HeLa Cell mRNA Research Articles

Expression profile screening and bioinformatics analysis of CircRNA, LncRNA, and mRNA in HeLa cells infected with Chlamydia muridarum.

We have previously shown that circRNAs in host cells are involved in the process of Chlamydia trachomatis infection. In this study we aimed to identify significantly altered circRNAs/lncRNAs/mRNAs in Chlamydia muridarum infected cells and investigate their biological functions in the interaction between Chlamydia muridarum and host cells. For this purpose, circRNA, lncRNA and mRNA expression profiles were screened and identified in HeLa cells with or without Chlamydia muridarum infection by microarray. Bioinformatics analyses including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis were then carried out and the circRNA-miRNA ceRNA network was constructed. The differentially expressed circRNAs and lncRNAs were selected for validation by RT-qPCR. The results shown that a total of 834 circRNAs, 2149 lncRNAs and 1283 mRNAs were found to be differentially expressed. Enrichment analysis of GO and KEGG showed that the dysregulated genes involved nuclear-transcribed mRNA catabolic process, protein binding, RNA catabolic process and translation, the MAPK signaling pathway, apoptosis, Toll-like receptor signaling pathway, cAMP signaling pathway and Notch signaling pathway may play important roles in Chlamydia infection. Our study provides a systematic outlook on the potential function of non-coding RNAs in the molecular basis of Chlamydia infection.

Live-Cell Imaging of Survivin mRNA by Using a Dual-Color Surface-Cross-Linked Nanoquencher.

Precise detection of cancer-related mRNAs can significantly benefit the early diagnosis and potential therapy of cancers. Herein, we report organic dark quencher-encapsulated surface-cross-linked micelles (qSCMs) as a new sort of nanoquencher for construction of potential multiple-color fluorescence imaging nanosensors. Such nanoquenchers featured simple preparation (one-pot), broad-spectrum quenching (450-800 nm), high quenching efficiency (>94%), good stability, negligible cargo leakage, facile covalent surface modification, and finally excellent modularity. As a proof-of-concept demonstration, a mRNA-detecting qSCM nanosensor was generated, capable of simultaneous live-cell imaging of endogenous actin mRNA (a house-keeping gene) and cancer-related survivin mRNA. This nanosensor was found to be GSH- and DNase I-resistant, and with actin mRNA as an intrinsic reference, it was used to image the precise survivin mRNA expression across different mammalian cells through convenient normalization of the signal readouts. Moreover, the nanosensor was further used to quantitatively image the downregulation of endogenous survivin mRNA in HeLa cells upon treatment of YM155 (an imidazolium bioactive compound known to suppresses endogenous survivin mRNA expression). These results clearly demonstrated the promising application of these qSCMs as new nanoquenchers in potential multicolor imaging of various endogenous biomarkers.

Pseudorabies virus UL24 antagonizes OASL-mediated antiviral effect

Oligoadenylate synthetases-like (OASL) protein exerts various effects on DNA and RNA viruses by inhibiting cGAS-mediated IFN production and by enhancing RIG-I-mediated IFN induction, respectively. In this study, we aimed to examine the role of OASL in pseudorabies virus (PRV) proliferation and investigate the function of the PRV UL24 protein in cellular innate immunity. We found that OASL regulates PRV proliferation by enhancing RIG-I signaling. PRV infection decreased the expression of OASL at both the mRNA and protein levels in PK15 and HeLa cells. OASL expression suppressed the proliferation of PRV in a RIG-I-dependent manner and boosted RIG-I-mediated IFN expression as well as IFN-stimulated gene (ISG) induction. In contrast, knockdown of OASL enhanced PRV proliferation and reduced RIG-I signaling. However, the PRV UL24 protein was found to impair RIG-I signaling, thus inhibiting transcription of IFN and ISGs. In addition, the UL24 protein reduced RIG-I-induced expression of endogenous OASL in an IRF3-dependent manner, thereby antagonizing the OASL antiviral effect. Taken together, our findings characterize the role of OASL in PRV proliferation and provide new insights into the role of UL24 in PRV pathogenesis.

GRK2-Dependent HuR Phosphorylation Regulates HIF1α Activation under Hypoxia or Adrenergic Stress.

Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses.

Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method.

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3’-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.

MiR-181a Reduces Platelet Activation via the Inhibition of Endogenous RAP1B.

