Malignant Biological Properties Research Articles

ING3 inhibits the malignant progression of lung adenocarcinoma by negatively regulating ITGB4 expression to inactivate Src/FAK signaling

Lung adenocarcinoma (LUAD) is the most commonly diagnosed subtype of lung cancer worldwide. Inhibitor of growth 3 (ING3) serves as a tumor suppressor in many cancers. This study aimed to elucidate the role of ING3 in the progression of LUAD and investigate the underlying mechanism related to integrin β4 (ITGB4) and Src/focal adhesion kinase (FAK) signaling. ING3 expression in LUAD tissues and the correlation between ING3 expression and prognosis were analyzed by bioinformatics databases. After evaluating ING3 expression in LUAD cells, ING3 was overexpressed to assess the proliferation, cell cycle arrest, migration and invasion of LUAD cells. Then, ITGB4 was upregulated to observe the changes of malignant activities in ING3-overexpressed LUAD cells. The transplantation tumor model of NCI-H1975 cells in nude mice was established to analyze the antineoplastic effect of ING3 upregulation in vivo. Downregulated ING3 expression was observed in LUAD tissues and cells and lower ING3 expression predicated the poor prognosis. ING3 upregulation restrained the proliferation, migration, invasion and induced the cell cycle arrest of NCI-H1975 cells. Additionally, ITGB4 expression was negatively correlated with ING3 expression in LUAD tissue. ING3 led to reduced expression of ITGB4, Src and p-FAK. Moreover, ITGB4 overexpression alleviated the effects of ING3 upregulation on the malignant biological properties of LUAD cells. It could be also found that ING3 upregulation limited the tumor volume, decreased the expression of ITGB4, Src and p-FAK, which was restored by ITGB4 overexpression. Collectively, ING3 inhibited the malignant progression of LUAD by negatively regulating ITGB4 expression to inactivate Src/FAK signaling.

Transcription factor LHX9 (LIM Homeobox 9) enhances pyruvate kinase PKM2 activity to induce glycolytic metabolic reprogramming in cancer stem cells, promoting gastric cancer progression

BackgroundGlycolytic metabolic reprogramming is a phenomenon in which cells undergo altered metabolic patterns during malignant transformation, mainly involving various aspects of glycolysis, electron transport chain, oxidative phosphorylation, and pentose phosphate pathway. This reprogramming phenomenon can be used as one of the markers of tumorigenesis and development. Pyruvate kinase is the third rate-limiting enzyme in the sugar metabolism process by specifically catalyzing the irreversible conversion of PEP to pyruvate.PurposeThis study aimed to reveal the critical mediator(s) that regulate glycolytic metabolism reprogramming in gastric cancer and their underlying molecular mechanism and then explore the molecular mechanisms by which LHX9 may be involved in regulating gastric cancer (GC) progression.MethodsFirstly, we downloaded the GC and glycolysis-related microarray datasets from TCGA and MSigDB databases and took the intersection to screen out the transcription factor LHX9 that regulates GC glycolytic metabolic reprogramming. Software packages were used for differential analysis, single gene predictive analysis, and Venn diagram. In addition, an enrichment analysis of the glycolytic pathway was performed. Immunohistochemical staining was performed for LHX9 and PKM2 protein expression in 90 GC patients, and the association between their expressions was evaluated by Spearman's correlation coefficient method. Three human GC cell lines (AGS, NCI-N87, HGC-27) were selected for in vitro experimental validation. Flow cytometry was utilized to determine the stem cell marker CD44 expression status in GCSCs. A sphere formation assay was performed to evaluate the sphere-forming capabilities of GCSCs. In addition, RT-qPCR and Western blot experiments were employed to investigate the tumor stem cell markers OCT4 and SOX2 expression levels in GCSCs. Furthermore, a lentiviral expression vector was constructed to assess the impact of downregulating LHX9 or PKM2 on the glycolytic metabolic reprogramming of GCSCs. The proliferation, migration, and invasion of GCSCs were then detected by CCK-8, EdU, and Transwell assays. Subsequently, the mutual binding of LHX9 and PKM2 was verified using chromatin immunoprecipitation and dual luciferase reporter genes. In vivo experiments were verified by establishing a subcutaneous transplantation tumor model in nude mice, observing the size and volume of tumors in vivo in nude mice, and obtaining fresh tissues for subsequent experiments.ResultsBioinformatics analysis revealed that LHX9 might be involved in the occurrence and development of GC through regulating glycolytic metabolism. High LHX9 expression could be used as a reference marker for prognosis prediction of GC patients. Clinical tissue assays revealed that LHX9 and PKM2 were highly expressed in GC tissues. Meanwhile, GC tissues also highly expressed glycolysis-associated protein GLUT1 and tumor cell stemness marker CD44. In vitro cellular assays showed that LHX9 could enhance its activity and induce glycolytic metabolic reprogramming in GCSCs through direct binding to PKM2. In addition, the knockdown of LHX9 inhibited PKM2 activity and glycolytic metabolic reprogramming and suppressed the proliferation, migration, and invasive ability of GCSCs. In vivo animal experiments further confirmed that the knockdown of LHX9 could reduce the tumorigenic ability of GCSCs in nude mice by inhibiting PKM2 activity and glycolytic metabolic reprogramming.ConclusionThe findings suggest that both LHX9 and PKM2 are highly expressed in GCs, and LHX9 may induce the reprogramming of glycolytic metabolism through transcriptional activation of PKM2, enhancing the malignant biological properties of GCSCs and ultimately promoting GC progression.

