Silenced Cell Lines Research Articles

Silencing Exosomal circ102927 Inhibits Foot Melanoma Metastasis via Regulating Invasiveness, Epithelial-Mesenchymal Transition and Apoptosis.

Exosomes contain abundant circular RNAs (circRNAs), playing an important role in intercellular communication. However, the function and underlying molecular mechanism of exosomal circRNAs in foot metastatic melanoma remain unclear. Twelve differentially expressed exosomal circRNAs between patients with metastatic and primary foot melanoma were screened through high-throughput sequencing, and their expression levels were detected by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). CircRNA102927 silencing and overexpression A2058 cell line was constructed, and the effects of circRNA102927 on cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) were assessed using cell counting kit-8 (CCK-8), flow cytometry, wound healing, Transwell, and Western blot assays, respectively. Twelve differentially expressed exosomal circRNAs were screened and ROC curve showed that six circRNAs could be used as the diagnostic biomarkers for metastatic melanoma. Melanoma-secreted exosomes induced the differentiation of CD4+ T cells into Treg cells. CircRNA102927 was highly expressed in metastatic melanomas. Functionally, circRNA102927 silencing inhibited proliferation, EMT, migration, and invasion in metastatic melanoma cells, while promoting apoptosis. Meanwhile, overexpression of circRNA102927 had the opposite effects. Our investigation suggests that silencing exosomal circRNA102927 may suppress foot melanoma metastasis by inhibiting invasiveness, EMT and promoting apoptosis.

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

BackgroundBreast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.ResultsIn this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.ConclusionsOur results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients’ outcome.

Caspases compromise SLU7 and UPF1 stability and NMD activity during hepatocarcinogenesis

Background & AimsThe homeostasis of the cellular transcriptome depends on transcription and splicing mechanisms. Moreover, the fidelity of gene expression, essential to preserve cellular identity and function is secured by different quality control mechanisms including nonsense-mediated RNA decay (NMD). In this context, alternative splicing is coupled to NMD, and several alterations in these mechanisms leading to the accumulation of aberrant gene isoforms have been involved in human disease including cancer. MethodsRNAseq, Westen blot, qPCR, co-IP in multiple silenced culture cell lines (replicates n≥4), primary hepatocytes and samples of animal models (Jo2, APAP, Mdr2-/- mice, n≥3). ResultsHere we describe that in animal models of liver injury and in human HCC (TCGA, NT =50 vs HCC =374), the process of NMD is inhibited. Moreover, we demonstrate that the splicing factor SLU7 interacts with and preserves the levels of the NMD effector UPF1, and that SLU7 is required for a correct NMD. Our previous findings demonstrated that SLU7 expression is reduced in the diseased liver, contributing to hepatocellular de-differentiation and genome instability during disease progression. However, little was known about the mechanisms underlying SLU7 downregulation. Here we provide evidence showing that caspases activated during liver damage would be responsible of the cleavage and degradation of SLU7. ConclusionsHere we identify the downregulation of UPF1 and the inhibition of NMD as a new molecular pathway contributing to the malignant reshaping of the liver transcriptome. Moreover, and importantly, we uncover caspases activation as the mechanism responsible for the downregulation of SLU7 expression during liver disease progression, which is a new link between apoptosis an hepatocarcinogenesis. Impact and implicationsThe mechanisms involved in the reshaping of hepatocellular transcriptome driving the progressive loss of cell identity and function in liver disease are not completely understood. In this context, we provide evidence on the impairment of a key mRNA surveillance mechanism known as nonsense-mediated mRNA decay (NMD). Mechanistically, we uncover a novel role for the splicing factor SLU7 in the regulation of NMD, including its ability to interact and preserve the levels the key NMD factor UPF1. Moreover, we demonstrate that the activation of caspases during liver damage mediates SLU7 and UPF1 proteins degradation and NMD inhibition. Our findings identify potential new markers of liver disease progression, and SLU7 as a novel therapeutic target to prevent the functional decay of the chronically injured organ.