AimSince RAP1B is critical for platelet functions, including hemostasis, this study was conducted to identify RAP1B regulating microRNAs (miRNAs) in ex vivo stored platelets.BackgroundPrevious studies with platelets identified factors affecting RAP1B activity but regulatory miRNAs that affect RAP1B protein expression have not been reported.ObjectiveTo understand the functional significance of miRNA mediated regulation of RAP1B in stored platelets, using microRNA, miR-181a as an example.MethodsA Tagged RNA Affinity approach (MS2-TRAP) was employed to identify miRNAs that bound to the 3` untranslated region (3`UTR) of the RAP1B mRNA in HeLa cells as an assay system. And subsequently, the mRNA 3’UTR:miRNA interactions were verified in platelets through the ectopic expression of miR-181a mimic and appropriate controls. The interaction of such miRNAs with RAP1B mRNA was also validated by qRT-PCR and Western analysis.ResultsSixty-two miRNAs from MS2 assay were then compared with already known 171 platelet abundant miRNAs to identify a common set of miRNAs. This analysis yielded six miRNAs (miR-30e, miR-155, miR-181a, miR-206, miR-208a and miR-454), which are also predicted to target RAP1B mRNA. From this pool, miR-181a was selected for further study since RAP1B harbors two binding sites for miR-181a in its 3′UTR. Ectopic expression of miR-181a mimic in platelets resulted in lowering the endogenous RAP1B at both mRNA and protein levels. Further, miR-181a ectopic expression reduced the surface expression of the platelet activation marker, P-selectin.ConclusionMicroRNA-181a can target RAP1B and this interaction has the potential to regulate platelet activation during storage.

Involvement of p53-dependent apoptosis signal in antitumor effect of Colchicine on human papilloma virus (HPV)-positive human cervical cancer cells.

Colchicine, a plant-derived alkaloid with relatively low toxicity on normal human epidermal keratinocytes (HEKn), has selective inhibitory effect on the growth of CaSki (HPV16-positive) and HeLa (HPV18-positive) human cervical cancer cell lines via the induction of apoptosis. Colchicine (2.5, 5.0 and 10.0 ng/ml) significantly reduced the expression of human papilloma virus (HPV) 16 E6/E7 mRNA and protein in CaSki and HeLa cells. Moreover, reduced expression of E6 and E7 induced by Colchicine resulted in the up-regulation of tumor suppressor proteins, p53 and Rb, as well as down-regulation of phospho Rb (pRb) protein. In addition, Bax, cytosolic cytochrome c and cleaved caspase-3 protein were increased while Bcl-2 protein was decreased significantly by 48 h of Colchicine treatment. These results implied that Colchicine could be explored as a potent candidate agent for the treatment and prevention of HPV-associated cervical cancer without deleterious effects.

Effect of CASC19 on proliferation, apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression

Objective: To investigate the effects of cancer susceptibility candidate gene 19 (CASC19) regulating the expression of microRNA-449b-5p (miR-449b-5p) on the proliferation, apoptosis and radiation sensitivity of cervical cancer cells. Methods: (1) HeLa cells of cervical cancer cell line were cultured. HeLa cells were irradiated with X-ray at different doses (0, 2, 4, 6, 8 Gy, respectively), then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR. (2) HeLa cell proliferation, apoptosis, radiation sensitivity (expressed as a survival fraction) and its related protein expression included cyclin D1, cleaved-caspase-3, and histone variant H2AX (γ-H2AX) were examined after different treatment including silencing CASC19 expression, over-expressing miR-449b-5p, down-regulating miR-449b-5p and silencing CASC19 expression. (3) The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p. Results: (1) With the increase of X-ray irradiation different dose (0, 2, 4, 6, 8 Gy), the expression level of CASC19 mRNA in HeLa cells gradually increased (F=502.681, P=0.000), and the expression level of miR-449b-5p gradually decreased (F=202.936, P=0.000).(2) After silencing CASC19 expression or over-expressing miR-449b-5p, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression level of cyclin D1 protein was significantly reduced (P<0.05), and the expression levels of cleaved-caspase-3 and γ-H2AX protein were significantly increased (P<0.05). After down-regulating miR-449b-5p and silencing CASC19 expression, the survival rate of HeLa cells was significantly reduced (P<0.05), the apoptosis rate was significantly increased (P<0.05), the survival fraction was significantly reduced (P<0.05), the expression levels of cyclin D1 and γ-H2AX protein were significantly increased (P<0.05), and the expression level of cleaved-caspase-3 was significantly decreased (P<0.05). (3) Over expression of miR-449b-5p could significantly reduce the luciferase activity of CASC19 wild type (1.00±0.09 versus 0.37±0.05, P<0.01), but there were no significant effect on the luciferase activity of CASC19 mutant type (0.92±0.07 versus 0.94±0.05, P>0.05). After the expression of CASC19 was silenced, the expression level of miR-449b-5p in HeLa cells increased significantly (1.00±0.12 versus 4.84±0.49, P<0.05). After overexpression of CASC19, the expression level of miR-449b-5p in HeLa cells was significantly reduced (1.00±0.09 versus 0.38±0.04, P<0.05). Conclusion: CASC19 in HeLa cells negatively regulates the expression of miR-449b-5p, and down-regulating the expression of miR-449b-5p could partially reverse the effects of silencing CASC19 on HeLa cell proliferation, apoptosis and radiation sensitivity.

