Stimulated Cell Apoptosis Research Articles

Transcriptomic landscape of sodium butyrate-induced growth inhibition of human colorectal cancer organoids.

Butyrate, a short-chain fatty acid, is predominantly produced by the decomposition of dietary fiber in the colon. Recent studies have shown that sodium butyrate (NaB) can inhibit cell proliferation and stimulate cell apoptosis in colorectal cancer (CRC) cells. However, the molecular mechanism behind NaB is still elusive. In this study, we aimed to explore the deeper mechanism of NaB in CRC by establishing a novel model - organoid. Organoids were generated from healthy and cancerous sites of CRC patients. RNA-seq experiments were undertaken using RNA isolated from the CRC organoids treated with NaB. Gene ontology analyses suggested that cell adhesion, cell-cell signaling, and extracellular matrix (ECM) organization were significantly enriched, with the manifested ECM related to the cell morphology variation in NaB-induced CRC organoids. The KEGG pathway and PPI analysis mainly focused on ECM-receptor interactions, as well as the PI3K-Akt signaling pathway. Interestingly, further analysis suggested that the upregulation of the expression of ITGB7 and ITGA2B was significantly associated with death by NaB-induced CRC organoids. In addition, NaB might further induce cell cycle arrest via the PI3K-Akt pathway, and thus cause cell death. Cumulatively, these results provide evidence that the ECM-integrin/PI3K axis may mediate phenotypic changes in the NaB-treated CRC organoid, which provides a broader perspective of the treatment and prognosis monitoring of CRC.

Effect of Conjugated Linoleic Acid (CLA) Supplementation on Expression of B-Cell Lymphoma-2 (Bcl-2) in the Bladder Epithelium of Wistar (Rattus norvegicus) Rats Exposed to Cigarette Smoke.

Bladder carcinoma is the 10th most common cancer in the world with an incidence about 3% of all cancers. The risk factor for smoking is found in 81% of all cases of bladder carcinoma. One of the protein groups associated with bladder urothelial carcinoma is B-Cell Lymphoma-2 (Bcl-2). Nicotine-derived nitrosamine ketone (NNK) contained in cigarette smoke would increase the proliferation of cancer cells through increased the expression of Bcl-2. The expression of Bcl-2 could be suppressed in the presence of Conjugated Linoleic Acid (CLA), a polyunsaturated fatty acid that has role in reducing the risk of cancer development which is reported in several studies, and then stimulate cell apoptosis. To determine the effect of CLA supplementation on Bcl-2 expression in the bladder of rats which is exposed to cigarette smoke. The study is an experimental study with true experimental posttest only control group design on Wistar rats. Sample was divided into 2 case groups: 0.5% of diet (125 mg) CLA supplementation in group A, 1% of diet (250 mg) CLA in group B; and 2 control groups: group without CLA supplementation (group C) as positive control and without cigarette smoke exposure (group D) as negative control. The study takes 60 days of exposure and then Bcl-2 expression on bladder epithelial was evaluated by immunohistochemistry staining. The results descriptively showed that rats in group C has an average Bcl-2 expression of 25.8±7.33%, while rats in group D has an average Bcl-2 expression 14.1±7.73% which means cigarette smoke exposure has been shown to increase the expression of Bcl-2 by 45.35% (p=0.019) in the bladder mucosa of experimental animals. Group B obtained an average Bcl-2 expression was 14.2±9.6% and has a significant difference when compared to group C, it shows that the addition of 1% CLA would reduce the expression of Bcl-2 by 44.96% (p=0.032). However, for group A, group with 0.5% diet of CLA supplementation did not showed decrease of Bcl-2 expression when compared to the group C (p=0.37). Conjugated Linoleic Acid (CLA) supplementation 1% of diet can reduce Bcl-2 expression in bladder epithelium of wistar rats (Rattus norvegicus) exposed to cigarette smoke.