Effect of trametinib and anti-PD-1 combo on tumor growth and survival of KRAS-mutant lung cancer by ID1 downregulation.

e14633 Background: Although anti-PD-1/PD-L1 antibodies represent a major breakthrough in NSCLC patients’ survival, this treatment efficacy is highly dependent on tumor PD-L1 expression. We reported the role of ID1 and the efficacy of a combined blockade of PD-1-ID1 in a KRAS mutant NSCLC mouse model (Baraibar et al; Cancers 2020). Trametinib, which is an oral MEK1/2 inhibitor, acts downstream of KRAS. Here we evaluate trametinib as an ID1 inhibitor able to enhance immunotherapy efficacy through PD-L1 overexpression. Methods: To explore whether MEK1/2 inhibition reduces ID1 expression in vitro and in vivo, experiments in NSCLC cells (human and murine) and in murine models were performed. RNAseq analysis was conducted to elucidate the pathways implicated in ID1 inhibition. Trametinib resistant (TR) cells were generated to analyze the effect of ID1 in the acquired resistance to treatment. Apoptosis was measured by flow cytometry. PD-L1 expression was evaluated by flow cytometry in control, ID1 silenced, ID1-overexpressing and TR cell lines. Treatment of trametinib combined with anti-PD1 was performed in KRAS-mutant LUAD models. Results: In vitro experiments in NSCLC cells showed ID1 inhibition at protein level after MEK1/2 blockade. However, we observe an increase in ID1 mRNA levels following treatment. In vivo experiments performed in C57BL6J and in ID1-deficient mice showed no significant differences in tumor growth between ID1 knock-out and ID1 wild-type mice treated with trametinib. IHC analysis of those tumor samples showed reduced ID1 levels (LLC p<0.001; CMT-167 p<0.0001). RNAseq analysis revealed an enrichment of ubiquitin-proteasome pathway after trametinib exposure. Blockade of proteasome activity with a specific inhibitor, restored ID1 levels. TR cells did not show MEKi induced ID1 inhibition, moreover, ID1 blockade rescued the sensitivity to trametinib in human and murine NSCLC TR cells (p<0.001). Flow cytometry analysis showed increased PD-L1 expression (p<0.001) in human and murine NSCLC cell lines after trametinib administration. In contrast, ID1-overexpressing cells and TR cells did not show PD-L1 upregulation after MEK inhibition. In vivo experiments confirmed the therapeutic efficacy of trametinib and anti-PD-1 combo, reducing tumor growth (p<0.05) and increasing mice survival (p<0.05), compared with trametinib monotherapy or immunotherapy alone. Conclusions: Pharmacological inhibition of MEK1/2 decreased ID1 expression in vitro and in vivo, replicating the effect of a specific shRNA against ID1, through proteasome activation. PD-L1 upregulation induced by ID1 blockade after trametinib treatment sensitized KRAS-mutant NSCLC cells to PD-1/PD-L1 immunotherapy, blocking tumor growth and improving overall survival.

Cell Cycle-Related FAM64A Could be Activated by TGF-β Signaling to Promote Glioma Progression.

Gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. FAM64A, a cell cycle-related gene, has been found to promote cell proliferation in various tumors, including gliomas. However, the regulatory mechanism and clinical significance of FAM64A in gliomas remain unclear. In this study, we investigated FAM64A expression in gliomas with different grades and constructed FAM64A silenced cell lines to study its functions. Our results demonstrated that FAM64A was highly expressed in glioblastoma(P <0.001)and associated with a poor prognosis (P <0.001).Expression profiles at the single-cell resolution indicated FAM64A could play a role in a cell-cycle-dependent way to promote glioma cell proliferation. We further observed that FAM64A silencing in glioma cells resulted in disrupted proliferation and migration ability, and increased cell accumulation in the G2/M phase (P = 0.034). Additionally, TGF-β signaling upregulates FAM64A expression, and SMAD4 and FAM64A co-localize in high-grade glioma tissues. We found FAM64A knockdown inhibited TGF-β-induced epithelial-mesenchymal transition in glioma. Our findings suggest that FAM64A could serve as a diagnostic and therapeutic target in gliomas.

S100A9 promotes glycolytic activity in HER2-positive breast cancer to induce immunosuppression in the tumour microenvironment