Clinical implications and immune implications features of TARS1 in breast cancer.

There has been an increase in the number of women suffering from breast cancer in recent years, and discovering new therapeutic targets and efficacy predictive markers is critical for comprehensive breast cancer treatment. First, we used bioinformatics methods to analyze TARS1(encoding cytoplasmicthreonyl-tRNA synthetase) expression, prognosis, and clinicopathological characteristics in TCGA database breast cancers, and then we collected breast cancer specimens from our center for validation. TARS1 was then subjected to GSEA (Gene Set Enrichment Analysis) enrichment analysis, GO/KEGG pathway enrichment analysis, and breast cancer immune infiltration characterization. As a last step, we examined TARS1's effects on breast cancer cell behavior with cellular assays. The overexpression of TARS1 has been found in several malignant tumors, including breast cancer, and has been linked to poor prognoses. Breast cancers with large primary tumors and negative hormone receptors are more likely to overexpress TARS1. Overexpression of TARS1 promotes the infiltration of T cells, such as Tregs and Th2s, while inhibiting the infiltration of NK cells and CD8+ T cells, which are anticancer cells in breast cancer. TARS1 was also found to be co-expressed with the majority of immune checkpoint-related genes, and breast cancer with TARS1 overexpression responded better to immunotherapy. By knocking down TARS1, breast cancer cells were prevented from proliferating and invading, as well as exhibiting other malignant biological properties. According to our study, TARS1 may be an oncogene in breast cancer and may be a biomarker of efficacy or a target of immunotherapy in breast cancer.

Inhibition of hyaluronic acid degradation pathway suppresses glioma progression by inducing apoptosis and cell cycle arrest

BackgroundAbnormal hyaluronic acid (HA) metabolism is a major factor in tumor progression, and the metabolic regulation of HA mainly includes HA biosynthesis and catabolism. In glioma, abnormal HA biosynthesis is intimately involved in glioma malignant biological properties and the formation of immunosuppressive microenvironment; however, the role of abnormal HA catabolism in glioma remains unclear.MethodsHA catabolism is dependent on hyaluronidase. In TCGA and GEPIA databases, we found that among the 6 human hyaluronidases (HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, SPAM1), only HYAL2 expression was highest in glioma. Next, TCGA and CGGA database were further used to explore the correlation of HYAL2 expression with glioma prognosis. Then, the mRNA expression and protein level of HYAL2 was determined by qRT-PCR, Western blot and Immunohistochemical staining in glioma cells and glioma tissues, respectively. The MTT, EdU and Colony formation assay were used to measure the effect of HYAL2 knockdown on glioma. The GSEA enrichment analysis was performed to explore the potential pathway regulated by HYAL2 in glioma, in addition, the HYAL2-regulated signaling pathways were detected by flow cytometry and Western blot. Finally, small molecule compounds targeting HYAL2 in glioma were screened by Cmap analysis.ResultsIn the present study, we confirmed that Hyaluronidase 2 (HYAL2) is abnormally overexpressed in glioma. Moreover, we found that HYAL2 overexpression is associated with multiple glioma clinical traits and acts as a key indicator for glioma prognosis. Targeting HYAL2 could inhibit glioma progression by inducing glioma cell apoptosis and cell cycle arrest.ConclusionCollectively, these observations suggest that HYAL2 overexpression could promote glioma progression. Thus, treatments that disrupt HA catabolism by altering HYAL2 expression may serve as effective strategies for glioma treatment.

α-Fetoprotein contributes to the malignant biological properties of AFP-producing gastric cancer.

