Nude Mice Assay Research Articles

LncRNA JPX promotes cervical cancer progression by modulating miR-25-3p/SOX4 axis

BackgroundThe long noncoding RNA (lncRNA) JPX is a molecular switch for X-chromosome inactivation. Accumulating studies have shown that the aberrant expression and function of lncRNAs are involved in the occurrence and development of tumors. However, the functional importance and mechanism of the action of lncRNA JPX in cervical cancer (CC) remain unknown.MethodIn this study, qRT-PCR and western blotting were used to evaluate the mRNA or protein expression of JPX, miR-25-3p and SOX4 in CC tissues and cell lines. StarBase v2.0 database, luciferase reporter assay and RNA immunoprecipitation assay were used to explore the relationship between JPX and miR-25-3p. EdU assay, CCK-8 assay and transwell assay were utilized to evaluate the proliferation, migration and invasion of CC cells. The tumor xenograft assay in nude mice was performed to demonstrate the role of the JPX/miR-25-3p/SOX4 axis in CC.ResultsWe found that JPX was markedly upregulated, whereas miR-25-3p was markedly downregulated in CC tissues and cell lines, and the expression of JPX was negatively correlated with miR-25-3p in CC tissues. Moreover, overexpression of JPX increased proliferation, migration and invasion of HeLa cells, whereas knockdown of JPX decreased proliferation, migration and invasion of HeLa cells. In contrast to JPX, overexpression of miR-25-3p decreased proliferation, migration and invasion of HeLa cells. In addition, knockdown of JPX was found to inhibit HeLa cell viability and tumor development via up-regulating the expression of miR-25-3p and inhibiting the expression of SOX4.ConclusionsOur study demonstrates that JPX promotes cervical cancer progression through modulating the miR-25-3p/SOX4 axis, and may serve as a potential target for CC therapy.

A novel epigenetic regulation of circFoxp1 on Foxp1 in colon cancer cells

Foxp1 is a tumor suppressor in colon cancer. However, circFoxp1 derived from Foxp1 is an oncogene. In this study, we aim to investigate the role of circFoxp1 in colon cancer and the regulatory mechanism between circFoxp1 and Foxp1. 78 human colon tumor tissues and the matched paracancerous tissues were collected. Quantitative polymerase chain reaction, immunohistochemistry, quantitative methylation-specific PCR, chromatin immunoprecipitation assay, CCK-8 assay, and Tumor xenograft in nude mice were performed. The expression of circFoxp1 was increased and Foxp1 was reduced in colon cancer tissues, which were associated with a poor overall survival rate of the patients with colon cancer. CircFoxp1 recruited DNMT1 to the promoter of Foxp1, leading to promotor hypermethylation, thereby inhibiting Foxp1 transcription. Interfering circFoxp1 by siRNA in SW620 cells significantly inhibited cell viability, while knockdown Foxp1 expression partially restored SW620 cell viability. In addition, knockdown of circFoxp1 significantly sensitized colon cancer cells to Capecitabine in vitro and vivo through regulating Foxp1. We discovered a novel epigenetic pathway that circFoxp1 regulated Foxp1 in colon cancer cells. CircFoxp1 may regulate DNA methylation and demethylation to coordinate colon cancer cell proliferation and participate in chemotherapy drug responses. Therefore, circFoxp1 may be a potential therapeutic target for colon cancer.

Enterotoxigenic Bacteroides fragilis induces the stemness in colorectal cancer via upregulating histone demethylase JMJD2B