PurposeThe purpose of this study was to investigate the correlation between S100 calcium binding protein A9 (S100A9), tumour glycolysis and tumour infiltrating lymphocytes (TIL) in human epidermal growth factor receptor 2 (HER2) - positive breast cancer (BRCA). Materials and methodsA total of 667 BRCA patients in Xiangya Hospital of Central South University were enrolled in this study. Haematoxylin and eosin (H&E) staining were used to count TIN in tissues. Human breast cancer cell lines (SK-BR-3 cells and BT474 cells) were transfected with S100A9 specific small interfering RNA (siRNA). The expressions of S100A9, glycolytic enzymes and lymphocyte markers were detected by immunohistochemistry (IHC) staining, Western blot and immunofluorescence. Lactate production, glucose consumption and the extracellular acidification rate (ECAR) were detected to assess glycolysis activity. ResultsS100A9 was significantly overexpressed in HER2+ cases. The expressions of phosphoglycerol kinase 1 (PGK1), lactate dehydrogenase A (LDHA) and enolase α (ENO1) were significantly up-regulated in S100A9 dominant tissues. The expressions of PGK1, LDHA and ENO1 detected in S100A9 silenced cell lines were significantly down-regulated. Moreover, S100A9 silencing significantly altered lactate production, glucose uptake and ECAR levels in HER2+ cell lines. Co-expression of S100A9 and c-Myc was detected in HER2+ tissues. The absence of S100A9 greatly hindered β-catenin expression in cell lines, which later induced the phosphorylation of c-Myc.The amount of TILs in cases with abundant S100A9 and LDHA was much greater than in cases with low S100A9 levels and poorer LDHA. TIL deficiency and elevated S100A9 intensity are factors affecting the survival rate of HER2+ BRCA cases. ConclusionsS100A9 overexpression upregulated the glycolysis activity of tumour cells through the c-Myc-related pathway, suppressing lymphocyte infiltration in the tumour stroma, affecting the efficacy of immune regulation and long-term survival of patients.

Cyclin A–CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry

Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken together, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.

Heme Oxygenase-1 Inhibition the Cytotoxicity of NK Cells to AML By Downregulating HLA-C

Backgroud and objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells with abnormal differentiation of myeloid primordial naive cells, which has the highest incidence in adult Acute leukemia with a low cure rate . Clinically, less than one-third of adult AML patients can achieve sustained remission after cytarabine combined with anthracycline chemotherapy, and over 50% relapse after remission. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is still the only way to cure leukemia clinically. However, due to age limitation, infection, graft-versus-host disease, and other complications, it is not suitable for all AML patients In recent years, numerous novel drugs have provided more options for the treatment of relapsed and refractory AML while the overall response rate is unsatisfactory and there is no significant advantage in improving overall survival Exploring the mechanism affecting AML relapse and refractory treatment, formulating effective coping strategies are the key to overcome the treatment bottleneck of AML Methods: Bone marrow nucleated cells were obtained from 15 newly diagnosed AML patients, 15 recurrent AML patients and 15 normal controls. The expressions of HO-1 and HLA-C were detected by quantitative reverse transcription PCR (RT-PCR) and Western blot. In vitro cell experiments, we constructed SiHO-1 lentiviral vector to transfect AML cell lines, cocultured NK cells with it, measured the change of CD107a expression on NK cell surface, and silenced HLA-C expression with small interfering RNA. The silenced AML cell lines were co-cultured with NK cells, and CD107a secretion on NK cells was measured by flow cytometry. Results: HO-1 expression in AML patients was higher than that in healthy donors (P<0.05), while HLA-C expression was lower than that in healthy donors (P<0.01). Additionally, the plasma HO-1 levels gradually increased, while the HLA-C levels gradually decreased from healthy donors to patients with relapsed AML. In addition, Pearman correlation analysis indicated that HLA-C expression was significantly correlated with HO-1 expression (r=0.635, P<0.001). In AML cell lines, HO-1 silencing enhanced the cytotoxicity of NK cells to AML cells (P＜0.01), while HO-1 overexpression weakened the cytotoxicity of NK cells to AML cells (P＜0.05). The expression of CD107a in NK cells after HLA-C silencing was lower than the control group in U937 and THP-1 cell line. (P＜0.05). Conclusion: decreased HLA-C expression is associated with increased HO-1 expression during the progression of AML. Besides, the inhibition of HO-1 promotes HLA-C expression, which can therefore promote cytotoxicity of NK cells to AML cells.

The regulation of proliferation and apoptosis in hepatocellular carcinoma via insulin-like growth factor 1 receptor

ObjectivesInsulin-like growth factor 1 receptor (IGF-1R) is a transmembrane tyrosine kinase receptor of the insulin receptor family. Its expression is consistently increased in hepatocellular carcinoma (HCC) tissue, and it participates in hepatic carcinogenesis. Targeting IGF-1R may be a potential therapeutic approach against hepatocellular carcinoma. This study therefore aimed to explore the effect of IGF-1R on hepatocellular carcinoma cells. MethodsIGF-1R silencing cell lines were established by small-interfering RNAs in hepatocellular carcinoma cell line SMMC7721, after which the proliferation, invasion, and apoptosis of SMMC7721 was evaluated. The activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and the expression of bone morphogenetic protein (BMP)-2 and BMP-7 were measured using Western blot analysis. ResultsThe results indicated that the knockdown of IGF-1R can inhibit the proliferation and invasion of HCC and promote the apoptosis of SMMC7721 by inhibiting the PI3K/AKT signaling pathway. Furthermore, depletion of IGF-1R was found to suppress the expression of BMP-2 and BMP-7. ConclusionsThe findings suggest that IGF-1R plays an important role in the progression of HCC. Therefore, IGF-1R is a potential target for the treatment of HCC in clinic.