This study aimed to investigate whether α-fetoprotein (AFP) could affect the malignant behavior of AFP-producing gastric cancer (AFP-GC) and to explore the relationship between AFP and mesenchymal-epithelial transition factor (c-Met) in AFP-GC. In this study, 23 patients with AFP-GC (AFP[+]) and 18 patients with common gastric cancer (AFP[-]) were evaluated for the c-Met expression using immunohistochemical analysis. The AFP-GC cell line, GCIY, was used. The AFP endoribonuclease-prepared small interfering RNA (siRNA) and eukaryotic AFP overexpression vector were used to increase/knockdown the expression of AFP. Afterward, the c-Met expression was evaluated by polymerase chain reaction and western blot. The proliferation, migration, and invasion of GCIY cells were estimated before and after the AFP overexpression/knockdown. The c-Met expression in both groups was the same (p > 0.05), and AFP[+] group had a higher positive incidence of the c-Met expression than the AFP[-] group (p < 0.01). Furthermore, the c-Met expression frequency was decreased by AFP knockdown and increased by AFP overexpression (p < 0.01). The cell counting kit-8 cell proliferation assay, cell invasion, and migration assays confirmed that the AFP could affect the malignant biological behavior of AFP-GC. These findings suggest that AFP contributes to the malignant biological properties of AFP-GC and the high expression of c-Met in AFP-GC.

Metastasis-related long non-coding RNAs AL359220.1, SH3BP5-AS1 and ZF-AS1 are significant for prognostic assessment of lung adenocarcinoma.

Metastasis of lung adenocarcinoma (LUAD) severely worsens prognosis. Genetic alteration in the tumor microenvironment (TME) is closely associated with metastasis and other malignant biological properties of LUAD. In this study, we establish a metastasis-related risk model to accurately predict LUAD prognosis. RNA-sequencing profiles and clinical data of LUAD patients including 503 tumor tissues and 54 adjacent normal tissues were collected in TCGA database. Additionally, the paired specimens from 156 LUAD patients were obtained in a single center. The metastatic relevance and clinical significance of metastasis-related long non-coding RNA (MRLNRs) was validated by series of in vitro experiments including western blotting, qPCR and transwell assays. Six MRLNRs were significantly correlated to prognoses of LUAD patients, of which AL359220.1, SH3BP5-AS1 and ZF-AS1 were further used to establish a metastasis-related risk scoring model (MRRS) due to the close associations with overall survival of LUAD patients. According to the MRRS, patients with higher scores in the high-risk group obtained poorer prognoses and survival outcomes. ZFAS1 expressed highly in tumor tissues and showed the inverse results compared to SH3BP5-AS1 and AL359220.1. In addition, the high expression of ZFAS1 was prominently correlated to the more advanced T-stage and distant metastasis. The reduction of ZFAS1 induced by siRNAs dramatically diminished the migration and invasion abilities of LUAD cells. In the present research, we elucidate the metastatic relevance and clinical significance of AL359220.1, SH3BP5-AS1 and ZF-AS1 in LUAD. Moreover, MRRS provide a promising assessing model for clinical decision making and prognosis of LUAD.

S100 Calcium-Binding Protein A8 Functions as a Tumor-Promoting Factor in Renal Cell Carcinoma via Activating NF-κB Signaling Pathway

Background Renal cell carcinoma (RCC), arising from the renal tubular epithelium, is one of the most common types of genitourinary malignancies. Based on the Gene Expression Omnibus (GEO) database (GSE100666), S100 calcium-binding protein A8 (S100A8) was highly expressed in RCC tissues. S100A8, an inflammatory regulatory factor, has emerged as an important mediator associated with the occurrence and development of cancer. Materials and Methods The Gene Expression Omnibus (GEO) database was used to identify the key genes and investigate the main signaling pathways in RCC. Human RCC samples and corresponding adjacent normal tissues were collected in our hospital. The expression of S100A8 in human RCC samples was detected using western blotting and immunohistochemical analysis. S100A8 overexpression or knockdown was mediated by using Lipofectamine 3000 in human renal cell carcinoma cell line 786-O and ACHN cells. Basic experiments, including MTT and cell apoptosis assays, were utilized for investigating the function of S100A8 in RCC. Furthermore, the levels of inflammation were also evaluated in 786-O and ACHN cells. Results In the current study, we found that downregulation of S100A8 inhibited proliferation and promoted apoptosis in 786-O and ACHN RCC cells. Of note, S100A8 silencing downregulated the phosphorylation of NF-κB p65, thereby decreasing the levels of TNF-α, cleaved caspase1, and MMP9. By contrast, S100A8 upregulation could increase these expressions. Conclusion Overall, S100A8 knockdown restrained RCC malignant biological properties, which was associated with the deactivation of the NF-κB signaling pathway. This present study demonstrates new insights that S100A8 may be a potential therapeutic target in RCC.

Terpinen-4-ol Induces Ferroptosis of Glioma Cells via Downregulating JUN Proto-Oncogene.