The function of ubiquitin-specific protease 31 in intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is the most serious type of stroke. High level of thrombin is found in the ICH. Ubiquitin-specific protease (USP) 31, a member of deubiquitinating enzymes family, has been found to negatively regulate the NF-κB pathway. However, the function of USP31 in ICH remains largely unknown. In the present study, the mRNA and protein expression levels of USP31 were measured by real-time PCR and western blot. Flow cytometry was used to measure cell apoptosis and the level of reactive oxygen species (ROS). In the current study, we found the mRNA level of USP31 was decreased in peripheral blood mononuclear cells (PBMCs) from the patients with ICH. Thrombin stimulated cell apoptosis, and increased the ROS level, the productions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1), but it decreased the expression level of USP31 in BV-2 cells. In agreement, USP31 overexpressing could alleviate these effects caused by thrombin. USP31 partially reversed the thrombin induced increase of nuclear factor kappa B (NF-κB) localization. Further, NF-κB inhibitor could alleviate the effects induced by USP31 knockdown. In addition, USP31 decreased the ubiquitination level of IκBα, which might contribute to the depression of NF-κB activation. In conclusions, USP31 played a role in the ICH might via regulating NF-κB signaling pathway.

Semi-Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity.

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC50 value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.

ETV4 mediated lncRNA C2CD4D-AS1 overexpression contributes to the malignant phenotype of lung adenocarcinoma cells via miR-3681-3p/NEK2 axis

ABSTRACT Lung adenocarcinoma (LUAD) is originated from the mucus-producing glands of the lungs. The involvement of long noncoding RNAs (lncRNAs) has been discovered in multiple diseases. In the present research, we aimed to unmask the role of C2CD4D and THEM5 antisense RNA 1 (C2CD4D-AS1) in LUAD. RT-qPCR or western blot analysis was respectively applied in the detection of RNA or protein expressions. The function of C2CD4D-AS1 in LUAD was assessed by functional assays. Through ChIP, RNA pull down, DNA pull down, RIP and luciferase reporter assays, the in-depth regulatory mechanism of C2CD4D-AS1 in LUAD was explored. C2CD4D-AS1 was dramatically overexpressed in LUAD tissues and cell lines. As a result, depletion of C2CD4D-AS1 significantly repressed cell proliferation, migration, invasion and stimulated cell apoptosis in LUAD. Mechanistically, ETS variant transcription factor 4 (ETV4) activated the transcription of C2CD4D-AS1 and stimulated its up-regulation in LUAD cells, thus affecting LUAD cell biological functions. Furthermore, C2CD4D-AS1 sponged microRNA-3681-3p (miR-3681-3p) and regulated NIMA-related kinase 2 (NEK2), thus participating in modulating LUAD cell biological behaviors. To conclude, C2CD4D-AS1 up-regulation induced by ETV4 enhanced NEK2 expression by sequestering miR-3681-3p to contribute to the malignant behaviors of LUAD cells.

Transcriptome comparative analysis of ovarian follicles reveals the key genes and signaling pathways implicated in hen egg production

BackgroundOvarian follicle development plays an important role in determination of poultry egg production. The follicles at the various developmental stages possess their own distinct molecular genetic characteristics and have different biological roles in chicken ovary development and function. In the each stage, several genes of follicle-specific expression and biological pathways are involved in the vary-sized follicular development and physiological events. Identification of the pivotal genes and signaling pathways that control the follicular development is helpful for understanding their exact regulatory functions and molecular mechanisms underlying egg-laying traits of laying hens.ResultsThe comparative mRNA transcriptomic analysis of ovarian follicles at three key developmental stages including slow growing white follicles (GWF), small yellow follicles (SYF) of recruitment into the hierarchy, and differentiated large yellow follicles (LYF), was accomplished in the layers with lower and higher egg production. Totally, 137, 447, and 229 of up-regulated differentially expressed genes (DEGs), and 99, 97, and 157 of down-regulated DEGs in the GWF, SYF and LYF follicles, including VIPR1, VIPR2, ADRB2, and HSD17B1 were identified, respectively. Moreover, NDUFAB1 and GABRA1 genes, two most promising candidates potentially associated with egg-laying performance were screened out from the 13 co-expressed DEGs in the GWF, SYF and LYF samples. We further investigated the biological effects of NDUFAB1 and GABRA1 on ovarian follicular development and found that NDUFAB1 promotes follicle development by stimulating granulosa cell (GC) proliferation and decreasing cell apoptosis, increases the expression of CCND1 and BCL-2 but attenuates the expression of caspase-3, and facilitates steroidogenesis by enhancing the expression of STAR and CYP11A1. In contrast, GABRA1 inhibits GC proliferation and stimulates cell apoptosis, decreases the expression of CCND1, BCL-2, STAR, and CYP11A1 but elevates the expression of caspase-3. Furthermore, the three crucial signaling pathways such as PPAR signaling pathway, cAMP signaling pathway and neuroactive ligand-receptor interaction were significantly enriched, which may play essential roles in ovarian follicle growth, differentiation, follicle selection, and maturation.ConclusionsThe current study provided new molecular data for insight into the regulatory mechanism underlying ovarian follicle development associated with egg production in chicken.