ABSTRACT The enrichment of Enterotoxigenic Bacteroides fragilis (ETBF) has been identified in CRC patients and associated with worse prognosis. Cancer stem cells (CSCs) play essential roles in CRC development. However, whether ETBF is involved in CSCs regulation is unknown. To clarify the role of ETBF in CSCs properties, we performed extreme limited dilution assays (ELDA) in nude mice injected with ETBF-treated or untreated CRC cells subcutaneously, tumor organoids culture in azoxymethane (AOM) mouse model after gavaging with or without ETBF, and cell sphere formation assay after incubating CRC cell lines with or without ETBF. The results indicated that ETBF increased the stemness of CRC cells in vivo and in vitro. Furthermore, ETBF enhanced the expression of core stemness transcription factors Nanog homeobox (NANOG) and sex determining region Y-box 2 (SOX2). Histone H3 Lysine 9 trimethylation (H3K9me3) is critical in regulating CSCs properties. As an epigenetic and transcriptional regulator, JmjC-domain containing histone demethylase 2B (JMJD2B) is essential for embryonic stem cell (ESC) transformation and H3K9me3 demethylation. Mechanistically, ETBF infection significantly upregulated JMJD2B levels in CRC cell lines and nude mice xenograft model. JMJD2B epigenetically upregulated NANOG expression via demethylating its promoter H3K9me3, to mediate ETBF-induced stemness of CRC cells. Subsequently, we found that the Toll-like receptor 4 (TLR4) pathway, activated by ETBF, contributed to the enhanced expression of JMJD2B via nuclear transcription factor nuclear factor of activated T cells 5 (NFAT5). Finally, in human CRC samples, the amount of ETBF positively correlated with nuclear NFAT5, JMJD2B, and NANOG expression levels. In summary, ETBF upregulated JMJD2B levels in a TLR4-NFAT5-dependent pathway, and played an important role in stemness regulation, which promoted colorectal carcinogenesis.

Exosome-transmitted linc00852 associated with receptor tyrosine kinase AXL dysregulates the proliferation and invasion of osteosarcoma.

BackgroundReceptor tyrosine kinase AXL has been found to be highly expressed in osteosarcoma and positively associated with poor prognosis. There are tumor groups with high or low AXL expression, which had different capabilities of invading vessels and forming distal metastases. Exosome‐transmitted lncRNA may be transferred intercellularly to promote tumor cells’ proliferation and invasion.MethodsExosomes were detected by electron microscopy, particle size analysis, and western blotting. High‐throughput sequencing helped to find the highest differentially expressed lncRNA in AXL‐associated exosomes. Clone formation, wound healing, transwell assay, and xenograft model in nude mice were performed to evaluate cells’ proliferation, migration, and invasion in vitro and in vivo. Lentiviral transfection was used to up‐ or down‐regulate the lncRNA levels in cell lines. Luciferase reporter assay and RNA FISH etchelped to indicate the molecular mechanisms. The results in the cell lines were proved in the osteosarcoma tissues with clinical analysis.ResultsThe exosomes derived from donor cells with high AXL expression could promote the proliferation and invasion and upregulate AXL expression of the receiver cells with low AXL. Linc00852 was the highest differentially expressed lncRNA in AXL‐associated exosomes and was also regulated by AXL expression. Although the mechanisms of linc00852 in nucleus were unrevealed, it could upregulate AXL expression partly by competitively binding to miR‐7‐5p. The AXL‐exosome‐linc00852‐AXL positive feedback loop might exist between the donor cells and the receiver cells. Clinically, linc00852 was significantly highly expressed in osteosarcoma tissues and positively associated with tumor volumes and metastases, which was also obviously related with AXL mRNA expression.ConclusionAXL‐associated exosomal linc00852 up‐regulated the proliferation, migration, and invasion of osteosarcoma cells, which would be considered as a new tumor biomarker and a special therapeutic target for osteosarcoma.

Overexpression of lncRNA TINCR is associated with high-grade, invasive, and recurring tumors, and facilitates proliferation in vitro and in vivo of urothelial carcinoma of the bladder

ObjectivesThe aberrant expression of long noncoding RNAs (lncRNAs) plays roles in cancer development. In this work, we measured the expression of lncRNA terminal differentiation-induced non-coding RNA (TINCR) in urothelial carcinoma of the bladder (UCB), determined its impact on the proliferation of UCB in vitro and in vivo and identified its possible targets. MethodsThe expression levels of genes were measured by Real-Time quantitative Polymerase Chain Reaction (qPCR). Cell proliferation, cell motility, and cell apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, wound healing assay, and ELISA, respectively. Tumor growth in vivo was determined by xenograft formation assay in nude mice. The predicted binding site between TINCR and hsa-miR-125b was confirmed by dual luciferase reporter assay. ResultsThe expression levels of TINCR were higher in cancerous tissues than that in paired noncancerous tissues of UCB. Higher expression levels of TINCR were positively associated with high-grade, invasive, and recurring tumors. Depletion of TINCR retarded proliferation, decreased motility, increased apoptosis in UCB cells, and markedly reduced tumor growth in xenograft nude mice. The predicted binding site between TINCR and hsa-miR-125b was functional. TINCR downregulated hsa-miR-125b in UCB cells. Hsa-miR-125b mimic reversed the proliferation caused by TINCR up-expression in UCB cells. ConclusionsUp-regulation of TINCR may act as an unfavorable indicator for the diseasing status of UCB. TINCR facilitates bladder cancer proliferation in vitro and in vivo. Hsa-miR-125b is a target for TINCR in UCB.

