CCK-8 Experiments Research Articles

Silencing of lncRNA KLF3-AS1 represses cell growth in osteosarcoma via miR-338-3p/MEF2C axis.

Osteosarcoma (OS) is a highly recurrent malignancy occurring among adolescents. The goal of this research was to scrutinize the role and action mechanism of KLF3-AS1 in OS. Western blotting and quantitative reverse transcription real-time PCR were conducted to ascertain the mRNA expressions of miR-338-3p, KLF3-AS1, and MEF2C in OS cell lines and tissue samples. Colony formation and CCK-8 experiments were done to evaluate the proliferative capacity of the cells. Western blotting was also executed to measure the relative expressions of the proteins Bcl-2 and Bax. RNA immunoprecipitation and dual luciferase reporter experiments were carried out to validate the target relationships among MEF2C, KLF3-AS1, and miR-338-3p. Mouse xenograft models were created to assess the influences of KLF3-AS1 on the growth of tumors in vivo. Elevated levels of KLF3-AS1 and MEF2C and reduced amounts of miR-338-3p were identified in OS. KLF3-AS1 targeted miR-338-3p, and miR-338-3p further targeted MEF2C. Silencing KLF3-AS1 induced apoptosis and attenuated proliferation in vitro and repressed the tumor growth in vivo. Inhibiting miR-338-3p inverted the cancer-suppressing effects of KLF3-AS1 silencing. Meanwhile, loss of MEF2C partially eliminated the effects brought about by miR-338-3p downregulation, namely the stimulation of cell growth and suppression of apoptosis. Silencing of KLF3-AS1 could repress the growth of cells and induce apoptosis by regulating miR-338-3p/MEF2C in OS. This suggests that the regulatory axis KLF3-AS1/miR-338-3p/MEF2C is a prospective target for OS treatment.

LINC01140 Targeting miR-452-5p/RGS2 Pathway to Attenuate Breast Cancer Tumorigenesis.

Background LINC01140 has been known to be involved in various cancers. However, its underlying molecular mechanism in breast cancer (BC) needs further exploration. Methods The LINC01140, miR-452-5p, and RGS2 levels in BC cells and tissues were evaluated by means of RT-qPCR and western blotting. The variations in the biological functions of BC cells were analyzed through CCK-8, transwell, western blotting, and xenograft experiments to observe cell viability, migration, levels of apoptosis-related proteins (Bax and Bcl-2), and tumor growth. The correlations existing among LINC01140, miR-452-5p, and RGS2 were validated through luciferase reporter and RIP assays. Results LINC01140 and RGS2 were remarkably downregulated in BC cells and tissues, whereas miR-452-5p was upregulated. LINC01140 overexpression diminished BC cell viability, migration, and tumor growth and facilitated apoptosis. MiR-452-5p upregulation enhanced cell viability and migration and suppressed apoptosis. Nevertheless, the additional upregulation of LINC01140 could reverse the promotive effects of miR-452-5p upregulation. Additionally, RGS2 overexpression inhibited the malignant phenotypes of BC cells, but miR-452-5p upregulation abolished this effect. In terms of mechanisms, LINC01140 acted as a miR-452-5p sponge. Moreover, RGS2 was determined to be miR-452-5p's downstream target gene in BC. Conclusion LINC01140 functioned as an antitumor agent in BC by sponging miR-452-5p to release RGS2. This hints that LINC01140 is a promising therapeutic target for BC.

Lncrna FEZf1-as1 negatively regulates ETNK1 to promote malignant progression of renal cell carcinoma.