LncRNA LINC00858 enhances cervical cancer cell growth through miR-3064-5p/ VMA21 axis.

Cervical cancer (CC) is the most common form of gynecological malignancy. Long intergenic non-protein coding RNA 858 (LINC00858) has been identified to participate in multiple cancers. However, the role and mechanism of LINC00858 in CC cells are still elusive. The aim of this study is to explore the biological functions and mechanisms of LINC00858 in CC cells. RT-qPCR analysis was used to examine the expression of LINC00858 in CC cells. EdU and colony formation assay were utilized to assess cell proliferation. TUNEL assay and flow cytometry assay were conducted to assess cell apoptosis. The mechanism regarding LINC00858 was certified through RNA pull down, RIP and luciferase reporter assays. The up-regulated LINC00858 was detected in CC cells. Reduction of LINC00858 effectively subdued CC cells proliferation and stimulated cell apoptosis. LINC00858 was determined to bind with miR-3064-5p and up-regulate VMA21 in CC cells. In rescue assays, miR-3064-5p down-regulation and VMA21 up-regulation were able to counteract the effect caused by LINC00858 decrease on CC cell proliferation and apoptosis. LINC00858 enhances cell proliferation, while restraining cell apoptosis in CC through targeting miR-3064-5p/VMA21 axis, implying that LINC00858 may serve as a promising therapeutic target for CC.

Why SNP rs3755955 is associated with human bone mineral density? A molecular and cellular study in bone cells.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p < 0.01), impeded cell proliferation (p < 0.05), stimulated cell apoptosis (p < 0.001) and decreased ALP enzyme activity, as compared with that of wild-type IDUA transfection. In RAW264.7 cells, transfection with variant-type IDUA significantly inhibited cell apoptosis (p < 0.01), promoted osteoclastic precursor cell migration (p < 0.0001), growth (p < 0.01), osteoclastic gene expression (TRAP, RANK, Inte-αv and Cath-K) (p < 0.05) and TRAP enzyme activity (p < 0.001), as compared with that of wild-type IDUA transfection. In both hFOB and RAW264.7 cells, the total protein and IDUA protein-specific phosphorylation levels were significantly reduced by variant IDUA transfection, as compared with that of wild-type IDUA transfection (p < 0.05). Variant allele A of phosSNP rs3755955 in IDUA gene regulates protein phosphorylation, inhibits osteoblast function and promotes osteoclastic activity. The SNP rs3755955 could alter IDUA protein phosphorylation, significantly regulates human osteoblastic and osteoclastic gene expression, and influences the growth, differentiation and activity of osteoblast and osteoclast, hence to affect BMD.

RETRACTED ARTICLE: Anti-breast Cancer Activity of Co(II) Complex by Inhibiting Cell Viability and Stimulating Cell Apoptosis

RETRACTED ARTICLE: Anti-breast Cancer Activity of Co(II) Complex by Inhibiting Cell Viability and Stimulating Cell Apoptosis

Knockdown of lncRNA C5orf66-AS1 inhibits osteosarcoma cell proliferation and invasion via miR-149-5p upregulation.