According to previous research, turmeric seeds exhibit anti-inflammatory, anti-malignancy, and anti-aging properties due to an abundance of terpinen-4-ol (T4O). Although it is still unclear how T4O works on glioma cells, limited data exist regarding its specific effects. In order to determine whether or not glioma cell lines U251, U87, and LN229 are viable, CCK8 was used as an assay and a colony formation assay was performed using different concentrations of T4O (0, 1, 2, and 4 μM). The effect of T4O on the proliferation of glioma cell line U251 was detected through the subcutaneous implantation of the tumor model. Through high-throughput sequencing, a bioinformatic analysis, and real-time quantitative polymerase chain reactions, we identified the key signaling pathways and targets of T4O. Finally, for the measurement of the cellular ferroptosis levels, we examined the relationship between T4O, ferroptosis, and JUN and the malignant biological properties of glioma cells. T4O significantly inhibited glioma cell growth and colony formation and induced ferroptosis in the glioma cells. T4O inhibited the subcutaneous tumor proliferation of the glioma cells in vivo. T4O suppressed JUN transcription and significantly reduced its expression in the glioma cells. The T4O treatment inhibited GPX4 transcription through JUN. The overexpression of JUN suppressed ferroptosis in the cells rescued through T4O treatment. Taken together, our data suggest that the natural product T4O exerts its anti-cancer effects by inducing JUN/GPX4-dependent ferroptosis and inhibiting cell proliferation, and T4O will hope-fully serve as a prospective compound for glioma treatment.

Karyopherin Subunit Alpha 1 Enhances the Malignant Behaviors of Colon Cancer Cells via Promoting Nuclear Factor-κB p65 Nuclear Translocation.

Aberrant nuclear factor-κB p65 (NF-κB p65) nuclear import commonly occurs in multiple cancers, including colon cancer. According to BioGRID, we noted that Karyopherin subunit alpha 1 (KPNA1), an important molecular transporter between the nucleus and the cytoplasm, may interact with NF-κB p65. KPNA1 itself is highly expressed in colon adenocarcinoma samples (N = 286) based on The Cancer Genome Atlas (TCGA) database. We aimed to explore the role of KPNA1 in colonic carcinogenesis and to determine whether NF-κB p65 nuclear translocation was involved. KPNA1 expressions at mRNA and protein levels were analyzed in colon cancer tissues. The regulatory effect of KPNA1 on malignant biological properties was detected in SW480 and HCT116 colon cancer cells. Coimmunoprecipitation and immunofluorescence were performed to verify the relationship between KPNA1 and NF-κB p65. KPNA1 ubiquitination was also preliminarily investigated. KPNA1 was firstly confirmed as a significantly upregulated gene in our collected clinical colon cancer samples (N = 35). KPNA1 depletion inhibited cell proliferation, induced cell cycle arrest, and diminished migratory and invasive capacity of SW480 and HCT116 cells. Colon cancer cells overexpressing KPNA1 acquired more aggressive behaviors. KPNA1 acted as a transporter to induce the nuclear accumulation of NF-κB p65, thereby activating NF-κB signaling pathway in colon cancer cells. Furthermore, HECT, C2, and WW Domain-Containing E3 Ubiquitin (HECW2) interacted with KPNA1 to induce its ubiquitination. KPNA1 labeled with polyubiquitins was degraded through ubiquitin-proteasome system. The present study uncovers a role of KPNA1-NF-κB p65 axis in promoting colonic carcinogenesis.

MicroRNA-627-5p inhibits colorectal cancer cell proliferation, migration and invasion by targeting Wnt2.