Rapid, facile, reagentless, and room-temperature conjugation of monolayer MoS2 nanosheets with dual-fluorophore-labeled flares as nanoprobes for ratiometric sensing of TK1 mRNA in living cells

Direct loading of fluorophore-labeled DNA molecules (named as flares) on gold nanoparticles (AuNPs) is a controllable and straightforward approach for intracellular imaging of target DNA molecules. However, the modification of AuNPs with flares requires a tedious and time-consuming procedure, additional reagents, or adenosine-rich DNA molecules. Here, we developed a rapid, simple, reagentless, and room-temperature approach for the modification of monolayer molybdenum disulfide nanosheets (M-MoS2 NSs) with dual-fluorophore-labeled flares, which were implemented for the ratiometric imaging of TK1 mRNA in living cells. The duplexes were prepared by hybridizing thiolated single-stranded DNA (ssDNA) to 6-carboxyfluorescein (FAM)- and 5-carboxytetramethylrhodamine (TAMRA)-labeled flares. The nanoflares were fabricated by conjugating the formed duplexes to the surface sulfur vacancy sites of the M-MoS2 NSs. The time required for preparing the nanoflares was found to be within 1 h. In the nanoflares, FAM stays away from TAMRA, leading to inefficient fluorescence resonance energy transfer (FRET). The presence of perfectly matched DNA (DNApm) molecules induces the liberation of the flares from the nanoflares. The liberated flares fold into hairpin-shaped structures, causing high FRET efficiency from FAM to TAMRA and efficient FAM-TAMRA static quenching. Following this mechanism, the nanoflares provided an effective platform for the ratiometric sensing of DNApm molecules with a limit of detection (at a signal-to-noise ratio of 3) of 8 nM and the linear range of 25-500 nM. Confocal microscopy experiments demonstrated that the nanoflares can be used to ratiometrically image TK1 mRA in HeLa and MCF-7 cells.

Effects of corticosteroid on mRNA levels of histamine H1 receptor in nasal mucosa of healthy participants and HeLa cells.

The purpose of this study is to examine the effect of intranasal corticosteroid (INCS) administration on histamine H1 receptor (H1R) gene expression in the nasal mucosa of healthy participants and the effects of dexamethasone on basal and histamine-induced H1R mRNA expression, and histamine-induced phosphorylation of extracellular signal-regulated kinase (ERK) in HeLa cells. Sixteen healthy participants were given INCS once daily for a week. After pretreatment of dexamethasone, HeLa cells were treated with histamine. Levels of H1R mRNA and phosphorylation of ERK were measured using real time PCR and immunoblot analysis, respectively. Levels of H1R mRNA in the nasal mucosa of healthy participants receiving INCS was significantly decreased. Dexamethasone suppressed basal levels of H1R mRNA, and histamine-induced up-regulation of H1R mRNA and ERK phosphorylation in HeLa cells. These data suggested that corticosteroid inhibited both basal transcription and histamine-induced transcriptional activation of H1R through its suppression of ERK phosphorylation in the signaling pathway involved in H1R gene transcription. It is further suggested that pre-seasonal prophylactic administration of INCS suppresses both basal and pollen-induced upregulation of H1R gene expression in the nasal mucosa of patients with pollinosis, leading to prevention of the exacerbation of nasal symptoms during peak pollen season. J. Med. Invest. 67 : 311-314, August, 2020.