To explore the role of LncFEZF1-AS1 in renal cell carcinoma (RCC) tissues and cells, and the possible molecular mechanism. Expressions of LncFEZF1-AS1 in RCC tissues and adjacent ones were detected. The association of LncFEZF1-AS1 level with clinical data of RCC patients was also analyzed. Besides, the differential expressions of LncFEZF1-AS1 in a variety of RCC cell lines were also determined. Then the LncFEZF1-AS1 knockdown model was constructed in RCC cell line to further determine the influences of LncFEZF1-AS1 on the proliferative ability and migration of RCC cells through CCK8 and Transwell experiments. Furthermore, luciferase reporter gene experiment were used to validate the combination of LncFEZF1-AS1 to ETNK1. Results suggested that expression of LncFEZF1-AS1 was noticeably higher in RCC tumor tissues and the RCC cells. Clinical pathological data analysis also suggested that high LncFEZF1-AS1 expression was in correlation with the pathological stage and the incidence of distant metastasis in RCC patients, and the poor overall survival rate. In vitro experiments demonstrated that knocking down of LncFEZF1-AS1 markedly repressed the proliferation and migration of RCC cell lines. Bioinformatics suggested that LncFEZF1-AS1 can interact with the downstream target gene ETNK1, which was confirmed by the luciferase reporter gene experiments. Western Blot results revealed that knocking down of LncFEZF1-AS1 markedly enhanced ETNK1. qRT-PCR analysis indicated that ETNK1 level was under-expressed in RCC tissues and in negative correlation with LncFEZF1-AS1. Further experiments suggested that knockdown of ETNK1 partially reversed the inhibitory effects of LncFEZF1-AS1 silencing on the proliferative and migrative abilities of RCC cells. LncFEZF1-AS1 could facilitation the proliferative and migration of RCC cells by regulating the expression of ETNK1. Therefore, FEZF1-AS1 might function as a cancer-promoting factor and possible new therapeutic target for RCC.

Expression and potential role of FOSB in glioma.

BackgroundFOSB is reported to be an oncogene in a variety of tumors. However, the expression and role of FOSB in glioma remain obscure. In this study, we aimed to explore the expression of FOSB in glioma and its biological role in glioblastoma multiforme (GBM).MethodsWestern blot, immunohistochemical staining, and quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression of FOSB in clinical samples. FOSB was knocked down in cells to determine the effects of FOSB on the phenotypic changes of tumors by plate cloning, CCK-8 assay, and Transwell assay. Finally, subcutaneous tumorigenesis in nude mice was used to observe the tumorigenesis of glioma cell lines after the knockdown of the FOSB gene.ResultsFOSB expression was higher in glioma compared with normal brain tissue. After the downregulation of FOSB, the expression of cleaved caspase-3 increased. Plate cloning and CCK-8 experiments showed that the proliferation of glioma cell lines decreased. The Transwell assay demonstrated that the glioblastoma cell lines had lower migration ability after the knockdown of FOSB. Finally, the tumor volume of U87 glioma cells in group sh-FOSB was smaller than that in the control group. The TUNEL staining in vitro showed that the apoptosis of sh-FOSB glioma cells increased.ConclusionFOSB was highly expressed in glioma tissues. The viability of glioma cells decreased, and the ability of glioma cells to proliferate and migrate was reduced when FOSB was downregulated. Hence, FOSB may promote the development and migration of gliomas.

Ferulic Acid Mitigates Growth and Invasion of Esophageal Squamous Cell Carcinoma through Inducing Ferroptotic Cell Death.

Objective Ferroptosis is an iron- and ROS-dependent form of cell death initiated by lipid peroxidation. The rapidly developing study of ferroptosis has facilitated its application in cancer therapeutics. The current study is aimed at investigating the functional property of ferulic acid (FA, a phenolic acid substance) on inducing ferroptosis in antiesophageal squamous cell carcinoma (ESCC). Methods ESCC cells were administrated with gradient doses of FA or with ferroptosis inhibitor deferoxamine. Cellular growth was measured with CCK-8 and colony formation experiments. LDH, caspase-3, MDA, SOD, GSH, and iron were assayed with corresponding kits. Apoptotic level was evaluated through Annexin V-FITC apoptosis staining, with migration and invasion utilizing Transwell assays. Through quantitative RT-PCR, angiogenesis-relevant genes VEGFA and PDGFB were detected. ROS generation was measured via DCFH-DA probe. Immunoblotting was conducted for monitoring ACSL4, SLC7A11, HO-1, and GPX4. Results FA administration observably mitigated cellular viability and colony formation capacity and motivated LDH release, caspase-3 activity, and apoptosis in EC-1 and TE-4 cells. In addition, migration and invasion together with angiogenesis of ESCC cells were restraint by FA. FA exposure led to the increase of MDA content, ROS production, and iron load as well as the reduction of SOD activity and GSH content. Also, FA augmented the activities of ACSL4 and HO-1, with lessening SLC7A11 and GPX4. Nonetheless, deferoxamine restrained the effect of FA on ESCC ferroptosis. Conclusion Altogether, FA may act as a ferroptosis inducer and thus attenuates cell growth and invasion of ESCC, which boosts the clinical application of FA in ESCC therapeutics.