microRNA-627-5p (miR-627-5p) dysregulation has been observed in several cancer types, such as hepatocellular carcinoma, oral squamous cell carcinoma, glioblastoma multiforme, and gastric cancer. The biological function of miR-627-5p in colorectal cancer (CRC) growth and metastasis is yet unclear. To investigate the effects of miR-627-5p on the malignant biological properties of colorectal malignant tumour cells by targeting Wnt2. The levels of miR-627-5p in colorectal tumour tissues were assessed in Gene Expression Omnibus datasets. In order to identify Wnt2 transcript expression in CRC tissues, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used. Luciferase reporter tests were used to explore whether miR-627-5p might potentially target Wnt2. Wnt2 transcript and protein levels were detected in CRC cells with high miR-627-5p expression. To learn more about how miR-627-5p affects CRC development, migration, apoptosis, and invasion, functional experiments were conducted. Cotransfection with the overexpression vector of Wnt2 and miR-627-5p mimics was utilized to verify whether overexpression of Wnt2 could cancel the impact of miR-627-5p in CRC. Western blot and qRT-PCR were conducted to investigate the effects of miR-627-5p on the Wnt/β-catenin signalling pathway. miR-627-5p was notably decreased in colorectal tumour tissues, while the gene level of Wnt2 was notably upregulated. A dual luciferase reporter assay revealed that miR-627-5p specifically targets the 3'-untranslated regions of Wnt2 and miR-627-5p upregulation markedly reduced the protein and gene expression of Wnt2 in CRC cells. In vitro gain-of-function assays displayed that miR-627-5p overexpression decreased CRC cells' capabilities to invade, move, and remain viable while increasing apoptosis. Wnt2 overexpression could reverse the suppressive functions of miR-627-5p. Moreover, upregulation of miR-627-5p suppressed the transcript and protein levels of the downstream target factors in the canonical Wnt/β-catenin signalling, such as c-myc, CD44, β-catenin, and cyclinD1. miR-627-5p acts as a critical inhibitory factor in CRC, possibly by directly targeting Wnt2 and negatively modulating the Wnt/β-catenin signalling, revealing that miR-627-5p could be a possible treatment target for CRC.

LSD1 silencing inhibits the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.

Hypopharyngeal cancer is a subtype of the head and neck malignancies. We aimed to explore the role of lysine-specific demethylase 1 (LSD1/KDM1A) in the progression of hypopharyngeal cancer and to identify the potential mechanisms. First, LSD1 expression in head and neck squamous cell carcinoma (HNSCC) tissues and the correlation between LSD1 and the stage of HNSC were analyzed by the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Following LSD1 silencing, proliferation of pharyngeal cancer cell line FaDu cells was evaluated by cell counting kit-8 and colony formation assays. Wounding healing and transwell assays were used to measure the capacities of migration and invasion. In addition, expression of proteins related to epithelial-to-mesenchymal transition (EMT), autophagy, and pyroptosis was tested by Western blot analysis or immunofluorescence. After treatment with autophagy inhibitor 3-methyladenine (3-MA) or NLR family pyrin domain containing 3 (NLRP3) inhibitor MCC950, the malignant biological properties were measured again. High LSD1 expression was observed in HNSC tissues, which was correlated with stage. LSD1 knockdown significantly suppressed the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. Moreover, autophagy and pyroptosis were induced by LSD1 depletion, observed by the enhanced fluorescence intensity of LC3, gasdermin-D (GSDMD)-N, and apoptosis-associated speck-like protein containing a CARD (ASC), accompanied by upregulated expression of LC3II/LC3I, Beclin-1, NLRP3, cleaved-caspase 1, ASC, interleukin (IL)-1β, and IL-18 and downregulated expression of p62. Importantly, 3-MA or MCC950 addition obviously reversed the inhibitory effects of LSD1 silencing on the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. To sum up, LSD1 silencing could restrain the progression of hypopharyngeal cancer cells by inducing autophagy and pyroptosis.

Starvation-induced long non-coding RNAs are significant for prognosis evaluation of bladder cancer.

Starving intratumoral microenvironment prominently alters genic profiles including long non-coding RNAs (lncRNAs), which further regulate bladder cancer (BCa) malignant biological properties, such as invasion and migration. Transcriptome RNA-sequencing data of 414 BCa tumor tissues and 19 normal tissues were obtained from TCGA database and paired samples of 132 BCa patients. A chain of in vitro validations such as qPCR, migration and invasion assays were performed to reveal the clinical relevance of AC011472.4 and AL157895.1. A total of 11 lncRNAs were identified as starvation-related lncRNAs, of which AC011472.4 and AL157895.1 were relevant to overall survival of BCa patients. Besides, a starvation-related risk score model was established based on the levels of AC011472.4 and AL157895.1. BCa patients with higher levels of AL157895.1 were divided into the high-risk group and usually obtained higher mortality rate, but AC011472.4 was contrary. AL157895.1 expressed highly in BCa cell lines and tumour tissues, especially in patients with the advanced grade, stage and T-stage, while AC011472.4 showed the reversed result. Moreover, increased level of AL157895.1 was remarkably correlated to T-stage, muscle invasion status and distant metastasis. SiRNAs-mediated silence of AC011472.4 and AL157895.1 respectively increased and diminished invasion and migration properties of BCa cells. In this study, we highlight the significant roles of AC011472.4 and AL157895.1 on evaluating prognoses of BCa patients and validate their correlation with various clinical parameters. These findings provide an appropriate risk score model for BCa clinical decision making.