TYROSINE KINASE PROTEINS TKS4 AND TKS5 PARTICIPATE IN THE INVASIVENESS OF SALIVARY GLAND MUCOEPIDERMOID CARCINOMA

Objectives The aim of this study was to verify the expression of Tks4 and Tks5 and the role played by these proteins in the invasiveness of human salivary mucoepidermoid carcinoma (HMC). Study Design Immunofluorescence was performed in HMC cells. Tks4 and Tks5 genes were silenced by short hairpin RNA (shRNA). Western blot and polymerase chain reaction were performed to confirm silencing of Tks4 and Tks5 in gene and protein expression levels. After silencing, 3 cell lines were obtained: HMC (Tks5-), HMC (Tks4-), and HMC-CT (control), and cell viability assays were performed. Cell invasion assays were performed by comparing silenced and nonsilenced cell lines. Mann-Whitney statistical test was performed to compare the cell lines. Results HMC-CT expresses Tks4 and Tks5. There was inhibition of Tks4 and Tks5 at gene and protein levels. Viable and stable cell lines were obtained after silencing. In the cell invasion assay, a greater invasion of cells was observed in the nonsilenced cell line (HMC-CT) when compared to the silenced cell lines (P < .001***). Conclusions Knockdown of Tks4 and Tks5 reduce invasiveness in HMC cell lines. This is the first in vitro evidence that shows the possible key role of Tks4 and Tks5 in the invasion of HMC. The aim of this study was to verify the expression of Tks4 and Tks5 and the role played by these proteins in the invasiveness of human salivary mucoepidermoid carcinoma (HMC). Immunofluorescence was performed in HMC cells. Tks4 and Tks5 genes were silenced by short hairpin RNA (shRNA). Western blot and polymerase chain reaction were performed to confirm silencing of Tks4 and Tks5 in gene and protein expression levels. After silencing, 3 cell lines were obtained: HMC (Tks5-), HMC (Tks4-), and HMC-CT (control), and cell viability assays were performed. Cell invasion assays were performed by comparing silenced and nonsilenced cell lines. Mann-Whitney statistical test was performed to compare the cell lines. HMC-CT expresses Tks4 and Tks5. There was inhibition of Tks4 and Tks5 at gene and protein levels. Viable and stable cell lines were obtained after silencing. In the cell invasion assay, a greater invasion of cells was observed in the nonsilenced cell line (HMC-CT) when compared to the silenced cell lines (P < .001***). Knockdown of Tks4 and Tks5 reduce invasiveness in HMC cell lines. This is the first in vitro evidence that shows the possible key role of Tks4 and Tks5 in the invasion of HMC.

Abstract 1284: The tumor scaffold protein SQSTM1 at the crossroads of DNA damage and immunotherapy responses in solid tumors