AXL, along with PROS1, is overexpressed in papillary thyroid carcinoma and regulates its biological behaviour

BackgroundAXL, a TAM tyrosine kinase receptor, plays an essential role in the pathogenesis of various solid tumours. This study explores the role of AXL and its ligand PROS1 in the generation and biological behaviour of papillary thyroid cancer (PTC).MethodsThe expression levels of AXL in PTC cancer tissue were analysed using immunohistochemistry (IHC) staining. The expression levels of AXL in PTC and normal thyroid cell lines were analysed using real-time quantitative polymerase chain reaction (RT-qPCR). CCK-8 was used to assess the proliferation of the PTC cell line with and without the effect of the AXL inhibitor (R428). Scratching assays played a role in evaluating the cell migration rate.ResultsPROS1 and AXL were expressed in TPC-1, B-CPAP, and Nthy-Ori 3–1 cells at different levels. Expression was significantly higher in PTC cell lines (TPC-1 and B-CPAP) than in the normal thyroid cell line (Nthy-Ori 3–1) (p < 0.05). In addition, AXL expression in PTC tissues was significantly higher than in adjacent normal tissues (p < 0.05). CCK-8 experiments confirmed that R428 suppresses the proliferation of PTC cell lines in a dose-dependent manner, with an increase in concentration from 0.5 to 4 μM, decreasing the inhibitory effect (p < 0.01). In addition, R428 inhibited PTC cell line migration to different degrees in a range of concentrations from 0.5 to 2 μM compared to control cells (p < 0.01).ConclusionPROS1 and its downstream receptor AXL expression were significantly higher in PTC than in normal thyroid cells. AXL expression was also higher in human PTC tissues than in normal thyroid tissues. Inhibiting the PROS1-AXL-mediated TAM signaling pathway via the AXL blocker R428 suppressed the proliferation and migration of human PTC cells, highlighting the role of this cascade in human PTC development and progression.

Preparation and drug release behavior of amphiphilic polyamino acids nanomicelles

To construct pH sensitive nanomicelles as novel drug delivery carriers, the synthesis of four triblock copolymer mPEG113-Lysinen-Leucine10 based on methoxypolyethylene glycol, lysine and leucine is described, and the structure of copolymer were characterized by FT-IR and 1H NMR. The polymer can be self-assembled into spherical or cubic shape in aqueous solution, and TEM revealed that the drug-loaded nanomicelles displayed uniformly dispersed spherical shape. The average particle size of nanomicelles is about 200 nm, which meets the requirements of material size in the field of sustained release. CCK-8 experiment proved that the synthesized material had good cytocompatibility, and the drug-loaded nanomicelles could be successfully uptaken by Hela cells, and model drug could be successfully released in cytoplasm and nucleus. Uv–vis results showed that the in vitro drug release of the nanomicelles was pH responsive and showed good dimensional stability at different temperatures.

The Study of Exosomes-Encapsulated mPEG-PLGA Polymer Drug-Loaded Particles for Targeted Therapy of Liver Cancer.

The emergence of targeted drugs brings hope to patients with advanced liver cancer. However, due to the complex and diverse environment in the human body, the overall response rate of targeted drugs is not high. Therefore, how to efficiently deliver targeted drugs to tumor sites is a major challenge for current research. The project intends to construct mPEG-PLGA nanoparticles loaded with Sora and encapsulate them with exosomes for targeted therapy of hepatocellular carcinoma. mPEG-PLGA drug-loaded nanoparticles were prepared by the dialysis method and characterized by TEM and DLS. The obtained nanoparticles were incubated with the exosomes of liver cancer cells, and the exosomes-encapsulated drug-loaded nanoparticles (Exo-Sora-NPs) were obtained under pulsed ultrasound conditions, and they were characterized by Western blot, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The toxic effect of Exo-Sora-NPs on liver cancer cells was detected by the CCK-8 experiment. The uptake efficiency of nanoparticles by liver cancer cells was detected by a confocal microscope. The accumulation and infiltration depth of nanomedicine in liver cancer tissues were observed by confocal microscope on frozen sections of liver cancer tissue after the H22 liver cancer subcutaneous tumor transplantation model was constructed. The tumor size, body weight, pathology, and serology analysis of mice were measured after administration. The mPEG-PLGA polymer drug-loaded particles encapsulated by exosomes have high targeting ability and biosafety. To a certain extent, they can target the drug to the tumor site with a smaller systemic response and have a highly effective killing effect on the tumor. Nanodrug-loaded particles encapsulated by exosomes have great potential as drug carriers.