CCL19 suppresses gastric cancer cell proliferation, migration, and invasion through the CCL19/CCR7/AIM2 pathway.

Absent in melanoma 2 (AIM2) has been reported to be an important inflammasome component that exerts tumor suppression in several tumors. However, whether CCL19/CCR7/AIM2 is involved in the progression of GC still remains unclear. Quantitative real-time and ELISA assay were used to determine the expressions of AIM2, CCL19 and CCR7 in GC tissues and cell lines. CCK-8, Edu staining, flow cytometry, Transwell assay, and tumorigenesis in nude mice were used to explore the function of AIM2 and CCL19 in vitro and in vivo. Apoptosis and inflammation-related biomarkers were detected by Western blot and ELISA assay. H&E staining was used to assess the histological changes in the subcutaneous tumor model. Immunohistochemistry (IHC) was used to evaluate the expression of Ki-67. We found that expression levels of AIM2, CCL19 and CCR7 were obviously lower in early GC tissues than those in progressive GC tissues. In vitro assays revealed that CCL19 treatment could enhance the suppressive effects of AIM2 overexpression on cell proliferation, migration, and invasion through CCR7. An in vivo assay also demonstrated that silencing of AIM2 reversed the suppressive effects of CCL19 on tumor growth. Collectively, CCL19 overexpression significantly inhibited GC cell proliferation and tumor growth in vitro and in vivo by up-regulating the CCR7/AIM2 pathway. Thus, CCL19 activated CCR7/AIM2 signaling pathway and it may be a potential therapeutic approach for GC therapy.

A novel function of IMPA2, plays a tumor-promoting role in cervical cancer

Discovery of genes and molecular mechanism involved in cervical cancer development would promote the prevention and treatment. By comparing gene expression profiles of cervical carcinoma in situ (CCIS) and adjacent normal tissues, we identified a potential cancer-promoting gene, IMPA2. This study aimed to elucidate the role of IMPA2 and underlying molecular mechanisms in cervical cancer progression. To do this expression of IMPA2 was compared between human cervical cancer and corresponding adjacent normal cervical tissues firstly. CCK-8 assay, clone formation assay, wound healing assay, transwell assay, and tumor formation in nude mice were performed to demonstrate the effect of IMPA2 in cervical cancer proliferation and metastasis. Further proteomic profiling and western blotting explored the molecular pathway involved in the IMPA2-regulating process. The results showed that IMPA2 gene expression was upregulated in cervical cancer. Consistently, silencing of IMPA2 suppressed tumor formation in BALB/c nude mice. Short hairpin RNA (shRNA)-mediated IMPA2 silencing significantly inhibited proliferation and colony-forming abilities of cervical cancer cells, while IMPA2 overexpression had little impact. Also, IMPA2 silencing suppressed cellular migration, but overexpression promoted migration. Proteomics analysis revealed the involvement of mitogen-activated protein kinase (MAPK) pathway in tumor-promoting action of IMPA2. Significantly, the inhibition of IMPA2 activated ERK phosphorylation, and its inhibitory effects can be restored by using selective ERK inhibitor, FR180204. In conclusion, IMPA2 acts as an oncogene in the proliferation and migration of cervical cancer. IMPA2 downregulated ERK phosphorylation to promote cervical cancer. These findings identify a new mechanism underlying cervical cancer and suggest a regulating effect of IMPA2 in MAPK signaling pathway.

ROR2 suppresses metastasis of prostate cancer via regulation of miR-199a-5p\u2013PIAS3\u2013AKT2 signaling axis