Osteosarcoma (OS) is the most common primary malignant bone tumor in the pediatric age group. Despite the various potential treatments for OS, the cure rate of patients with OS remains very low. An increasing number of long non-coding RNAs (lncRNAs) have been identified as key regulators of the progression of malignant human tumors. However, the biological functions of the lncRNA C5orf66-antisense 1 (C5orf66-AS1) in OS are yet to be fully elucidated. The present study aimed to investigate the functions and underlying mechanisms of C5orf66-AS1 in OS tissues and cell lines. Expression levels of C5orf66-AS1 and microRNA (miRNA/miR)-149-5p in tissues from patients with OS and OS cells lines were evaluated using reverse transcription quantitative (RT-q)PCR. The miRNA target interaction between C5orf66-AS1 and miR-149-5p was predicted and verified using StarBase and dual-luciferase reporter assays. Cell viability, migration, invasion and apoptosis were analyzed using Cell Counting Kit-8, Transwell assays and flow cytometry, respectively. In addition, the expression levels of migration- and apoptosis-associated proteins [matrix metalloproteinase-9 (MMP-9), Bcl-2 and Bax] were determined using western blotting and RT-qPCR. The results demonstrated that C5orf66-AS1 was significantly upregulated and miR-149-5p was significantly downregulated in OS tissues and cells (MG63 and U2OS). Bioinformatics analysis further confirmed that miR-149-5p could directly bind to C5orf66-AS1. Furthermore, it was revealed that C5orf66-AS1 negatively regulated the expression of miR-149-5p in OS cells, as confirmed by the inhibition of C5orf66-AS1 expression and miR-149-5p upregulation in cells transfected with small interfering (si RNA targeting C5orf66-AS1. In addition, C5orf66-AS1 silencing significantly inhibited the proliferation, invasion and migration of U2OS cells, and stimulated cell apoptosis. These findings were reversed using miR-149-5p inhibitor. Increased Bax expression and decreased Bcl-2 and MMP-9 expression were also observed in C5orf66-AS1-siRNA transfected U2OS cells, compared with the control group. In summary, the results from the present study indicated that C5orf66-AS1 knockdown inhibits OS cell proliferation and invasion via the upregulation of miR-149-5p. This findings may provide a promising novel target for the treatment of OS.

Camostat mesilate inhibits pro-inflammatory cytokine secretion and improves cell viability by regulating MFGE8 and HMGN1 in lipopolysaccharide-stimulated DF-1 chicken embryo fibroblasts.

Camostat mesilate (CM) possesses potential anti-viral and anti-inflammatory activities. However, it remains unknown whether CM is involved in lipopolysaccharide (LPS)-mediated inflammatory responses and cell injury. In this project, differentially expressed proteins (DEPs, fold change ≥ 1.2 or ≤ 0.83 and Q value ≤ 0.05) in response to LPS stimulation alone or in combination with CM were identified through tandem mass tags (TMT)/mass spectrometry (MS)-based proteomics analysis in DF-1 chicken embryo fibroblasts. The mRNA expression levels of filtered genes were determined by RT-qPCR assay. The results showed that CM alleviated the detrimental effect of LPS on cell viability and inhibited LPS-induced TNF-α and IL-6 secretions in DF-1 chicken embryo fibroblasts. A total of 141 DEPs that might be involved in mediating functions of both LPS and CM were identified by proteomics analysis in DF-1 chicken embryo fibroblasts. LPS inhibited milk fat globule EGF and factor V/VIII domain containing (MFGE8) expression and induced high mobility group nucleosome binding domain 1 (HMGN1) expression, while these effects were abrogated by CM in DF-1 chicken embryo fibroblasts. MFGE8 knockdown facilitated TNF-α and IL-6 secretions , reduced cell viability, stimulated cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. HMGN1 loss did not influence TNF-α and IL-6 secretions, cell viability, and cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. In conclusion, CM exerted anti-inflammatory and pro-survival activities by regulating MFGE8 in LPS-stimulated DF-1 chicken embryo fibroblasts, deepening our understanding of the roles and molecular basis of CM in protecting against Gram-negative bacteria.

Functional miR-142a-3p Induces Apoptosis and Macrophage Polarization by Targeting tnfaip2 and glut3 in Grass Carp (Ctenopharyngodon idella).

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.

SNHG17 Serves as an Oncogenic lncRNA by Regulating the miR-361-3p/STC2 Axis in Rectal Cancer

Long noncoding RNA (lncRNA) have been reported to be crucial regulators for carcinogenesis, including rectal cancer. This work aimed to explore the roles and associated mechanisms of small nucleolar RNA host gene 17 (SNHG17) in rectal cancer. A quantitative real-time polymerase chain reaction was performed to measure the expression level of SNHG17 in rectal cancer tissues and cells. Cell counting kit-8 (CCK-8) assay and flow cytometry assay were conducted to measure the biological roles of SNHG17 in rectal cancer. In addition, luciferase activity reporter assay, RNA immunoprecipitation (RIP) assay, and rescue experiments were conducted to explore the mechanisms of SNHG17 in rectal cancer. The upregulation status of SNHG17 was identified in rectal cancer tissues and cells. Functionally, knockdown the expression of SNHG17 inhibits rectal cancer cell proliferation via stimulating cell apoptosis. In vivo assay showed that the knockdown of SNHG17 inhibits tumor growth. Furthermore, we showed that microRNA-361-3p (miR-361-3p) has decreased expression in tumor tissues and cells, and SNHG17 functions as a sponge for miR-361-3p. The upregulation status of stanniocalcin 2 (STC2) was also found in rectal cancer, and the knockdown of STC2 hinders cancer progression. In conclusion, lncRNA SNHG17 functions as an oncogenic lncRNA in rectal cancer by regulating the miR-361-3p/STC2 axis.