Abstract Introduction After the recent advent of Immune Checkpoint Inhibitors (ICIs), one of current challenge of clinical cancer trials is certainly to develop the optimal combinations of ICIs with DNA-Damaging Agents (chemotherapy and/or radiotherapy, named DDAs hereafter). Elucidating resistance mechanisms to these different treatments is pivotal to propose new predictive biomarkers and to develop therapeutic strategies to improve ICI efficiency. We hypothesized that resistance to DDA and ICIs is mediated in part by intrinsic tumor mechanisms, some of which may be shared. Methods To address this knowledge gap, we compared RNA expression signatures, antigen presentation, and PD-L1 expression from cohorts of cancer patients treated with radiotherapy, chemotherapy, and immunotherapy to identify shared molecular pathways that may mediate cross-resistance. Using a panel of lung cancer cell lines, we then confirmed that the tumor-cell-intrinsic expression of SQSTM1 is positively correlated with antigen presentation, and inversely with DNA damage repair and DDA/ICIs resistance gene signatures. This SQSTM1/HERV pathway was then supported in vitro using engineered silenced cell-lines (SQSTM1, MAVS, and STING ShRNA) treated with FDA-approved DDA (cisplatin, oxaliplatin, doxorubicin, and ionizing radiations) or synthetic double-stranded RNA (5’ppp dsRNAs and poly IC). Results Through three complementary approaches (in silico, ex vivo on patient cohorts, and in vitro), we identify the p62/SQSTM1 scaffold protein as a key molecular mediator able of predicting and controlling sensitivity for DDA and ICIs. Mechanistically, in response to DNA damage, we found that SQSTM1 is essential for the inhibition of DNA repair and the reactivation of human endogenous retroviruses (hERV). Analogous to a “viral alarm”, the hERV are sensed and activate IFN responses that drive the expression of MHC-I and PD-L1, leading to tumor immune evasion. Targeting the hERV pathways in SQSTM1-depleted tumor cells by poly(I:C)/Lyovec, or docetaxel treatment can rescue the hERV, IFN, and MHC pathway, providing a promising therapeutic avenue turning a “cold” into a “hot” tumor in non-responders cancer patients. Conclusion Depending on its levels, we thus propose SQSTM1 as a predictive biomarker for guiding treatment decisions from i) ICI alone, ii) ICI combined with cisplatin, or; iii) ICI combined with poly(I:C)/Lyovec or docetaxel, with the aim to increase immunotherapy efficacy. Citation Format: Iris Grosjean, Grégoire D’Andréa, Nathalie Yazbeck, Amine Belaid, Barnabé Roméo, Marie Angela Domdom, Nicolas Guillot, Eric Gilson, Emmanuel Chamorey, Gérard Milano, Véronique Hofman, Marius Ilié, Isabelle Benard-Thiery, Simon Heeke, Patrick Brest, François Ghiringhelli, Paul Hofman, Baharia Mograbi. The tumor scaffold protein SQSTM1 at the crossroads of DNA damage and immunotherapy responses in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1284.

Abrogation of ARF6 in promoting erastin-induced ferroptosis and mitigating capecitabine resistance in gastric cancer cells.

ADP ribosylation factor 6 (ARF6) is a member of the Rat sarcoma virus (RAS) superfamily that is involved in the regulation of vesicular trafficking, membrane lipid remodeling, and signaling pathways. Our earlier work discovered that ARF6, as a downstream effector of the Kirsten rat sarcoma viral oncogene (Kras)/extracellular signal-regulated kinases (ERK) signaling pathway, may increase proliferation and induce the Warburg effect in gastric cancer (GC) cells. Additionally, ARF6 appears to be a potential biomarker for predicting the prognosis of GC. Ferroptosis has recently been described as a type of nonapoptotic iron-dependent cell death that is strongly associated with the Kras mutation. Therefore, it is critical to continue investigating the link between ARF6 and ferroptosis. We first created ARF6 silenced cancer cell lines with lentivirus transfection. The knockdown efficiency was confirmed through quantitative polymerase chain reaction (qPCR) and western blotting. Subsequently, we used Cell Counting Kit-8 (CCK-8) and malondialdehyde (MDA) assay for lipid peroxidation measurement. Following this, qPCR and western blotting were conducted to clarify the mechanism involved. Finally, immunohistochemistry was used to stain human GC samples. Our findings established that, whereas ARF6 did not directly regulate lipid peroxidation, it did render GC cells susceptible to oxidative stress, particularly erastin-induced lipid peroxidation. Additionally, our research demonstrated that ARF6 may control capecitabine resistance via several routes. ARF6 may play a critical role in the development of GC.

SIPL1, Regulated by MAZ, Promotes Tumor Progression and Predicts Poor Survival in Human Triple-Negative Breast Cancer.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer owing to a lack of effective targeted therapy and acquired chemoresistance. Here, we explored the function and mechanism of shank-interacting protein-like 1 (SIPL1) in TNBC progression.MethodsSIPL1 expression was examined in human TNBC tissues and cell lines by quantitative reverse transcription PCR, western blot, and immunohistochemistry. SIPL1 overexpression and silenced cell lines were established in BT-549 and MDA-MB-231 cells. The biological functions of SIPL1 in TNBC were studied in vitro using the CCK-8 assay, CellTiter-Glo Luminescent Cell Viability assay, caspase-3/8/9 assay, wound healing assay, and transwell assay and in vivo using a nude mouse model. The potential mechanisms underlying the effects of SIPL1 on TNBC progression were explored using bioinformatics analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by qPCR.Results SIPL1 expression was higher in human TNBC tissues and cell lines than in adjacent normal tissues and a breast epithelial cell line (MCF10A). High expression of SIPL1 was positively correlated with poor overall and disease-free survival in patients with TNBC. SIPL1 overexpression elevated and SIPL1 silencing repressed the malignant phenotypes of TNBC cells in vitro. SIPL1 overexpression promoted xenograft tumor growth in vivo. Myc-associated zinc-finger protein (MAZ) transcriptionally activated SIPL1. Finally, we found that SIPL1 promoted TNBC malignant phenotypes via activation of the AKT/NF-κB signaling pathways.ConclusionsThese results indicate that the MAZ/SIPL1/AKT/NF-κB axis plays a crucial role in promoting the malignant phenotypes of TNBC cells.