Bones are the most common metastatic sites for prostate cancer (PCa). Receptor tyrosine kinase-like orphan receptor 2 (ROR2), a noncanonical Wnt receptor, plays crucial roles in skeletal morphogenesis, osteoblast differentiation, and bone formation. The role of ROR2 in PCa metastasis is unclear. We analyzed online datasets from Oncomine as well as using IHC staining on tissue array to determine the relationship between ROR2 expression level and disease outcome of PCa. To investigate how ROR2 regulates migration and invasion of PCa cells, we performed transwell assay and orthotopic xenograft model in nude mice. We then applied the Micro-Western Array (MWA), a high-throughput western blotting platform to analyze the downstream signaling pathways being regulated by ROR2. Compared with nonmalignant PZ-HPV-7 and RWPE-1 cells, PCa cell lines express lower level of ROR2 protein. Constitutive expression of ROR2 in PC-3, DU-145, or C4-2B PCa cells significantly suppressed the cell migration, invasion, and epithelial–mesenchymal transition (EMT) proteins. MWA, western blotting, and microRNA analysis showed that elevation of ROR2 suppressed the expression of miR-199a-5p, which in turn increased the expression of PIAS3. The upregulation of PIAS3 then decreased AKT2 and the phosphorylation of AKT, resulting in the inhibition of migration and invasion of PCa cells both in vitro and in orthotopic xenograft mice model. IHC staining of tissue array and Oncomine datasets analysis indicated that the gene and protein level of ROR2 is much lower in metastatic prostate tumors as compared with primary tumors or adjacent normal prostate tissues. Low level of ROR2 correlated to poor survival and high recurrent frequency in PCa patients. In conclusion, we discovered that ROR2 suppresses PCa metastasis via regulation of PIAS3–PI3K–AKT2 signaling axis.

Long non-coding RNA PRRT3-AS1 silencing inhibits prostate cancer cell proliferation and promotes apoptosis and autophagy.

What is the central question of this study? What is the role of lncRNA PRRT3-AS1 in the regulation of peroxisome proliferator-activated receptor γ (PPARγ) gene-mediated mechanistic target of rapamycin (mTOR) signalling pathway in proliferation, apoptosis and autophagy of prostate cancer cells? What is the main finding and its importance? The targeting relation between lncRNA PRRT3-AS1 and PPARγ was verified, and it was demonstrated that silencing of lncRNA PRRT3-AS1 can upregulate apoptosis and autophagy yet downregulate proliferation, migration and invasion of prostate cancer cells through the mTOR signalling pathway. Further work is needed to consolidate the therapeutic value of lncRNA PRRT3-AS1 in clinical trials and treatment of prostate cancer. Although long non-coding RNAs (lncRNAs) are correlated with multiple cancers, their molecular mechanisms in prostate cancer (PC) remain inadequately understood. This study investigated the effects of lncRNA PRRT3-AS1 on the progression of prostate cancer (PC) with involvement of peroxisome proliferator-activated receptor γ (PPARγ). Microarray analysis was used to identify the differentially expressed genes and lncRNAs associated with PC. RT-qPCR and western blot analysis were employed to test the expression of lncRNA PRRT3-AS1, mammalian target of rapamycin (mTOR) signalling pathway-, apoptosis- and autophagy-related genes. A scratch test, Transwell assay, CCK-8 assay, colony formation assay, flow cytometry and monodansylcadaverine staining were employed to identify the migration, invasion, proliferation activity, cell cycle and apoptosis and autophagy of PC3 cells, respectively. Tumorigenicity assays in nude mice were used to detect the tumorigenic ability. GSE55945 and GSE46602 datasets indicated that lncRNA PRRT3-AS1 was highly expressed in PC. PPARγ was predicted as a target gene of lncRNA PRRT3-AS1. Ectopic overexpression of PPARγ or lncRNA PRRT3-AS1 silencing led to inhibited cell viability, migration and invasion, and accelerated apoptosis. Furthermore, the delivery of si-PRRT3-AS1 or PPARγ vector to PC3 cells resulted in the regression of xenografts in nude mice. Based on the in vitro and in vivo experiments, silencing of lncRNA PRRT3-AS1 was observed to activate the PPARγ gene, which in turn could inhibit PC cell proliferation and promote apoptosis and autophagy by blocking the mTOR signalling pathway.

PFND1 Predicts Poor Prognosis of Gastric Cancer and Promotes Cell Metastasis by Activating the Wnt/β-Catenin Pathway.

BackgroundPrefoldin (PFDN) subunits have recently been found to function importantly in various tumor types, while the role of PFDN subunit 1 (PFDN1) in gastric cancer (GC) remains largely unknown. Herein, we aimed to investigate the clinical significance, the biological role and the underlying mechanism of PFDN1 in GC development.Materials and MethodsPFDN1 expression levels were measured in human GC specimens by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Furthermore, the effects of aberrant PFDN1 expression on GC cells behavior were assessed by wound-healing assay and transwell assay in vitro, and metastasis assay in nude mice, as well as Wnt/β-catenin signaling-induced epithelial–mesenchymal transition (EMT)-related markers by qRT-PCR and Western blot.ResultsPFDN1 levels were significantly upregulated in GC tissues compared with those in matched adjacent normal tissues. PFDN1 upregulation correlated strongly with clinical metastasis and unfavorable prognosis for GC patients. In vitro and in vivo studies revealed that PFDN1 facilitated GC cell migration, invasion and metastasis. Mechanically, PFDN1 modulated GC cell behavior by activating Wnt/β-catenin signaling-mediated EMT.ConclusionThese results suggested a central role of PFDN1 in GC metastatic development via the Wnt/β-catenin pathway, thus providing a potential therapeutic target for patients with GC.