Design BH3 domain fusion protein as targeting pro-apoptotic self-assembling nanoparticles

Cancer is a serious global health issue, and apoptosis is a logical and practical cancer therapeutic strategy. Apoptosis responses to internal and external signals. Both BH3 domain in the pro-apoptotic proteins and truncated BH3 domain can stimulate cell apoptosis. However, the faults of peptides in systemic administration restrict the applications of truncated BH3 domain. Ferritin, as an attractive nanoparticle with the capacity of self-assemble to unique hollow spherical structure, could display truncated BH3 domain an N-terminal. Thus, in this study, we designed a pro-apoptosis self-assembling protein nanoparticle by BH3 domain fusion at N-terminal of ferritin. We evaluated the size, cytotoxicity and pro-apoptosis effect of these nanoparticles. The results showed that RGD-BH3-HFn, BH3-HFn and HFn had uniformly spherical structure with sizes at 26.08 ± 0.11 nm, 22.07 ± 0.67 nm, and 16.81 ± 0.88 nm, respectively; RGD-BH3-HFn has stronger cytotoxicity against tumor cells than BH3-HFn and HFn. The total apoptosis ratios (including necrosis) of C6 cells induced by RGD-BH3-HFn, BH3-HFn, and HFn proteins were 15.24%, 10.13% and 2.14%, respectively; those of bEnd.3 cells were 15.47%, 7.33% and 1.70%, respectively; while the total apoptosis rate (including necrosis) of MCF-7 cells were 3.24%, 4.9% and − 1.68%, respectively. The results suggested self-assembling RGD-BH3-HFn could target to C6 cells and bEnd.3 cells, and enhance tumor cells apoptosis, its apoptosis effect against C6 cells was 7.11-fold that of HFn, and apoptosis effect against bEnd.3 cells was 9.08-fold that of HFn. These results indicated BH3 domain can be designed as targeting pro-apoptotic nanoparticles.

Long non-coding RNA FAM201A promotes lung squamous cell carcinoma progression through interaction with miR-101.

This study aimed to investigate the mechanism of LncRNA FAM201A mediating lung squamous cell carcinoma progression through interaction with miR-101. NCI-H520 cells and SK-MES-1 cells were transfected with miRNA-101-mimics and miRNA-101-inhibitor, the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect FAM201A and miR-101 expression. CCK-8, Wound healing assay and transwell assay were utilized to detect the influence of FAM201A on the malignancy of NCI-H520NCI-H520 and SK-MES-1SK-MES-1 cells. Cell apoptosis was determined by flow cytometry. The underlying pathways of FAM201A were measured using Western blot. Xenograft tumor experiments were conducted to detect tumor growth and metastasis in vivo.NCI-H520SK-MES-1 Kaplan-Meier method calculated patient survival. (1) Silencing of FAM201A inhibited the proliferation, migration and invasion of NCI-H520 and SK-MES-1cells and stimulated cell apoptosis significantly. Furthermore, FAM201A elimination hindered tumor growth and metastasis in vivo. (2) Compared with the si-control group, the protein expression of Ki67, Vimentin, Cleaved-caspase-3 and N-cadherin were decreased in the si-FAM201A group. (3) After transfection of miR-101-mimics, the expression level of Vimentin protein was significantly increased, while the expression level of Vimentin protein was significantly decreased after miR-101-inhibitor transfection. (4) MiR-101 mimics could alleviate FAM201A silencing-induced inhibitive effects on cell proliferation, migration, invasion and promotive effects on cell apoptosis. FAM201A could target miR-101 and upregulate Vimentin to inhibit lung cancer progression. FAM201A was expected to be a potential biomarker and therapeutic target for lung cancer.