The Human Papillomavirus (HPV) E1 protein regulates the expression of cellular genes involved in immune response

The Human Papillomavirus (HPV) E1 protein is the only viral protein with enzymatic activity. The main known function of this protein is the regulation of the viral DNA replication. Nevertheless, it has been demonstrated that the ablation of HPV18 E1 mRNA in HeLa cells promotes a deregulation of several genes, particularly those involved in host defense mechanisms against viral infections; however, the specific contribution of E1 protein in HPV-independent context has not been studied. The aim of this work was to determine the effect of the HPV E1 protein in the regulation of cellular gene expression profiles evaluated through RNA-seq. We found that E1 proteins from HPV16 and 18 induced an overexpression of different set of genes associated with proliferation and differentiation processes, as well as downregulation of immune response genes, including IFNβ1 and IFNλ1 and Interferon-stimulated gene (ISG), which are important components involved in the antiviral immune response. Together, our results indicate that HR-(High-Risk) and LR-(Low-Risk) HPV E1 proteins play an important role in inhibiting the anti-viral immune response.

MiR-29a function as tumor suppressor in cervical cancer by targeting SIRT1 and predict patient prognosis.

IntroductionCervical cancer is the second most frequently malignant tumors in females and metastasis is a challenge of the treatment of cervical cancer. MiR-29a is usually low expressed in several tumors and its functions in cervical cancer remain unclear.Patients and methodsThe quantitative real-time polymerase chain reaction was employed to assess the expression of miR-29a and the Sirtuin-1 (SIRT1). Cell metastatic ability was assessed using Transwell and Western blot assays. The dual-luciferase reporter assay was performed to verify that miR-29a targeted to the 3’-untranslated region (UTR) of SIRT1 mRNA.ResultsMiR-29a was low expressed in cervical cancer and downregulation of miR-29a was associated with poor outcome. MiR-29a regulated the expression of SIRT1 by targeting to its 3’-UTR of mRNA in HeLa cells. SIRT1 was upregulated in cervical cancer tissues and cells in comparison with the non-tumor tissues and normal cells. Upregulation of SIRT1 predicted worse outcome of cervical cancer patients. MiR-29a was participated in the migration, invasion and epithelial–mesenchymal transition (EMT) in cervical cancer through directly targeting to the 3’-UTR of SIRT1 mRNA. SIRT1 reversed partial roles of miR-29a on metastasis in cervical cancer.ConclusionmiR-29a suppressed migration, invasion and EMT by directly targeting to SIRT1 in cervical cancer. The newly identified miR-29a/SIRT1 axis provides novel insight into the pathogenesis of cervical cancer.

MicroRNA-101-5p Suppresses the Expression of the Ras-Related Protein RAP1A.

MicroRNAs (miRs) are small, non-coding RNAs that negatively regulate gene expression. The stem-loop sequence miR-101-1 generates mature miR-101-5p and miR-101-3p. The function and target mRNA of miR-101-5p have not yet been elucidated in detail. Here, we demonstrate that miR-101-5p inhibits the expression of RAP1A, a member of the RAS gene family. Transfection of a miR-101-5p mimic significantly inhibited the expression of RAP1A mRNA in HeLa, HEK293, A549, and COLO201 cells. The same treatment significantly inhibited cell proliferation. The cytostatic effect with transfection of miR-101-5p was antagonized by treatment with the RAP inhibitor salirasib. These results suggested that miR-101-5p inhibits RAP1A, and thus, the expression levels of miR-101-5p regulate cell proliferation.

Visualizing mRNA in live mammalian cells.

mRNAs play essential role in gene expression and regulation. Direct visualization of specific intracellular mRNAs can provide key information about mRNA distribution and dynamics that accelerating study of their functionality. This highlights the necessity of characterizing mRNA in mammalian cells with good spatial and temporal resolution. Live cell imaging and tracking of mRNA remains a challenge due to their relatively short half-life and lack of versatile imaging probes. This review provides an overview of current methods of live-cell mRNA imaging, including exogenous hybridization probes and genetically encoded strategies. It then focuses on the development and application of aptamer initiated fluorescence complementation (AiFC) method for real-time imaging of mammalian mRNAs and includes a detailed protocol for observing β-actin mRNA in HeLa cells.