Flufenamic Acid Inhibits Adipogenic Differentiation of Mesenchymal Stem Cells by Antagonizing the PI3K/AKT Signaling Pathway.

Objectives Flufenamic acid (FFA) is a representative of the fenamic acids, an important group of NSAIDs. In the present study, we study the effects of FFA on adipogenesis of human mesenchymal stem cells (MSCs) and we explore the potential mechanism. Methods To investigate the effects of FFA on adipogenic differentiation of hMSCs, human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs), representative of hMSCs, were treated with FFA during adipogenic differentiation in vitro. The effects of FFA in vivo were evaluated using a heterotopic adipose formation assay in nude mice as well as ovariectomized (OVX) and aged mice. To explore the mechanism of FFA, Western blot was used to determine activation of the PI3K/AKT signaling pathway. Results Our results demonstrate that, at certain concentrations, FFA inhibited adipogenesis of human MSCs both in vitro and in vivo. Mechanistically, FFA inhibited adipogenesis of human MSCs by inhibiting the PI3K/AKT pathway. Conclusions The present study indicated that FFA could be used to inhibit adipogenesis of human MSCs in tissue engineering and diseases related to excessive adipogenic differentiation of MSCs.

Circ_0001971 regulates oral squamous cell carcinoma progression and chemosensitivity by targeting miR-194/miR-204 in vitro and in vivo.

Circular RNAs (circRNAs) play a wide role in human cancers, including oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate the biological functions of circ_0001971 and associated mechanisms in OSCC. The expression of circ_0001971, miR-194, and miR-204 was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell proliferation and viability were assessed using cell counting kit-8 (CCK-8) assay. Cell migration and invasion were examined using the transwell assay. Cell apoptosis was monitored by flow cytometry assay. The protein levels of proliferation marker (CyclinD1), epithelial mesenchymal-transition (EMT) markers (E-cadherin (E-cad) and N-cadherin (N-cad)) and apoptosis markers (Cleaved-caspase-3 (Cleaved-cas-3) and Cleaved-caspase-9 (Cleaved-cas-9)) were measured by Western blot. The relationship between circ_0001971 and miR-194 or miR-204 was predicted by online tool starBase and verified by the Dual-Luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Tumor formation assay in nude mice was conducted to observe the role of circ_0001971 in vivo. The expression of circ_0001971 was significantly increased in tumor tissues and cell lines. Circ_0001971 knockdown inhibited cell proliferation, migration, and invasion but promoted cisplatin (DDP) sensitivity and cell apoptosis. It was confirmed that miR-194 and miR-204 were targets of circ_0001971, and miR-194 inhibition or miR-204 inhibition reversed the effects of circ_0001971 knockdown in OSCC cells. Moreover, circ_0001971 knockdown impeded tumorigenesis and development in vivo. Circ_0001971 regulates cell proliferation, migration, invasion, apoptosis, and chemosensitivity of OSCC by interacting with miR-194 and miR-204 in vitro and in vivo. We provided a theoretical basis for the action mechanism of circ_0001971 on OSCC progression and chemosensitivity.

LINC00114 promoted nasopharyngeal carcinoma progression and radioresistance in vitro and in vivo through regulating ERK/JNK signaling pathway via targeting miR-203.