LncRNA TP73-AS1 Exacerbates the Non-Small-Cell Lung Cancer (NSCLC) Process via Regulating miR-125a-3p-Mediated ACTN4.

Background LncRNA TP73-AS1 has been revealed to exert a noteworthy impact on the occurrence and advancement of different cancers. In this study, we explored the function of TP73-AS1 in tumor growth, cell progression as well as the relevant molecular mechanism in non-small-cell lung cancer (NSCLC). Methods QRT-PCR was employed to assess the expression of TP73-AS1, miR‐125a-3p, and actinin alpha 4 (ACTN4) in NSCLC cells. The biological effect of TP73-AS1 on NSCLC cells was assessed by cell transfection, CCK8, and transwell experiments. We further predicted the interaction among RNAs (TP73-AS1, miR-125a-3p, and ACTN4) through bioinformatics online tools and verified via luciferase reporter, RNA immunoprecipitation, and qRT-PCR assays. Xenograft models of SPC-A1 cells were conducted to test how TP73-AS1 regulates tumorigenesis. Western blot, as well as the immunohistochemistry (IHC) assays, was utilized to measure the expression levels. Functions of TP73-AS1 in NSCLC progression through the miR-125a-3p/ACTN4 axis were investigated by rescue experiments. Results Knockdown of TP73-AS1 suppressed the growth and simultaneously attenuated the migration and invasion ability of NSCLC SPC-A1 and A549 cells. Bioinformatics and molecular mechanism assays demonstrated that TP73-AS1 could bind to miR-125a-3p/ACTN4 and regulate their expression. Moreover, the rescued‐function experiment demonstrated that suppressing miR-125a-3p or elevating ACTN4 turned around the suppression effect of sh-TP73-AS1 on NSCLC progression. TP73-AS1 inhibition could also inhibit the NSCLC tumor growth and correspondingly regulated the expression of miR-125a-3p and ACTN4 in the tumor xenograft model. Conclusion The present study indicated that TP73-AS1 affects NSCLC progression through a new competitive RNA (ceRNA) regulatory network of miR-125a-3p/ACTN4, providing an underlying target for NSCLC treatment in the future.

Brusatol inhibits the growth of prostate cancer cells and reduces HIF-1α/VEGF expression and glycolysis under hypoxia

Prostate cancer (PCa) has a high rate of morbidity and mortality, which urges us to find a unique and effective drug for treatment. Brusatol, a triterpenoid-degraded derivative, possesses antitumor activities. However, the significance of Brusatol in prostate cancer has not yet been completely elucidated. Therefore, this study aimed to explore how Brusatol affected prostate cancer cells. DU145 and PC-3 cell lines were chosen as experimental models. After Brusatol was added to relevant cells in culture, CCK-8 and colony formation experiments were used to assess cell viability; apoptosis rates were calculated using flow cytometry; and transwell assays were utilized to assess cell migration and invasion ability. Vimentin, N-cadherin, E-cadherin, zonula occludens-1 (ZO-1), hypoxia- inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase (LDHA) protein expression were evaluated by western blotting, and glucose consumption in cells was assessed using related equipment. In DU145 and PC-3 cells, Brusatol drastically reduced cell proliferation, promoted apoptosis, hindered migration and invasion. Considerably decreased HIF-1α and VEGF protein levels under hypoxia were detected. Furthermore, the expression of GLUT1, HK2, and LDHA was diminished, resulting in decreased glucose consumption in a Brusatol concentration-dependent manner. These findings demonstrate that Brusatol serves as a potent antitumor drug that suppresses DU145 and PC-3 cancer cell growth, metastasis, and glycolysis. This discovery could provide a possible clinical treatment strategy for prostate cancer.