Augmented cytotoxic effects of paclitaxel by curcumin induced overexpression of folate receptor-α for enhanced targeted drug delivery in HeLa cells

BackgroundNew targeted therapies are intended to minimize the toxic effects and maximize destruction of tumor cells. Folate is a membrane-bound receptor that plays a vital role in the uptake of anti-folate molecules aimed for efficient drug delivery of anti-folate drugs. PurposeThe present study is aimed at the modulation of the expression of folate receptor by curcumin that enhances the intake, cytotoxicity and anticancer effects of paclitaxel in HeLa cells. Materials and methodsHeLa cells were pretreated with curcumin and treated with paclitaxel. We measured the cell viability, uptake of radiolabelled folic acid and paclitaxel, Folate receptor –alpha (FR-α) protein expression by immunocytochemistry and western blot and FR-α mRNA expression by qualitative and quantitative analysis. ResultsThis study shows that curcumin (10 - 50 µM) causes significantly increased cytotoxicity in a dose and time dependent manner. It also enhances the intake of radiolabeled folic acid and paclitaxel 3–4 folds in HeLa cells. The pretreatment of HeLa cells with curcumin shows statistically significant of cell death by paclitaxel. The quantitative RT-PCR demonstrates the expression of FR- α mRNA upon curcumin treatment. Furthermore, immunochemistry and western blotting analysis proved that curcumin enhances expression the FR- α in HeLa cells. ConclusionOur study proved that the molecular mechanism of curcumin enhances the upregulation of FR – α mRNA and protein expression in HeLa cells. Therefore, a combination of curcumin and paclitaxel at less concentration may be a targeting strategy for FR-targeted drug delivery providing a better therapeutic intervention of cancer.

MiR-501 is upregulated in cervical cancer and promotes cell proliferation, migration and invasion by targeting CYLD

BackgroundCervical cancer is the common gynecological deadly malignancy worldwide. Here we attempted to evaluate the effects and mechanisms of microRNA-501-5p (miR-501) on the cell proliferation, migration, invasion and the clinical significance in the cervical cancer. MethodsCervical cancer HeLa cells were transfected with miR-501 mimic or inhibitor or siRNA against Cylindromatosis (CYLD) using Lipofectamine 2000. miR-501 expression was assessed in HeLa cells and cervical cancer specimens by real-time qRT-PCR. The functional roles of miR-501 were determined by CCK-8, colony formation, scratch wound healing and transwell assays. The apoptosis rate was detected by flow cytometry assay. CYLD, BCL-2, BAX, NF-κB p65 and phosphorylated p65 (p-p65) proteins were examined by Western blotting. CYLD expression was evaluated by immunohistochemistry in cervical cancer tissues. ResultsmiR-501 was upregulated, whereas CYLD protein was downregulated in cervical cancer tissues compared to normal cervical tissues. miR-501 overexpression and CYLD protein downregulation were positively correlated with poor differentiation, tumor size, International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. CYLD was downregulated by miR-501 at both mRNA and protein levels in HeLa cells. miR-501 promoted cell proliferation, migration and invasion in cervical cancer, while inhibited the apoptosis. This is possibly due to the downregulation of CYLD and subsequent activation of NF-κB p65. ConclusionsmiR-501 upregulation and CYLD downregulation are associated with the development and progression of cervical cancer. miR-501 promotes cervical cancer cell proliferation, migration and invasion possibly via downregulating CYLD and subsequently activating NF-κB p65. miR-501 might be a potential therapeutic target for cervical cancer.

MiR-128 inhibits telomerase activity by targeting TERT mRNA.

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes.