Colorectal Adenocarcinoma Cell Culture in a Microfluidically Controlled Environment with a Static Molecular Gradient of Polyphenol.

To study the simultaneous effect of the molecular gradient of polyphenols (curcumin, trans-resveratrol, and wogonin) and biological factors released from tumor cells on apoptosis of adjacent cells, a novel microfluidic system was designed and manufactured. The small height/volume of microfluidic culture chambers and static conditions allowed for establishing the local microenvironment and maintaining undisturbed concentration profiles of naturally secreted from cells biochemical factors. In all trials, we observe that these conditions significantly affect cell viability by stimulating cell apoptosis at lower concentrations of polyphenols than in traditional multiwell cultures. The observed difference varied between 20.4–87.8% for curcumin, 11.0–37.5% for resveratrol, and 21.7–62.2% for wogonin. At low concentrations of polyphenols, the proapoptotic substances released from adjacent cells, like protein degradation products, significantly influence cell viability. The mean increase in cell mortality was 38.3% for microfluidic cultures. Our research has also confirmed that the gradient microsystem is useful in routine laboratory tests in the same way as a multiwell plate and may be treated as its replacement in the future. We elaborated the new repetitive procedures for cell culture and tests in static gradient conditions, which may become a gold standard of new drug investigations in the future.

LncRNA DLEU1 Contributes to the Growth and Invasion of Colorectal Cancer via Targeting miR-320b/PRPS1.

Growing evidences suggest that long non-coding RNAs (lncRNAs) are closely correlated to the development of human cancer, such as colorectal cancer (CRC). A previous report suggested that DLEU1 accelerated CRC development. However, DLEU1’s underlying mechanism in CRC remains unclear. In our study, the level of DLEU1 in CRC tissues is investigated by qRT-PCR. Our data exhibited that DLEU1 level was observably increased in CRC tissues and CRC cell lines and was closely associated with bad prognosis of CRC patients. CRC cell proliferation was repressed by sh-LncRNA DLEU1, whereas cell apoptosis was markedly stimulated. Moreover, knockdown of DLEU1 inhibited cell migration and invasion. Mechanistically, through interacting with miR-320b in CRC, DLEU1 promoted the level of PRPS1 which was a target of miR-320b. The rescue experiment confirmed that knockdown of DLEU1 repressed cell proliferation, migration and invasion while stimulated cell apoptosis via miR-320b/phosphoribosyl pyrophosphate synthetase 1 (PRPS1) axis. Meanwhile, the data of xenograft model exhibited that inhibition of DLEU1 suppressed tumor growth in vivo. In summary, DLEU1 knockdown may repress PRPS1 expression via miR-320b, and then repress cell proliferation, migration and invasion while stimulate cell apoptosis. Our research may provide a novel target for the treatment of CRC.

LINC00665 activates Wnt/β-catenin signaling pathway to facilitate tumor progression of colorectal cancer via upregulating CTNNB1

BackgroundLINC00665 is a newly identified oncogene, which has been reported to be oncogene in various cancers. Nevertheless, its role in the progression of colorectal cancer (CRC) remains obscure to the extent. This study aimed at exploring the role and mechanism of LINC00665 in CRC progression.Materials and methodsRNA and protein expression were detected via qRT-PCR and western blot. Functional assays were conducted to investigate the role of LINC00665 in the CRC cellular processes. TOP/FOP assay was performed to detect the activity of Wnt/β-catenin signaling pathway. Mechanism investigations were carried out to explore the regulatory relationship among genes.ResultsLINC00665 was overtly expressed in CRC cell lines at high levels. Functionally, silencing of LINC00665 could curb in vitro CRC cell growth, migration and invasion, while stimulating cell apoptosis. Mechanically, LINC00665 sponged miR-214-3p to up-regulate CTNNB1 expression, consequently activating Wnt/β-catenin signaling pathway. Furthermore, LINC00665 could bind to U2AF2 and enhance the association between U2AF2 and CTNNB1, increasing the stability of CTNNB1. CTNNB1 overexpression could reverse the suppressive effects of LINC00665 downregulation.ConclusionLINC00665 stimulates CRC progression through the activation of Wnt/β-catenin signaling pathway, which hopefully might be a therapeutic target for CRC.