STAT3 regulates SRGN and promotes metastasis of nasopharyngeal carcinoma through the FoxO1-miR-148a-5p-CREB1 axis

AbstractNasopharyngeal carcinoma (NPC), which is marked by a distinct distribution, is a common subtype of epithelial carcinoma arising from the nasopharyngeal mucosal lining. SRGN acts as an important and poor prognostic factor of NPC through multiple different mechanisms. However, the biological role and mechanism of SRGN in NPC remain unknown. Expression levels of miR-148a-5p, CREB1, FoxO1, and SRGN in NPC tissues and cell lines were tested by qRT-PCR or/and Western blot. The impacts of miR-148a-5p, CREB1, FoxO1, and SRGN on NPC cell viability, proliferation, migration, and invasion were estimated in vitro by CCK-8, colony formation, wound healing and Transwell experiments, and in vivo by a xenograft tumor model. JASPAR analysis was used to predict the binding activity of Foxo1 (CREB1) with the miR-148a-5p (SRGN) promoter, and the interaction was validated by EMSA and ChIP assays. The miR-148a-5p-CREB1 interaction was validated by a dual-luciferase reporter and RIP assays. CREB1 and SRGN were increased while miR-148a-5p was decreased in NPC. Silencing of SRGN and CREB1, as well as miR-148a-5p overexpression, repressed NPC tumor progression in vitro and in vivo. CREB1 promoted SRGN expression in NPC by targeting the promoter area of SRGN. Silencing of FoxO1 facilitated NPC tumor progression, while silencing of STAT3 repressed NPC tumor progression. FoxO1 bound to and regulated miR-148a-5p in NPC, and miR-148a-5p targeted CREB1. Additionally, FoxO1 knockdown abolished the downregulation of CREB1 and SRGN induced by STAT3 silencing. Our results suggest that STAT3 regulates SRGN and promotes the growth and metastasis of NPC through the FoxO1-miR-148a-5p-CREB1 axis.

Significance of lncRNA CDKN2B-AS1 in Interventional Therapy of Liver Cancer and the Mechanism under Its Participation in Tumour Cell Growth via miR-199a-5p.

Methods Totally 34 LC patients admitted to our hospital between January 2020 and March 2021 (Obs group) and 32 healthy individuals over the same time span (Con group) were enrolled. CDKN2B-AS1 and miR-199a-5p in the two groups were PCR quantified, and their association and value for the diagnosis and therapy of LC were analyzed. In addition, purchased LC cells were adopted for in vitro assays, and the influences of CDKN2B-AS1 and miR-199a-5p on biological behaviours of LC cells were assessed through CCK-8, Transwell, and flow cytometry experiment, and their regulatory association was verified by the dual luciferase reporter (DLR) assay and rescue assay. And the autophagic protein expression was tested by the western blot to confirm the effect of both on the autophagic capacity of LC cells. Results CDKN2B-AS1 in LC cases presented high expression and dropped after therapy (P < 0.05), and the opposite situation of miR-199a-5p was found in the LC cases (P < 0.05). In vitro assays, after silencing of CDKN2B-AS1 and upregulation of miR-199a-5p, LC cells presented weaker viability, invasion and migration activities, and stronger apoptotic activity (all P < 0.05). The DLR assay revealed suppressed fluorescence activity of CDKN2B-AS1-WT by miR-199a-5p (P < 0.05). Moreover, according to the rescue assay, the impacts of silencing CDKN2B-AS1 on LC cells could be completely offset by silencing miR-199a-5p (P < 0.05). According to the clone formation and WB assay, the growth and autophagy of LC cells were under the regulation of CDKN2B-AS1 targeting miR-199a-5p (P < 0.05). Conclusion With high expression in LC cases, CDKN2B-AS1 is implicated in the development and progression of LC by suppressing cell autophagy through targeting miR-199a-5p.

Legumain affects the PI3K/AKT tumor progression pathway in retinoblastoma

Known as a common malignant tumor among children, retinoblastoma (RB) is highly malignant and has poor prognosis, damages children's vision and degrades quality of life. To identify a potential molecular mechanism of RB, we conducted this study on legumain (LGMN), which is highly expressed in multiple tumors. In this study, we found that LGMN was significantly upregulated in RB cells and was positively expressed in RB tissues. We confirmed that LGMN overexpression (LGMN-OE) can promote RB cell proliferation and inhibit cell apoptosis through CCK8 experiments and flow cytometry. In addition, real-time quantitative polymerase chain reaction (RT‒qPCR) and Western blot results showed that LGMN-OE could regulate the expression of epithelial-mesenchymal transformation-related genes and proteins, related to tumor invasion and metastasis. Moreover, after LGMN knock down, the result was the opposite., RNA sequence analysis revealed 1159 differentially expressed genes between LGMN-OE and the negative control (NCOE), of which 564 were upregulated and 595 were downregulated. The first 10 genes were verified by RT‒qPCR based on P value and fold change. Interestingly, we found that LGMN could regulate the expression of recoverin (RCVRN)through a gene responsible for cancer-related retinopathy. We also screened and verified that LGMN partially activated the PI3K/AKT pathway in RB. Furthermore, we evaluated the effect of legumain inhibitors (e.g., esomeprazole) on RB, and the results suggest that esomeprazole may provide a reference for the clinical adjuvant treatment of RB. In conclusion, legumain can serve as an attractive target for RB therapy and hopefully provide new insights and ideas for the development of targeted drugs and precise personalized clinical therapy.