CircRNF144B/miR-342-3p/FBXL11 axis reduced autophagy and promoted the progression of ovarian cancer by increasing the ubiquitination of Beclin-1

Circular RNAs (circRNAs) can regulate autophagy and ovarian cancer (OC) progression. However, autophagy-associated circRNAs involved in OC progression are largely unknown. Bioinformatics, RNA sequencing, and qRT-PCR were conducted to detect circRNF144B expression in OC as well as its relationship with patient prognosis. Functional experiments were used to determine the effects of circRNF144B on the proliferation, mobility and autophagy of OC. Double luciferase reporter assays, immunoprecipitation, and ubiquitination detection were performed to determine the molecular mechanisms of circRNF144B in autophagy and OC progression. CircRNF144B was elevated in OC tissues with low autophagy levels, and associated with poor prognosis. CircRNF144B promoted the malignant biological properties of OC cells, and inhibited the autophagy. Mechanistically, circRNF144B acts as a sponge for miR-342-3p and inhibits miR-342-3p-induced degradation of lysine demethylase 2 A (FBXL11) mRNA, leading to elevated FBXL11 protein levels. Elevated FBXL11 promoted the ubiquitination and degradation of Beclin-1, thus inhibiting autophagy. In conclusion, CircRNF144B increased FBXL11 level by sponging miR-342-3p, whereas elevated FBXL11 promoted the ubiquitination and protein degradation of Beclin-1, thus suppressing autophagy flux and promoting OC progression. Thus, circRNF144B may be an effective target for OC therapy.

Rutaecarpine prevents the malignant biological properties of breast cancer cells by the miR-149-3p/S100A4 axis.

BackgroundBreast cancer (BC) is a frequent malignancy that endangers women’s health, and its fatality rate ranks 1st among female malignancies. Research has shown that rutaecarpine (RUT), which is a Chinese herbal medicine, blocks the proliferation of cancer cells by a variety of molecular mechanisms. However, the possible effects and mechanism of RUT in the autophagy and angiogenesis of BC cells has not been clearly articulated.MethodsMiR-149-3p and S100A4 expression levels were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the optimal concentration and time of RUT was confirmed by Cell Counting Kit-8 (CCK-8) assays of the BC cells. After treatment, changes in cell proliferation and the cell cycle were evaluated by CCK-8 assays, clone formation assays, and flow cytometry, and the levels of apoptosis, autophagy, and angiogenesis-related proteins were identified by Western blot. The targeted regulation of miR-149-3p on S100A4 was also examined by luciferase reporter assays.ResultsWe found that RUT inhibited cell growth and upregulated miR-149-3p in MDA-MB-231 cells. In relation to the biological function activity, RUT attenuated proliferation and angiogenesis, and induced cell-cycle arrest and autophagy by miR-149-3p in the MDA-MB-231 cells. Additionally, miR-149-3p downregulated S100A4 by targeting binding to S100A4, and S100A4 was required for miR-149-3p to play a role in BC progression. We also discovered that an autophagy agonist (rapamycin) or an angiogenesis inhibitor (TNP-470) changed BC progression mediated by the RUT/miR-149-3p/S100A4 axis.ConclusionsRUT blocks the malignant behaviors of BC cells through the miR-149-3p/S100A4 axis and thus alters autophagy and angiogenesis. Thus, the RUT-mediated miR-149-3p/S100A4 axis might be an underlying therapeutic agent and target for BC.

Human bone marrow mesenchymal stem cell-derived exosomes containing microRNA-425 promote migration, invasion and lung metastasis by down-regulating CPEB1.

ObjectiveBone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) could mediate the malignancy of tumor cells by transmitting targeted cargo. Therein, this study intends to explore the function of BMSC-Exos transmitting microRNA-425 (miR-425)/cytoplasmic polyadenylation binding protein 1 (CPEB1) in lung cancer growth.MethodsmiR-425 and CPEB1 levels in cancer tissues and cells were measured. BMSCs and their exosomes were collected and identified. After intervention with BMSC-Exos, miR-425 or CPEB1, invasion and migration of A549 and NCI-H1299 cells in vitro, and lung metastasis of A549 cells in vivo were observed. The relationship between miR-425 and CPEB1 was verified.ResultsmiR-425 was highly expressed while CPEB1 was lowly expressed in lung cancer tissues of patients. CPEB1 was the direct target of miR-425. Down-regulating miR-425 or up-regulating CPEB1 decreased cell invasion and migration ability of A549 and NCI-H1299 cells, as well as decreased the number of lung metastasis lesions in vivo. After co-culture with BMSC-Exos, A549 and NCI-H1299 cells showed promoted migration and invasion in vitro and A549 cells demonstrated increased lung metastasis in vivo. Down-regulated miR-425 or up-regulated CPEB1 reversed the promotion of BMSC-Exos on lung cancer cell invasion, migration and lung metastasis.ConclusionBMSC-Exos could deliver miR-425 to inhibit CPEB1 expression in lung cancer cells, thereby promoting the malignant biological properties of lung cancer cells and their metastasis in vivo.