Nasopharyngeal carcinoma (NPC) is a malignancy and is prone to distant metastasis and radioresistance. Long non-coding RNAs (lncRNAs) play vital roles in human cancers. The purpose of this study was to explore the role and the action mechanism of intergenic lncRNA (LINC00114) in NPC. The expression of LINC00114 and microRNA-203 (miR-203) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). NPC cells were exposed to X-ray as radiation treatment. Cell proliferation, migration, cell survival fraction and apoptosis were assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), transwell, colony formation, and flow cytometry assays, respectively. The expression of Cleaved-cas-3, Cleaved-cas-9, phosphor-ERK (p-ERK) and phosphor-JNK (p-JNK) was quantified by Western blot. The interaction between miR-203 and LINC00114 was predicted by bioinformatics tool microRNA.org and verified by dual-luciferase reporter assay. Tumor formation assay in nude mice was conducted to examine the role of LINC00114 in vivo. LINC00114 was upregulated in serums from NPC patients, tissues and cell lines of NPC. LINC00114 knockdown inhibited proliferation, migration, and radioresistance of NPC cells. MiR-203 was a target of LINC00114, and miR-203 inhibition eliminated the effects of LINC00114 knockdown. Besides, the extracellular signal-regulated kinases (ERK)/c-Jun N-terminal kinases (JNK) pathway was inactivated by LINC00114 knockdown but recovered by miR-203 inhibition. Moreover, LINC00114 knockdown suppressed tumor growth and radioresistance in vivo. LINC00114 contributed to NPC development and radioresistance through the regulation of ERK/JNK signaling pathway and the mediation of miR-203, suggesting that LINC00114 was a promising biomarker to defense NPC progression and radioresistance.

Therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and neuroblastoma progression.

As a hallmark of metabolic reprogramming, aerobic glycolysis contributes to tumorigenesis and aggressiveness. However, the mechanisms and therapeutic strategies regulating aerobic glycolysis in neuroblastoma (NB), one of leading causes of cancer-related death in childhood, still remain elusive.Methods: Transcriptional regulators and their downstream glycolytic genes were identified by a comprehensive screening of publicly available datasets. Dual-luciferase, chromatin immunoprecipitation, real-time quantitative RT-PCR, western blot, gene over-expression or silencing, co-immunoprecipitation, mass spectrometry, peptide pull-down assay, sucrose gradient sedimentation, seahorse extracellular flux, MTT colorimetric, soft agar, matrigel invasion, and nude mice assays were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. Survival analysis was performed by using log-rank test and Cox regression assay.Results: Transcription factor myeloid zinc finger 1 (MZF1) was identified as an independent prognostic factor (hazard ratio=2.330, 95% confidence interval=1.021 to 3.317), and facilitated glycolysis process through increasing expression of hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1). Meanwhile, a 21-amino acid peptide encoded by upstream open reading frame of MZF1, termed as MZF1-uPEP, bound to zinc finger domain of Yin Yang 1 (YY1), resulting in repressed transactivation of YY1 and decreased transcription of MZF1 and downstream genes HK2 and PGK1. Administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP inhibited the aerobic glycolysis, tumorigenesis and aggressiveness of NB cells. In clinical NB cases, low expression of MZF1-uPEP or high expression of MZF1, YY1, HK2, or PGK1 was associated with poor survival of patients.Conclusions: These results indicate that therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and NB progression.

NALP3 orchestrates cellular bioenergetics to facilitate non-small cell lung cancer cell growth

AimsPrevious work has reported the closely correlation between inflammation and carcinogenesis, while the role of NALP3, the key component of inflammasome activation in NSCLC remains elusive. This study was to unravel the mechanism of NALP3 on modulating NSCLC cancer cell growth. MethodsIHC and immuno-blot were performed to analyze expression of NALP3 and indicated molecules. CCK-8 and xenograft nude mice assay were used to evaluate cell growth in vitro and in vivo. Bioenergetics assay was performed to measure OXPHOS and aerobic glycolysis. siRNA and shRNA were constructed to knockdown endogenous NALP3 and DNMT1. Co-immunoprecipitation was applied to confirm the interaction between NALP3 and DMAP1. BioProfile FLEX analyzer and Lactate Reagent Kit were used to measure relative level glucose uptake and lactate production. Key findingsWe reported NALP3 were up-regulated in NSCLC tumor tissues. NALP3 depletion suppressed cancer cell growth in vitro and in vivo. Moreover, data showed depletion of NALP3 promoted cell bioenergetics switch from aerobic glycolysis to OXPHOS. Additionally, we found NALP3 interacted with DMAP1 and alteration of NALP3 increased DNMT1 level. Subsequently, we clarified depletion of DNMT1 significantly suppressed NSCLC cell growth and orchestrated cellular metabolism which was similar to the effects of NALP3 knockdown. Finally, our data showed high NALP3 was associated with poor outcomes, and correlated with TNM stage and differentiation. SignificanceCurrent study elucidated NALP3 could promote metabolic reprogramming to regulate NSCLC cell growth and suggested that NALP3 may be considered as a novel biomarker and therapeutic target for NSCLC patients.