LncRNA GAS6-AS1 facilitates tumorigenesis and metastasis of colorectal cancer by regulating TRIM14 through miR-370-3p/miR-1296-5p and FUS

BackgroundLong non-coding RNAs (lncRNAs) are essential regulators of tumorigenesis and the development of colorectal cancer (CRC). Here, we aimed to investigate the role of lncRNA GAS6-AS1 in CRC and its potential mechanisms.MethodsBioinformatics analyses evaluated the level of GAS6-AS1 in colon cancer, its correlation with clinicopathological factors, survival curve and diagnostic value. qRT-PCR were performed to detect the GAS6-AS1 level in CRC samples and cell lines. The CCK8, EdU, scratch healing, transwell assays and animal experiments were conducted to investigate the function of GAS6-AS1 in CRC. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene analyses were carried out to reveal interaction between GAS6-AS1, TRIM14, FUS, and miR-370-3p/miR-1296-5p.ResultsGAS6-AS1 was greatly elevated in CRC and positively associated with unfavorable prognosis of CRC patients. Functionally, GAS6-AS1 positively regulates CRC proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and induces CRC growth and metastasis in vivo. Moreover, GAS6-AS1 exerted oncogenic function by competitively binding to miR-370-3p and miR-1296-5p, thereby upregulating TRIM14. Furthermore, we verified that GAS6-AS1 and TRIM14 both interact with FUS and that GAS6-AS1 stabilized TRIM14 mRNA by recruiting FUS. Besides, rescue experiments furtherly demonstrated that GAS6-AS1 facilitate progression of CRC by regulating TRIM14.ConclusionCollectively, these findings demonstrate that GAS6-AS1 promotes TRIM14-mediated cell proliferation, migration, invasion, and EMT of CRC via ceRNA network and FUS-dependent manner, suggesting that GAS6-AS1 could be utilized as a novel biomarker and therapeutic target for CRC.

BMAL1 promotes colorectal cancer cell migration and invasion through ERK- and JNK-dependent c-Myc expression.

Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT-like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial-to-mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK-8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial-to-mesenchymal transition (EMT) pathway and induce the β-catenin release further promotes the expression of oncogene c-Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK-signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c-Myc in vitro. Taken together, these results suggested that the BMAL1/ c-Myc-signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK-dependent pathway. Our study showed that BMAL1 promotes CRC metastasis through MAPK-c-Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

Circ_0000467 modulates malignant characteristics of colorectal cancer via sponging miR-651-5p and up-regulating DNMT3B

Circular RNAs (circRNAs) are widely expressed in cancer tissues and participate in modulating the progression of malignant tumors, playing a pro- or anti-cancer role. This work is conducted to probe the precise role of circ_0000467 in colorectal cancer (CRC) and its regulatory mechanism. The differentially expressed circRNAs in CRC tissues and paracancerous tissues were screened by bioinformatics analysis. The expression levels of circ_0000467, miR-651-5p and DNA methyltransferases 3B (DNMT3B) mRNA in CRC tissues and cells were detected by qRT-PCR. circ_0000467 knockdown cell model was constructed to investigate the effects of circ_0000467 on CRC cell growth, migration and invasion by CCK-8 and Transwell experiments. Western blot was performed to examine DNMT3B protein expression in CRC cells. Dual-luciferase reporter gene experiment was executed to validate the targeting relationship between circ_0000467 and miR-651-5p, miR-651-5p and DNMT3B. Circ_0000467 expression and DNMT3B mRNA expression were increased and miR-651-5p expression was down-regulated in CRC tissues and cell lines. Knockdown of circ_0000467 repressed CRC cell growth, migration and invasion. Dual-luciferase reporter gene experiments validated that miR-651-5p was a direct target of circ_0000467 and miR-651-5p could specifically bind with DNMT3B 3’UTR. Functional compensation experiments showed that the regulatory effect of circ_0000467 on CRC cells’ behaviors could be partially counteracted by miR-651-5p. Circ_0000467 may enhance the growth and metastasis of CRC cells by targeting miR-651-5p and up-regulating DNMT3B expression. Circ_0000467 may be a potential diagnostic biomarker and therapeutic target for CRC.