Hsa_circ_0076931 suppresses malignant biological properties, down-regulates miR-6760-3p through direct binding, and up-regulates CCBE1 in glioma.

The function of circular RNAs (circRNAs) in gliomas is as yet unknown. The present study explored role of hsa_circ_0076931 in glioma. circRNA expression profiles were identified via RNA-seq followed by qRT-PCR validation in three pairs of glioma and normal brain tissues (NBT). The function of hsa_circ_0076931 was investigated in vitro using cell lines as well as in vivo using a xenograft tumor. Hsa_circ_0076931 was up-regulated by overexpression and an mRNA profile compared with wild-type was identified by RNA-seq. The relationship between miR-6760-3p and hsa_circ_0076931 or CCBE1 was confirmed via luciferase reporter or AGO2-RIP assays. A total of 507 circRNAs were identified in glioma tissues that were differentially expressed compared with that in NBT, and the sequencing data were deposited in BioProject (ID: PRJNA746438). Hsa_circ_0007694 and hsa_circ_0008016 were memorably increased whereas hsa_circ_0076931 and hsa_circ_0076948 decreased in glioma compared with those in NBT. Additionally, hsa_circ_0076931 expression was negatively correlated with histological grade. Overexpression of hsa_circ_0076931 inhibited proliferation, migration, and invasion while promoting apoptosis of glioma cells. A total of 4383 and 537 aberrantly expressed genes were identified between the hsa_circ_0076931-overexpressed and control groups in H4 and U118-MG cells, respectively; the sequencing data were deposited in BioProject (ID: PRJNA746438). These differentially expressed genes were mainly enriched in cancer-related pathways. In addition, elevated hsa_circ_0076931 levels induced the expression of CCBE1 while suppressing miR-6760-3p expression. miR-6760-3p can bind to hsa_circ_0076931. The experimental evidence supports using hsa_circ_0076931 as a marker for glioma and to help prevent malignant progression. The mechanism might be relevant to miR-6760-3p and CCBE1.

Sinapine Thiocyanate Inhibits the Proliferation and Mobility of Pancreatic Cancer Cells by Up-Regulating GADD45A.

Background: Sinapine thiocyanate (ST), an alkaloid isolated from the seeds of cruciferous species, has exhibited anti-inflammatory, anti-malignancy, and anti-angiogenic effects in previous studies. However, the effects and molecular mechanisms of action of ST in pancreatic cancer (PC) are still limited.Materials and methods: PC cells were treated with different concentrations (0, 20, 40, and 80 μM) of ST. The proliferative ability of PC cells in vitro was determined using cell count kit-8 (CCK-8), 5-ethynyl-2ʹ deoxyuridine, colony formation, and flow cytometry assays. The mobility of PC cells in vitro was analyzed using wound healing assay, transwell assay, Western blotting, and immunofluorescence. High-throughput sequencing followed by bioinformatics analysis, reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), and Western blotting were performed to identify the key targets of ST. Finally, CCK-8 assay, wound healing assay, and xenograft tumor model were used to determine the relationship between ST and growth arrest and DNA damage-inducible alpha (GADD45A; the key target of ST) and malignant biological properties of PC in vitro and in vivo.Results: ST significantly repressed the PC cell proliferation rate and colony formation in vitro and arrested cells in the G2/M phase. ST inhibited PC cell mobility in vitro and increased E-cadherin expression (an epithelial biomarker). GADD45A was considered the key target of ST in PC and was elevated in PC cells treated with ST. The inhibition of GADD45A significantly alleviated the suppressive effects of ST on PC cell proliferation and mobility in vitro. ST suppressed PC cell proliferation in vivo and increased GADD45A expression in tumor tissues.Conclusion: ST exhibited significant anti-tumor effects on PC cells by upregulating GADD45A. ST may be a potential drug for PC treatment.

Lidocaine suppresses the malignant behavior of gastric cancer cells via the c-Met/c-Src pathway.