Vangl2 participates in the primary ciliary assembly under low fluid shear stress in hUVECs.

The pattern of blood fluid shear stress (FSS) is considered the main factor that affects ciliogenesis in human umbilical vein endothelial cells (hUVECs), the underlying mechanism is unclear. Microfluidic chamber experiments were carried out to load hUVECs with low fluid shear stress (LSS, 0.1 dynes/cm2) or high fluid shear stress (HSS, 15 dynes/cm2). Van Gogh2 (Vangl2), a core protein in the planar cell polarity (PCP) pathway, was silenced and overexpressed in hUVECs. Immunofluorescence analysis showed that primary cilia assemble under LSS while disassembling under HSS. Vangl2 expression was consistent with cilia assembly, and its localization showed a polar distribution under LSS. Furthermore, the average number of ciliated cells and primary cilia length were increased in the Vangl2 overexpressing cell lines (the OE group) but decreased in the Vangl2 silenced cell lines (the SH group). When these cells were loaded with different FSS, more ciliated cells with longest primary cilia were observed in the LSS loaded OE group compared with those in the other groups. Immunoprecipitation showed that the interaction between Bardet-Biedl syndrome8 (BBS8) and Vangl2 was enhanced following LSS loading compared to that under HSS. However, the interactions between phosphorylated dishevelled segment polarity protein 2 (pDvl2), kinesin family member 2a (Kif2a), and polo-like kinase 1 (Plk1) and Vangl2 were restrained following LSS loading. Overall, the results indicated that Vangl2 played a significant role during LSS-induced primary cilia assembly by recruiting BBS to promote the apical docking of basal bodies and by restraining Dvl2 phosphorylation from reducing primary cilia disassembly.

The Role of NPM1 in the Invasion and Migration of Drug Resistant Bladder Cancer.

To study the difference of tumor progression caused by differential expression of NPM1 in drug-resistant bladder cancer. The expression of NPM1 was analyzed by PCR and Western blot. NPM1 silencing bladder cancer cells (T24/DDP Lv-NPM1, PUMC-91/DDP Lv-NPM1) and overexpressing bladder cancer cells (T24/ DDP Lv5-NPM1, PUMC-91/DDP Lv5-NPM1) were established by lentivirus and limited dilution method. The efficiency of gene interference was detected by fluorescence microscopy and Western blot. The migration ability and invasion ability of tumor in vitro were analyzed by wound healing assay and transwell cell invasion test, and the tumorigenic ability in vivo was judged by nude mouse tumorigenicity assay. Compared with the corresponding negative control group, both NPM1 silencing cell lines T24/DDP Lv-NPM1 and PUMC-91/DDP Lv-NPM1 showed strong migration ability and high invasive ability. At the same time, there was no significant difference in migration ability and the invasive cells proportion between NPM1 overexpressing cell line and related negative control group. NPM1 silencing bladder cancer cells had obvious tumorigenicity in vivo. NPM1 silencing cells had significant migration and invasion ability. The silencing of NPM1 will accelerate tumorigenicity of drug resistant bladder cancer. Differential expression of NPM1 is of great value in monitoring the progression of drug-resistant bladder cancer.