Circ_0000003 promotes the proliferation and metastasis of non-small cell lung cancer cells via miR-338-3p/insulin receptor substrate 2

ABSTRACT Background Circular RNAs (circRNAs) play a pivotal regulatory role in a variety of tumors.Nevertheless, the detailed function of circ_0000003 in non-small cell lung cancer (NSCLC) and its regulatory mechanism remain elusive. Methods RT-PCR was carried out to detect the expressions of circ_0000003, miR-338-3p and insulin receptor substrate 2 (IRS2)in NSCLC tissues. Besides, western blot was done to monitor IRS2 expression in NSCLC cells. The correlation between circ_0000003 and clinicopathologic characteristics of NSCLC patients was analyzed as well.CCK8 and BrdUassays were used to monitor cell proliferation; flow cytometry was used to detect apoptosis; and transwell assay was conducted to detect its migration and invasion.Moreover, dual luciferase reporter gene assay was done to verify the targeting relationship between circ_0000003 and miR-338-3p.Additionally, the effect of circ_0000003 on the growth of NSCLC cells in vivo was evaluated by tumorigenesis assay in nude mice. Results The expression of circ_0000003 was significantly high in NSCLC tissues and cell lines, and its high expression level was notably correlated with lymph node metastasis andTNM staging.In vitro experiments showed that overexpression of circ_0000003 facilitated the proliferation, migration, invasion and inhibited the apoptosis of NSCLC cells, while the knockdown of circ_0000003 had the opposite effect.In vivo experiments revealed that knockdown of circ_0000003 impeded tumor growth and metastasis. Further, the underlying mechanism showed that circ_0000003 functioned as endogenous competitive RNA and directly targeted miR-338-3p to positively regulated IRS2 expression. Conclusion Circ_0000003 promotes the proliferation and metastasis of NSCLC cells via modulating miR-338-3p/IRS2 axis.

Upregulated LINC00565 Accelerates Ovarian Cancer Progression By Targeting GAS6.

BackgroundLong noncoding RNAs (lncRNAs) have been identified to participate in tumorigenesis. However, the underlying mechanisms of differentially expressed lncRNAs engaged in diseases remain indistinct and need further exploration.MethodsRaw data files downloaded from TCGA and GEO dataset were used to analyze the differentially expressed lncRNAs and LINC00565 was picked out as the potential oncogene. qRT-PCR was used to analyze the LINC00565 level in ovarian tissues and cell lines. Subsequently, the selected ovarian tumor cells were then transfected with LINC00565 siRNA by Lipofectamine 2000 and the cell cycle was detected by flow cytometry. Effect of LINC00565 on tumor growth and cell cycle was verified by tumor formation assay in nude mice. The mechanism of LINC00565 involving in cell cycle regulation was further explored by Western blot.ResultsIn this research, we discovered that LINC00565, a novel lncRNA, was highly expressed in ovarian cancer (OC). LINC00565 expression level was negatively associated with outcomes of OC patients. Further analysis showed that LINC00565 expression was closely correlated to tumor size, FIGO stage, but not related to other clinical features. In vitro experiments indicated that knockdown of LINC00565 significantly inhibited proliferative, invasive and migratory abilities of ovarian cancer cells. Besides, knockdown of LINC00565 can induce cell cycle arrest in G0/G1 phase. In addition, in vivo assay showed that low expression of LINC00565 inhibited the growth of OC. Further study found that LINC00565 knockdown markedly downregulated the protein expressions of CyclinD1, CyclinE1 and CDK4, but upregulated the expression of P16 and P21. Subsequently, we confirmed that LINC00565 promoted the progression of OC via upregulating GAS6, which has been confirmed to promote tumor progression.ConclusionIn summary, our study firstly reported that the LINC00565 functioned as an oncogene to promote the progression of OC by interacting with GAS6.

Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma.