Upregulation of miR-151-5p promotes the apoptosis of intestinal epithelial cells by targeting brain-derived neurotrophic factor in ulcerative colitis mice

ABSTRACT Ulcerative colitis (UC) is the most prevalent form of chronic inflammatory bowel disease, the etiology of which is poorly understood. This study investigated the role of miR-151-5p on UC and explored the role of brain-derived neurotrophic factor (BDNF) in a UC mouse model and cell model. A UC mouse model was engineered by dextran sulfate sodium (DSS) induction. Primary mouse intestinal epithelial cells (IECs) were isolated. Colitis mice were intraperitoneally injected with miR-151-5p antagomir and antagomir negative control, and weight loss, disease activity index, and colon length of mice were measured. Colon tissues of mice were histologically analyzed. A UC cell model was constructed by treating MODE-K cells with DSS. miR-151-5p expression in the cell model was modulated by transfection. The exogenous BDNF effect on the UC cell model and intestinal cell apoptosis, viability and proliferation was detected by flow cytometry, CCK-8 and EdU experiment. The expression of miR-151-5p and apoptosis-related proteins was assessed through q-PCR and western blotting. miR-151-5p was upregulated in the colon tissues and primary IECs of colitis mice. miR-151-5p directly inhibited the expression of BNDF. miR-151-5p upregulation promoted apoptosis in UC MODE-K cells. miR-151-5p upregulation repressed the viability of UC MODE-K cells. Exogenous BNDF treatment reversed the effect of miR-151-5p on UC MODE-K cells. miR-151-5p knockdown improved UC symptoms in mice, including alleviating weight loss, reducing disease activity index and improving colon length and damaged colon tissues. miR-151-5p contributed to intestinal epithelial cells apoptosis in colitis mice via inhibiting BNDF expression.

The Role of CD28 and CD8+ T Cells in Keloid Development.

Background: A keloid is a benign skin tumor that extends beyond the initial injury area, and its pathologic mechanism remains unclear. Method: High-throughput sequencing data were obtained from normal skin tissue of patients with keloids (Group N) and healthy controls (Group C). Important genes were mined by bioinformatics analysis and identified by RT–qPCR, Western blotting, immunohistochemistry and immunofluorescence assays. The CIBERSORT algorithm was used to convert gene expression information into immune cell information. Flow cytometry was used to verify the key immune cells. Fluorescence-activated cell sorting coculture and CCK8 experiments were used to explore the effect of CD8+ T cells on keloid-associated fibroblasts. Neural network models were used to construct associations among CD28, CD8+ T cells and the severity of keloids and to identify high-risk values. Result: The expression levels of costimulatory molecules (CD28, CD80, CD86 and CD40L) in the skin tissue of patients with keloids were higher than the levels in healthy people (p < 0.05). The number of CD8+ T cells was significantly higher in Group N than in Group C (p < 0.05). The fluorescence intensities of CD28 and CD8+ T cells in Group N were significantly higher than those in Group C (p = 0.0051). The number and viability of fibroblasts cocultured with CD8+ T cells were significantly reduced compared with those of the control (p < 0.05). The expression of CD28 and CD8+ T cells as the input layer may be predictors of the severity of keloids with mVSS as the output layer. The high-risk early warning indicator for CD28 is 10–34, and the high-risk predictive indicator for CD8+ T cells is 13–28. Conclusions: The abnormal expression of costimulatory molecules may lead to the abnormal activation of CD8+ T cells. CD8+ T cells may drive keloid-associated immunosuppression. The expression of CD28 and CD8+ T cells as an input layer may be a predictor of keloid severity. CD28 and CD8+ T cells play an important role in the development of keloids.