The present study was designed to investigate the role and mechanism of action behind the action of lidocaine in gastric cancer cells. Lidocaine was tested for its potential role in affecting the viability of cells using Cell Counting Kit-8 (CCK-8) assays. It was found that there was a decreased MKN45 cell viability upon lidocaine treatment in a dose-dependent manner. Phosphorylated c-Met, phosphorylated c-Src, c-Met and c-Src levels were detected using western blotting following lidocaine or hepatocyte growth factor (HGF) intervention. It was found that the phosphorylation levels of c-Met and c-Src were markedly reduced by lidocaine treatment, with this effect being further relieved by the addition of HGF. Subsequently, whether lidocaine repressed the malignant biological properties of gastric cancer cells through the c-Met/c-Src axis was further investigated through the detection of epithelial-mesenchymal transition markers (N-caderin and vimentin), wound healing and transwell assay analysis. In addition, cell apoptosis and the levels of apoptosis-related proteins were determined using TUNEL and western blot assays, respectively. The results demonstrated that the malignant behavior of cells were notably repressed upon lidocaine treatment, but the addition of HGF markedly reversed these effects, indicating that the effects of lidocaine on supressing the malignant behaviour of cells could be mediated through the c-Met/c-Src axis. Subsequently, whether lidocaine affected the sensitivity of cells to cisplatin or 5-FU was analyzed using a CCK-8 assay. Enhanced sensitivity of cells to cisplatin or 5-FU was observed when treated in combination with lidocaine. The present study concluded that the involvement of the c-Met/c-Src pathway in the biological behaviour of MKN45 cells was mediated by lidocaine. Therefore, lidocaine may have the potential to suppress the malignant behaviour and proliferation of gastric cancer cells.

CBX8 acts as an independent RNA-binding protein to regulate the maturation of miR-378a-3p in colon cancer cells.

CBX8 is the core component of the PCG family protein PRC1 complex. It is overexpressed in many solid tumors and plays an important role in the prognosis and biological behaviors of tumors such as occurrence, development, invasion, and metastasis. However, exploration of the role and molecular mechanism of CBX8 in tumors is still in its infancy. Our study found that the down-regulation of CBX8 expression by RNA interference induced differential expression of several microRNAs in human colon cancer cells. The 5 most differentially expressed miRNA precursors (pre-miRNA) (hsa-miR-363-3p, hsa-miR-378a-3p, hsa-miR-371b-3p, hsa-miR-361-3p, and hsa-miR-576-3p) share a common motif sequence: ARAAAKUGCMC. We selected miR-378a-3p and further revealed that the negative regulation of miRNA expression by CBX8 mainly occurs in the processing of pre-miRNA to mature miRNA. CBX8 uses its own RNA-binding domain to interact with pre-miRNA, and is dependent on its own nuclear localization characteristics to limit nucleoplasmic transport of pre-miRNA. Changing the characteristic sequence of pre-miRNA or mutating the RNA-binding domain and nuclear localization signal of CBX8 can effectively weaken the regulation of miR-378a-3p expression by CBX8. However, our experimental results showed that miR-378a-3p inhibited the malignant expression of human colon cancer cells by targeting PDIA4, resulting in increased activity of caspases-3 and -7. In summary, our study suggests that CBX8 acts as an independent RNA-binding protein to regulate miRNA expression. Simultaneously, this study shows the correlation between the CBX8/miR-378a-3p/PDIA4 pathway and the malignant biological properties of colorectal cancer, suggesting this proposed pathway as a possible therapeutic target for human cancers.

Overexpression of centromere protein K (CENPK) gene in Differentiated Thyroid Carcinoma promote cell Proliferation and Migration

ABSTRACT Differentiated thyroid carcinoma (DTC) is one of the most common malignant tumors. Increasing evidence indicates that centromere protein K(CENPK) may play a key role in promoting carcinogenesis. The expression, biological functions, and clinical significance of CENPK in DTC are still unclear. The CENPK expression in the DTC specimen was confirmed using quantitative real-time PCR and Western blot. The expression of CENPK was silenced and promoted by lentivirus-mediated transfection with shRNA sequences or CENPK plasmid targeting CENPK in TPC1 and FTC-133 cells, respectively. Colony formation, Cell Counting Kit-8 (CCK-8), Transwell invasion, and scratch assays were performed to assess the malignant biological properties of FTC-133 and TPC1 cells. Tumorigenicity assay was performed using C57BL/6 mice to explore the influence of CENPK on the growth of TPC1. The present work suggested that the expression of CENPK remarkably increased in follicular thyroid cancer and papillary thyroid cancer tissue samples at the mRNA level. Immunohistochemical staining also showed consistent results at the protein level. In addition, CENPK mRNA expression level showed great value in diagnosis of DTC. Knockdown of CENPK significantly inhibited the invasion and migration of TPC1 and FTC-133 cells. In contrast, CENPK overexpression promoted invasion and migration of TPC1 and FTC-133 cells. Knockdown and overexpression of CENPK showed consistent effect on DTC tumor growth and expression of Ki-67 invivo. Our results indicated that CENPK was evidently upregulated in DTC. Knocking down CENPK suppressed TPC1 cell proliferation, invasion and migration. Targeting the CENPK may be anovel therapeutic method for DTC.