WITHDRAWN: The TLR4/MyD88/NF-κB pathway promotes infl ammation in HPV-related cervical cancer cells

// Ninghong Jiang 1,2 , Feng Xie 1,2 , Qisang Guo 1,2 , Limei Chen 1,2 , Fang Chen 1,2 , Luopei Guo 1,2 and Long Sui 1,2 1 Medical center for diagnostics and treatments of cervical disease, Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China 2 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China Correspondence to: Long Sui, email: suilong@fudan.edu.cn Keywords : cervical carcinoma; HPV; TLR4/NF-κB signaling pathway; infl ammation; tumor promotion Received: July 18, 2017 Accepted: January 02, 2018 Epub: January 08, 2018 Abstract Toll-like receptor 4 (TLR4) promotes proinfl ammatory cytokine production and activation of innate immunity. It is also highly expressed in human papillomavirus (HPV)-related cervical cancer. In our study, we investigated the role of the TLR4/MyD88/NF-κB pathway in HPV-related cervical cancer. RT-qPCR and western blotting were used to detect mRNA and protein levels in HeLa and CaSki cells. Levels of IL-6, IL-8, IL-1β, VEGF, TGF-β1, IFN-α, MIP-3α and TNF-α in cell supernatants were detected using ELISAs. Expression levels of MyD88, IRAK1, IRAK4, TRAF-6 and MAPK as well as phosphorylation of IKKα/IKKβ, IκBα and p65 were all higher than control in LPS-stimulated groups of HeLa and CaSKi cells. By contrast, lower expression was detected in groups transfected with shRNA targeting TLR4, though expression of IKKα/IKKβ, IκBα and p65 remained unchanged. Levels of COX-2, iNOS,MIP-3α, IL-6, IL-8, VEGF and TGF-β1 were higher in LPS-stimulated than in negative control groups, but were lower in cells transfected with TLR4-shRNA.These data suggest the TLR4/MyD88/NF-κB pathway promotes expression of proinfl ammatory cytokines in HPVrelated cervical cancer cells, which involves in the formation of microenvironment for tumor development. Targeting the TLR4/MyD88/NF-κB pathway can provide a rationale for treatment of HPV-related cervical cancer.

PCC0208009 enhances the anti-tumor effects of temozolomide through direct inhibition and transcriptional regulation of indoleamine 2,3-dioxygenase in glioma models.

Indoleamine 2,3-dioxygenase (IDO), which is highly expressed in human glioblastoma and involved in tumor immune escape and resistance to chemotherapy, is clinically correlated with tumor progression and poor clinical outcomes, and is a promising therapeutic target for glioblastoma. IDO inhibitors are marginally efficacious as single-agents; therefore, combination with other therapies holds promise for cancer therapy. The aim of this study was to investigate the anti-tumor effects and mechanisms of the IDO inhibitor PCC0208009 in combination with temozolomide. The effects of PCC0208009 on IDO activity inhibition, and mRNA and protein expression in HeLa cells were observed. In the mouse glioma GL261 heterotopic model, the effects of PCC0208009 on l-kynurenine/tryptophan (Kyn/Trp), tumor growth, flow cytometry for T cells within tumors, and immunohistochemistry for IDO and Ki67 were examined. In the rat glioma C6 orthotopic model, animal survival, flow cytometry for T cells within tumors, and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and IDO were examined. The results show that PCC0208009 is a highly effective IDO inhibitor, not only directly inhibiting IDO activity but also participating in the gene regulation of IDO expression at the transcription and translation levels. PCC0208009 significantly enhanced the anti-tumor effects of temozolomide in GL261 and C6 models, by increasing the percentages of CD3+, CD4+, and CD8+ T cells within tumors and suppressing tumor proliferation. These findings indicate that PCC0208009 can potentiate the anti-tumor efficacy of temozolomide and suggest that combination of IDO inhibitor-based immunotherapy with chemotherapy is a potential strategy for brain tumor treatment.

Pre-mRNA processing includes N6 methylation of adenosine residues that are retained in mRNA exons and the fallacy of "RNA epigenetics".

By using a cell fraction technique that separates chromatin-associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown in a previous study that residues in exons are methylated (m6A) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called “epigenetic” demethylation. The turnover rate of mRNA molecules is faster, depending on m6A content in HeLa cell mRNA, suggesting that specification of mRNA stability may be the major role of m6A exon modification. In mouse embryonic stem cells (mESCs) lacking Mettl3, the major mRNA methylase, the cells continue to grow, making the same mRNAs with unchanged splicing profiles in the absence (>90%) of m6A in mRNA, suggesting no common obligatory role of m6A in splicing. All these data argue strongly against a commonly used “reversible dynamic methylation/demethylation” of mRNA, calling into question the concept of “RNA epigenetics” that parallels the well-established role of dynamic DNA epigenetics.