Long Intergenic Non-Coding RNA LINC00885 Promotes Tumorigenesis of Cervical Cancer by Upregulating MACC1 Expression Through Serving as a Competitive Endogenous RNA for microRNA-432-5p.

PurposeLong intergenic non-protein coding RNA 885 (LINC00885) has been well studied in breast cancer; however, its contribution in cervical cancer remains unclear. In this study, we aimed to determine the detailed functions of LINC00885 in cervical cancer and elucidate the underlying molecular regulation mechanism.MethodsThe expression status of LINC00885 in cervical cancer was determined using reverse transcription-quantitative polymerase chain reaction and by searching The Cancer Genome Atlas database. The detailed functions of LINC00885 in cervical cancer cells were confirmed using Cell Counting Kit 8 assay, flow cytometry analysis, Transwell cell migration and invasion assays, and tumor xenograft assay. Mechanistic experiments included bioinformatics prediction, RNA immunoprecipitation, luciferase reporter assay, and rescue experiments.ResultsLINC00885 was clearly overexpressed in cervical cancer, which was linked with unfavorable clinical outcomes. Functionally, LINC00885 deficiency suppressed cervical cancer cell proliferation, migration, and invasion but stimulated cell apoptosis in vitro. Furthermore, loss of LINC00885 restricted the growth of cervical cancer cells in vivo. Mechanistically, LINC00885 functioned as a competitive endogenous RNA for microRNA-432-5p (miR-432-5p) in cervical cancer. Furthermore, metastasis-associated colon cancer 1 (MACC1) was confirmed as the direct target of miR-432-5p, and LINC00885 could enhance MACC1 expression by sequestering miR-432-5p. Rescue experiments revealed that silencing of miR-432-5p or upregulation of MACC1 expression could effectively counteract the restrained aggressive properties of cervical cancer cells induced by LINC00885 deficiency.ConclusionLINC00885 upregulated MACC1 expression in cervical cancer cells by sponging miR-432-5p, thereby promoting cancer progression. The LINC00885/miR-432-5p/MACC1 pathway may help in the identification of potential prognostic biomarkers and therapeutic targets in cervical cancer.

Serum Exosomal miRNA-1226 as Potential Biomarker of Pancreatic Ductal Adenocarcinoma.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality and it is urgent to find biomarkers for early detection of PDAC. Exosomal miRNAs are useful biomarkers for cancer detection. The aims of this study were to investigate the potential role of serum exosomal miRNA in detection of PDAC and to analyze the correlation between the levels of exosome miRNA and the tumor biological behaviors.Materials and MethodsThirteen serum samples were collected from five patients with PDACs, three healthy individuals (HIs) and five benign pancreatic lesions (BP) for a high throughput profiling analysis to identify an altered miRNA expression patterns in PDAC. Candidate exosomal miRNAs were filtered based on a second independent cohort that included 17 PDACs and 12 benign pancreatic lesions by quantitative real-time polymerase chain reaction (qRT-PCR). Four miRNAs were selected for miRNA validation as PDAC biomarkers in a subsequent set of samples. The association between candidate exosomal miRNA and tumor behavior (tumor invasion or metastases) was evaluated in 17 PDACs. In vitro studies were performed to evaluate the role of candidate exosomal miRNA on cell viability, apoptosis and cell migration in two PDAC cell lines.ResultsThe expression of 11 miRNAs showed same trend between PDAC and BP, and between PDAC and HIs. Six of them were upregulated (miR-203b-5p, miR-342-5p, miR-337-5p, miR-149-5p, miR-877-5p, miR-203a-3p), and five were downregulated (miR-1226-3p, miR-3182, miR-625-3p, miR-624-5p, miR-664a-5p). miR-1226-3p was selected as the candidate exosomal biomarker for the PDAC detection. The expression of serum exosomal miRNA-1226-3p was downregulated in PDACs compared to the BPs (p = 0.025). miR-1226-3p had acceptable performance in predicting [area under the curve (AUC) = 0.74] PDAC. Exosomal miRNA-1226-3p level in PDAC with invasion or metastases was lower than that without invasion or metastases (p = 0.028). Transfection of miRNA-1226-3p significantly inhibited the proliferation of PANC-1 and BXP-3 cells, stimulated cell apoptosis and inhibited cell migration.ConclusionSerum exosomal miRNA-1226-3p is a potential biomarker in diagnosing and predicting the tumor invasion or metastases of PDAC.