Identification of Possible Driver Gene in Patients with Acquired Pure Red Cell Aplasia

OBJECTIVE: Acquired pure red cell aplasia (aPRCA) is a disorder characterized by normocytic anemia associated with reticulocytopenia and an absence of erythroblasts. The pathogenesis of aPRCA is believed to be the induction of autoimmunity, although many underline mechanisms are still not known. In this study, we aimed to identify the gene mutations in aPRCA patients and its possible effects on the origin of the disease.METHODS: Blood genome DNA extracted from ten newly diagnosed patients with aPRCA was checked with whole-exome sequencing (WES). Candidate gene (nonsynonymous, splicing single-nucleotide variants (SNVs) or insertion/deletions (INDELs)) which met the criteria of high frequency in aPRCA but rare in 1000 Genomes were first confirmed by Sanger sequencing and checked the mRNA level in PRCA patients by quantitative real-time polymerase chain reaction. Next the pGreen-CMV-puro system was employed to generate lentivirus expressing short hairpin RNAs to target the candidate genes, and stable transfected K562 cell lines with silenced candidate genes was constructed. Finally the erythroid and megakaryocytic differentiation was evaluated for the transfected K562 cell lines either by benzidine staining hemoglobin, CD235a or CD41 expression on the cell surface.RESULTS:MKI67, SEC22B and STK10 genes who met the selection criteria were selected for further study. Of the three genes, mRNA level were down-regulated in the mutated patients. The mRNA expression of the three genes was all found decreased in the transfected K562 cell lines. Only STK10 silenced K562 cell lines showed significantly reduction in number of benzidine-positive cells and expression of CD235a stimulated by hemin compared with non-transfected cell lines. However, CD41 expression was similar in STK10 silenced K562cells to control K562 cells after inducted by phorbol 12-myristate 13-acetate. No difference was found between the transfected and non-transfected cell lines with MKI67 or SEC22B gene either in erythropoiesis or megakaryocyte hematopoiesis.CONCLUSION:STK10 gene mutation is common in patients with aPRCA and causes the reduction of mRNAs expression in the mutated patients. Silence of STK10 gene in K562 cell lines might inhibit erythroid, but not megakaryocytic differentiation. STK10 might be one of the driver genes for the abnormal erythropoiesis in patients with aPRCA. DisclosuresNo relevant conflicts of interest to declare.

Clinicopathological Significance of FOXO4 Expression and Correlation with Prx1 in Head and Neck Squamous Cell Carcinoma.

Objective Forkhead box O 4 (FOXO4), a key albumen in the forkhead box O (FOXOs) family, plays crucial roles as a tumor suppressor in the cancer development. In our previous study, Peroxiredoxin1 (Prx1) promoted the development of oral cancer and was predicted to bind to FOXO4. The aim of this study was to investigate the clinicopathological significance of FOXO4 expression and its potential mechanism in head and neck squamous cell carcinomas (HNSCC). Methods The function of FOXO4 correlation with HNSCC prognosis was analyzed via ONCOMINE, UALCAN, Human Protein Atlas, and cBioPortal. The expression of FOXO4 was detected in Prx1 silenced CaL27 and SCC9 cell lines by Western blot. FOXO4 protein expression was observed via immunohistochemistry (IHC) and the binding of Prx1 to FOXO4 measured by Duolink analysis in a 4-nitro-quinoline-1-oxide- (4NQO-) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/− mice. Results By the analysis of Bioinformation Databases, there was a significant interaction of FOXO4 down expression to clinical tumor stages and pathological grades in the patients with HNSCC. Reduced mRNA and protein expression of FOXO4 were found to be significantly correlated with the poor overall survival (OS) of HNSCC patients. FOXO4 expression is negatively related to Prx1 significantly in HNSCC tissues. By employing a 4NQO-induced oral carcinogenesis mouse model, we confirmed that FOXO4 expression was reduced in 4NQO-induced squamous cell carcinoma (SCC) tongue tissues compared with those in normal tissues. Prx1 knockdown resulted in the upregulation of FOXO4 expression in the SCC tissues and CaL27 and SCC9 cell lines. Furthermore, the interaction of Prx1 with FOXO4 was observed in mouse tongue tissues by Duolink analysis. Conclusion FOXO4 plays an important role in the development of HNSCC. The lower expression of FOXO4 is significantly correlated with the shorter OS in patients with HNSCC. FOXO4 is negatively regulated via interaction with Prx1. FOXO4 could be a potential molecular target for the treatment and prognosis of HNSCC.