Eukaryotic initiation factor 3c (eIF3C) is involved in the initiation of protein translation. Aberrant eIF3C expression has been reported in different types of human cancer. The present study aimed to assess the role of eIF3C in the malignant behavior of renal cell carcinoma invitro and invivo. eIF3C expression was assessed in 16 pairs of renal cell carcinoma (RCC) and matched distant normal tissues, and in RCC cell lines using immunohistochemistry. Subsequently, eIF3C was depleted using lentiviral short hairpin RNA and cell proliferation, cell cycle distribution and apoptosis of these eIF3C‑depleted cells were examined. Additionally, tumor cell xenograft assays in nude mice, Affymetrix microarrays and ingenuity pathway analyses were performed. eIF3C expression was upregulated in RCC tissues and cell lines. Depletion of eIF3C reduced tumor cell proliferation and arrested them at the G1 stage, thus promoting their apoptosis invitro. Depletion of eIF3C also inhibited the formation and growth of tumor cell xenografts in nude mice. In addition, depletion of eIF3C altered the expression levels of 994 differentially expressed genes in RCC cells (516 genes were upregulated and 478genes were downregulated). The expression levels of phosphorylated‑AKT, c‑JUN and NFKB inhibitor α were lower in the shorth hairpin RNA eIF3C‑transfected RCC cells compared with in the control group. In conclusion, the present study demonstrated that upregulated eIF3C expression contributed to the development and progression of RCC. Future studies should further evaluate whether eIF3C could be used as a potential strategy for RCC targeting therapy.

MiR-129-5p inhibits liver cancer growth by targeting calcium calmodulin-dependent protein kinase IV (CAMK4)

This study was designed to investigate the mechanism by which miR-129-5p affects the biological function of liver cancer cells. The expression levels of miR-129–5p in liver cancer tissues and cells were, respectively, determined. Crystal violet staining and flow cytometry were used to detect cell proliferation and apoptosis. Wound healing assay and transwell assay were performed to test cell migration and invasion. The target gene of miR-129–5p was analyzed and verified by bioinformatics analysis and luciferase reporter assay. Tumorigenicity assays in nude mice were used to test the antitumor ability of calcium calmodulin-dependent protein kinase IV (CAMK4). miR-129–5p was found to be underexpressed in hepatocellular cancer tissues and cells and also to inhibit liver cells proliferation, migration, and invasion and promote apoptosis. CAMK4 was a direct target for miR-129–5p and was lowly expressed in liver cancer tissues and cells. CAMK4 was also found to inhibit liver cells proliferation, migration and invasion, and promote apoptosis. CAMK4 might exert an antitumor effect by inhibiting the activation of mitogen-activated protein kinase (MAPK). MiR-129–5p was a tumor suppressor with low expression in liver cancer tissues and cells. CAMK4, which is a direct target gene of miR-129–5p, could inhibit tumor by inhibiting the activation of MAPK signaling pathway.

PYCR1 interference inhibits cell growth and survival via c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathway in hepatocellular cancer

BackgroundLiver cancer is the second leading causes of cancer-related death globally. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays a critical role in metabolic profiles of tumors. Therefore, it is necessary to explore the mechanisms of PYCR1 on cell growth and survival in hepatocellular carcinoma (HCC).MethodsProtein and mRNA expression levels of PYCR1 in 140 pairs of tumor and adjacent normal liver tissues of HCC patients were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). Expressions of PYCR1 were inhibited in BEL-7404 cells and SMMC-7721 cells using gene interference technology. The cell proliferation was detected by Celigo and MTT assay. The colony formation assay was also performed. The cell apoptosis was measured by flow cytometric assay. The effect of PYCR1 interference on tumor growth was observed by xenograft nude mice assay in vivo. The downstream pathway of PYCR1 interference was searched by microarray and bioinformatics analysis, and validated by qRT-PCR and western blot.ResultsPYCR1 levels were significantly up-regulated in HCC tumor tissues than adjacent normal liver tissues in both protein and mRNA levels (P < 0.01). In vitro, the cell proliferation was significantly slower in shPYCR1 group than shCtrl group in BEL-7404 and SMMC-7721 cells (P < 0.001). The colony number was significantly smaller after PYCR1 interference (P < 0.01). The percentage of apoptosis cells significantly increased in shPYCR1 group (P < 0.01). In vivo, PYCR1 interference could obviously suppress tumor growth in xenograft nude mice. The volume and weight of tumors were significantly smaller via PYCR1 interference. The c-Jun N-terminal kinase (JNK) signaling pathway significantly altered, and insulin receptor substrate 1 (IRS1) were significantly down-regulated by PYCR1 interference in both mRNA and protein levels (P < 0.001).ConclusionPYCR1 interference could inhibit cell proliferation and promote cell apoptosis in HCC through regluting JNK/IRS1 pathway. Our study will provide a drug target for HCC therapy and a potential biomarker for its diagnosis or prognosis.