7-Difluoromethyl-5,4’-dimethoxygenistein inhibited the angiogenesis induced by cervical cancer SiHa cells via inhibiting TLR4/VEGF-A axis (217)

<b>Objectives:</b> VEGF inhibitors, such as bevacizumab, inhibit angiogenesis and improve the prognosis of patients with recurrent or metastatic cervical cancer. Antiangiogenic drugs reduce the formation of immature and poorly functional blood vessels and induce the normalization of blood vessels. However, in many cases, features of the vascular normalization brought by VEGF inhibitors are quickly replaced by marked vascular regression, which in turn will exacerbate the degree of hypoxia in the tumor microenvironment. In addition, a variety of adverse effects of antiangiogenic drugs have also been found at present. Therefore, it is a very attractive work to extract natural molecules that are non-toxic or low in toxicity from plants as antiangiogenic drugs. 7-Difluoromethyl-5,4'-dimethoxygenistein (DFMG) is a new chemical entity synthesized from the genistein extracted from the natural plant fruit soybean by ourselves. Previously, DFMG has been found to maintain plaque stability in atherosclerosis through antiangiogenesis. This study explored the effect of DFMG on angiogenesis in cervical cancer and its possible mechanism. <b>Methods:</b> Cell proliferation was detected by the CCK-8 experiment. The expressions of TLR4 and VEGF-A were detected by Q-PCR and Western blot. Angiogenesis was detected by tubule formation test and chorioallantoic membrane (CAM) test. TLR4 was overexpressed and silenced on SiHa cells to detect its effect on VEGF-A secretion and angiogenesis. The effects of DFMG on VEGF-A secretion and angiogenesis were detected on TLR4-overexpressed or -silenced SiHa cells. <b>Results:</b> 1) 50 μM DFMG can inhibit the activity of Siha cells, but not HUVE-12 cells. 2) DFMG inhibits the formation of tubules induced by Siha cells, while bevacizumab can inhibit the ability of HUVE-12 cells to form tubules, but DFMG and genistein have no effect on it. 3) DFMG inhibits the expression and secretion of VEGF-A protein in Siha cells. 4) Overexpression of TLR4 in Siha cells can antagonize the angiogenesis-inhibiting effect caused by bevacizumab, and the silent expression of TLR4 can antagonize the angiogenesis-promoting effect caused by VEGF-A. 5) The numbers of tubules in the tubule formation experiment and new blood vessels in CAM in the DFMG-treated Siha cells with TLR4 overexpression was significantly higher than that in the DFMG-treated Siha cells with TLR4 silence. 6) The overexpression of TLR4 could antagonize the effect of DFMG on the secretion and expression of VEGF-A in SiHa cells, while the silent expression of TLR4 could cooperate with DFMG to inhibit the secretion and expression of VEGF-A.Fig. 1 <b>Conclusions:</b> DFMG inhibits the angiogenesis induced by cervical cancer Siha cells which could be regulated by TLR4/VEGF-A signaling pathway. DFMG reduces angiogenesis by inhibiting VEGF-A secretion of SiHa cells, which is not observed in normal cells.

Evaluation of Biocompatibility of 316 L Stainless Steels Coated with TiN, TiCN, and Ti-DLC Films

In this study, TiN, TiCN, and Ti-diamond-like carbon (Ti-DLC) films were coated on 316 L stainless steel (AISI 316 L) substrate surface by physical vapor deposition. The biocompatibility of the three films (TiN, TiCN, and Ti-DLC) and three metals (AISI 316 L, Ti, and Cu) was compared on the basis of the differences in the surface morphology, water contact angle measurements, CCK-8 experiment results, and flow cytometry test findings. The biocompatibility of the TiN and TiCN films is similar to that of AISI 316 L, which has good biocompatibility. However, the biocompatibility of the Ti-DLC films is relatively poor, which is mainly due to the inferior hydrophobicity and large amount of sp2 phases. The presence of TiC nanoclusters on the surface of the Ti-DLC film aggravates the inferior biocompatibility. Compared to the positive Cu control group, the Ti-DLC film had a higher cell proliferation rate and lower cell apoptosis rate. Although the Ti-DLC film inhibited cell survival to a certain extent, it did not show obvious cytotoxicity. TiN and TiCN displayed excellent performance in promoting cell proliferation and reducing cytotoxicity; thus, TiN and TiCN can be considered good orthodontic materials, whereas Ti-DLC films require further improvement.