Silencing long non-coding RNA HNF1A-AS1 inhibits growth and resistance to TAM of breast cancer cells via the microRNA-363/SERTAD3 axis

Long non-coding RNAs (lncRNAs) can exert effects on drug resistance of cancer cells. This study investigated the role of lncRNA HNF1A-antisense 1 (HNF1A-AS1) in growth and Tamoxifen (TAM) sensitivity of breast cancer (BC) cells. HNF1A-AS1 expression was promoted in BC cells and tissues. BC cells with HNF1A-AS1 silencing were constructed to detect cell proliferation. TAM resistant cell line with HNF1A-AS1 silencing and parent cell line with overexpressed HNF1A-AS1 were constructed to measure drug resistance. Silencing HNF1A-AS1 reduced proliferation and TAM resistance of BC cells. The downstream microRNAs (miRs) of HNF1A-AS1 and its targets were figured out and their functions in TAM resistance of BC cells were identified. HNF1A-AS1 sponged miR-363 to promote SERTAD3 expression. Downregulation of miR-363 or upregulation of SERTAD3 stimulated TAM resistance of BC cells. The findings in vitro were reproduced in in vivo experiments. It could be concluded that silencing HNF1A-AS1 inhibited growth and drug resistance to TAM of BC cells through the miR-363/SERTAD3 axis and the inactivation of the TGF-β/Smad pathway.

Epigenetic identification of mitogen-activated protein kinase 10 as a functional tumor suppressor and clinical significance for hepatocellular carcinoma.

BackgroundMitogen-activated protein kinase 10 (Mapk10) is a member of the c-jun N-terminal kinases (jnk) subgroup in the MAPK superfamily, and was proposed as a tumor suppressor inactivated epigenetically. Its role in hepatocellular carcinoma (HCC) has not yet been illustrated. We aimed to investigate the expression and epigenetic regulation of mapk10 as well as its clinical significance in HCC.ResultsMapk10 was expressed in almost all the normal tissues including liver, while we found that the protein expression of MAPK10 was significantly downregulated in clinical samples of HCC patients compared with these levels in adjacent normal tissues (29/46, P < 0.0001). Clinical significance of MAPK10 expression was then assessed in a cohort of 59 HCC cases, which indicated its negative expression was significantly correlated with advanced tumor stage (P = 0.001), more microsatellite nodules (P = 0.025), higher serum AFP (P = 0.001) and shorter overall survival time of HCC patients. Methylation was further detected in 58% of the HCC cell lines we tested and in 66% of primary HCC tissues by methylation-specific PCR (MSP), which was proved to be correlated with the silenced or downregulated expression of mapk10. To get the mechanisms more clear, the transcriptional silencing of mapk10 was reversed by pharmacological demethylation, and ectopic expression of mapk10 in silenced HCC cell lines significantly inhibited the colony formation ability, induced apoptosis, or enhanced the chemosensitivity of HCC cells to 5-fluorouracil.ConclusionMapk10 appears to be a functional tumor suppressor gene frequently methylated in HCC, which could be a valuable biomarker or a new diagnosis and therapy target in a clinical setting.

MiR-146a Enhances the Sensitivity of Breast Cancer Cells to Paclitaxel by Downregulating IRAK1.

Objective: To investigate the effect of miR-146a on the sensitivity of breast cancer cells to paclitaxel (PTX). Materials and Methods: The mRNA expressions of miR-146a in normal breast cancer cells, MCF-7, and PTX-resistant breast cancer cells, MCF-7/PTX, were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). MTS was used to analyze the cytotoxicity treated with different concentrations of PTX. Overexpressed and silenced cell lines of miR-146a and interleukin-1 receptor-associated kinase 1 (IRAK1) were constructed, respectively. Cells were treated with PTX and observed the changes of cell morphology. Proliferation was detected by clone formation assay. Invasion and migration were measured by transwell. RT-PCR was applied to detect the expression of IRAK1 gene. Dual luciferase report was performed to validate the target relationship between miR-146a and IRAK1. Salvage experiments were used to further verify the relationship between miR-146a and IRAK1. Results: PTX reduces the viability of MCF-7 and MCF-7/PTX cells in a dose-dependent manner. The IC50 of PTX in MCF-7 cells was significantly lower compared with MCF-7/PTX cells (p < 0.05). Compared with MCF-7/PTX cells, the expression of miR-146a gene in MCF-7 cells was significantly increased, while the expression of IRAK1 gene was significantly reduced (p < 0.05). Cell proliferation, invasion, and migration were decreased after miR-146a overexpression or IRAK1 silencing. Whereas, miR-146a silencing and IRAK1 overexpression can increase cell proliferation, invasion, and migration ability. Salvage experiments further verify that IRAK1 can weaken the role of miR-146a. Conclusion: miR-146a can enhance the sensitivity of breast cancer cells to PTX; the mechanism may be related to the downregulation of IRAK1.