechanism of asthma and lung cancer through its regulation of the Toll-like receptor signaling pathway. Furthermore, TREM1 may serve as a potential treatment target and prognostic indicator for patients with lung cancer.

UBC9 Deficiency Accelerates AML Progression By Downregulating PARP1 Sumoylation and Subsequently Activating the NF-ΚB Pathway

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a disease of heterogeneous clonal hematopoietic progenitor cells. Despite the relentless development of therapeutic options, including chemotherapy, and identification of many new therapeutic targets, the prognosis of AML is still poor, with a 5-year survival rate of approximately 35% in patients younger than 60 years old and 10% in patients older than 60 years, which may be due to the high degree of AML heterogeneity. Targeting the shared mechanisms of action among subclones has emerged led to find novel strategies, such as targeting protein post post-translational modification. Here we show that the impact of the UBC9 (the only E2 conjugation enzyme) and its related SUMOylation, a critical and reversible posttranslational modification as ubiquitination, on AML progression, to provide novel and potent sight for AML therapy. To identify the role of UBC9 expression in the clinical outcome of AML patients, we measured the expression of key SUMOylation genes, including SAE1, UBA2, UBC9, SUMO1, SUMO2, SUMO3, PIAS1, PIAS2, PIAS3, PIAS4, CBX4, and RANBP2, in the TCGA dataset (n=164) by using a multivariate survival curve based on the online tool SurvExpress. Our results showed that the survival probability curves of the low- and high-risk groups were significantly different due to the differences in the levels of expression of key SUMOylation genes ( p = 0.0011), and we found that the expression level of UBC9 was lower in the high-risk group than in the low-risk group. We next analyzed the UBC9 expression level and the prognosis of AML patients from the Therapeutically Applicable Research To Generate Effective Treatment dataset (n=156), which showed that AML patients with low UBC9 expression showed shorter overall survival ( p = 0.018)(Figure 1). In addition, real-time PCR showed that the UBC9 expression level in bone marrow samples from patients with de novo AML was significantly downregulated in both the high-risk patients based on National Comprehensive Cancer Network guidelines and the liver or spleen enlargement patients ( p = 0.01 and p = 0.001, respectively) (Figure 1). We used RNA interference system to knock out Ubc9 in a mouse model of MLL-AF9 and MLL-ENL induced AML. We observed the survival of AML mice and found that Ubc9 fl/+;Vav-Cre recipient mice showed a significantly shorter survival time than Ubc9 fl/+ mice (Figure 2). In addition, apoptosis experiments revealed reduced apoptosis of leukemia cells and leukemia stem cells in Ubc9 fl/+;Vav-cre mice. HE staining showed significantly higher leukemia cell infiltration in the livers, meninges, and kidneys of Ubc9 fl/+;Vav-cre mice compared to Ubc9 fl/+ mice. Transwell experiments indicated that Ubc9 deficiency resulted in increased migration of leukemia cells. Colony forming assay demonstrated that Ubc9-deficient leukemia cells formed significantly larger colonies. Flow cytometry analysis showed that Ubc9-deficient leukemia cells expressed high levels of c-kit and low levels of Mac1. To further elucidate the role of UBC9 in human AML cells, we constructed UBC9 deficient KG1 and K562 cell lines by using CRISPR-Cas9 technology. The UBC9 KD cell lines presented the same phenotype such as the increase of cell viability and the enhancement of invasiveness. To better understand the role that UBC9 plays in AML cell biology, we performed a system-level approach based on quantitative SUMO proteomic analysis to identify potential UBC9 substrates and then identified PARP1 with SUMOylation site lysine residue 486 (K486) by multiomics analysis. Finally, we validated that UBC9-mediated PARP1 SUMOylation played an important role in AML progression by activating the NF-kappa B pathway via the site-directed mutation of PARP1 (K486R) in UBC9-KO AML cells by using western blot and Co-immunoprecipitation. Together, our study revealed that UBC9 KO accelerated AML progression and enabled us to contrast global SUMOylation profiles of UBC9 in AML cells. In addition, we found that the PARP1 SUMOylation-mediated NF-κB pathway activation is a positive regulatory mechanism in the tumorigenesis of AML. These findings may provide potential clinical implications for AML treatment.