shRNA Plasmid Research Articles

Protein kinase D1 regulates the growth and metabolism of oral squamous carcinoma cells in tumor microenvironment

To observe the effect of protein kinase D1 (PKD1) on the growth and metabolism of oral squamous cell carcinoma HSC-4 cells and related molecular mechanisms in the tumor microenvironment. HSC-4 cell lines were transfected with shRNA plasmids. Three groups (Wild, control-shRNA, and PKD1-shRNA) were cultured under acidic or hypoxic environment for a certain time. Western blot was used to detect the expression of autophagy-related and glycolytic-related proteins. The proliferation changes were detected by CCK-8 kits. The PKD1-knockdown HSC-4 cell line was established. PKD1 silencing increased autophagy activity. Under hypoxic and acidic conditions, the PKD1-knockdown HSC-4 cells showed lower proliferation than the parental cells. PKD1-knockdown also decreased the expression of hypoxia induciblefactor 1α (HIF-1α) and pyruvate kinase M2 (PKM2). Under hypoxic and acidic conditions, PKD1 gene silencing can increase apoptotic autophagy activity. Downregulated PKD1 gene expression can reduce the glycolysis of oral squamous cell carcinoma cells and inhibit tumor cell proliferation. This study revealed the important role of PKD1 in the metabolism and growth of oral squamous cell carcinoma, making it a possible target for the treatment of oral squamous cell carcinoma.

Corrigendum. Decreased PECAM1-mediated TGF-β1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

Study question Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window? Summary answer The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. What is known already RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. Study design, size, duration This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. Participants/materials, setting, methods We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1. Main results and the role of chance Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit β2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-β1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-β1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-β1 might play a pivotal role in modulating endometrial receptivity. Limitations reasons for caution Although we have shown that PECAM1 and TGF-β1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear. Wider implications of the findings Our findings provide insight into the contribution of PECAM1 and TGF-β1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF. Study funding/competing interest(s) This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.

DC-SIGN mediates gastric cancer progression by regulating the JAK2/STAT3 signaling pathway and affecting LncRNA RP11-181G12.2 expression

BackgroundThe molecular mechanisms of gastric cancer (GC) development are very complicated. Recent studies revealed that DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein (DC-SIGNR) is involved in colon cancer and GC biological processes. However, the exact roles of DC-SIGN in GC remain unrevealed. MethodsDC-SIGN overexpression and knockdown experiments were performed by using DC-SIGN shRNA or DC-SIGN plasmid to investigate the biological roles of DC-SIGN in proliferation, cell cycle progression, migration and invasion of GC cells in vitro. Furthermore, the lncRNA profiles of SGC-7901 cells with control shRNA and DC-SIGN shRNA were generated by using microarray analysis. Mechanistically, the relationship between DC-SIGN, RP11-181G12.2 and the JAK2/STAT3 signaling pathway was then investigated using qRT-PCR and western blot assays. Additionally, we analyzed DC-SIGN and RP11-181G12.2 expression levels in GC specimens based on the Cancer Genome Atlas database. ResultsIn this study, the results showed that DC-SIGN was highly expressed in GC cells and significantly correlated with advanced clinical stage and lymphatic metastasis. Downregulation of DC-SIGN significantly inhibited the proliferation, cell cycle progression, migration and invasion of GC cells in vitro. The reverse results could partly be seen with the upregulation of DC-SIGN. Mechanistically, knockdown of DC-SIGN inactivated the JAK2/STAT3 signaling pathway, and overexpression of DC-SIGN activated the JAK2/STAT3 signaling pathway. In addition, through LncPath microarray analysis, we identified a lncRNA, RP11-181G12.2, that was significantly upregulated after knockdown of DC-SIGN; this was also confirmed by qRT-PCR. Furthermore, RP11-181G12.2 knockdown enhanced DC-SIGN expression in GC cells, further activating the JAK2/STAT3 signaling pathway. In contrast, DC-SIGN overexpression suppressed RP11-181G12.2 expression. ConclusionsOur study suggests that DC-SIGN might be involved in the progression of GC by regulating the JAK2/STAT3 signaling pathway and affecting lncRNA RP11-181G12.2 expression.

Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed >1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p< 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Suppression of KIF22 Inhibits Cell Proliferation and Xenograft Tumor Growth in Colon Cancer.

Background: Kinesin family member 22 (KIF22) is known as a regulator of cell mitosis and cellular vesicle transport. The alterations of KIF22 are associated with a series of tumors; however, its possible role in the progression of colon cancer is still unclear. Materials and Methods: This retrospective research collected 82 paired tissues with colon cancer. KIF22 protein and mRNA expression levels were detected by immunohistochemistry assays and Immunoblot assays, respectively. Short hairpin RNA (shRNA) plasmids were used to suppress the expression of KIF22 in HCT116 and HT29 cells, and the silencing efficiencies of shRNA plasmids targeted KIF22 were detected by quantitative PCR assays and immunoblot assays. In addition, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays and xenograft tumor growth assays were performed to observe cell proliferation in vitro and in vivo. Results: In human colon cancer tissues, the expression level of KIF22 was increased and correlated with clinical pathological features, including tumor stage and clinical stage (p = 0.034, and p = 0.015, respectively). Suppression of KIF22 inhibited cell proliferation and xenograft tumor growth. Conclusion: KIF22 might play an important role in the regulation of cell proliferation in colon cancer and might therefore serve as a promising therapeutic target.

C-Fos separation from Lamin A/C by GDF15 promotes colon cancer invasion and metastasis in inflammatory microenvironment.

Inflammatory microenvironment is an important factor for promoting cancer invasion and metastasis, but the underlying molecular mechanisms remain unclear. Here, we mimicked an inflammatory microenvironment both in vitro and in vivo and investigated its effects on the invasion and metastasis of colon cancer. Moreover, colon cancer patient samples were also analyzed statistically. Conditioned medium from the differentiated macrophages induced invasion and migration of colon cancer cells in vitro, which could be reversed by the treatment of a neutralizing anti-growth differentiation factor 15 (GDF15) antibody, indicating GDF15 involvement in inflammation-induced invasiveness. Also, we observed similar effects of human recombinant GDF15 on colon cancer cells. Mechanistically, GDF15 activated c-Fos by separating it from Lamin A/C, increasing transcriptional activity of c-Fos and regulating EMT gene expressions. However, c-Fos knockdown using lentivirus shRNA plasmid inhibited GDF15-triggered invasion and migration in vitro. In vivo, inflammation caused by lipopolysaccharides obviously increased GDF15 secretion, and c-Fos knockdown reduced the lung metastasis of colon cancer cells in mice model. In addition, c-Fos expressions in patient samples were found to be associated with colon cancer metastasis and TNM stages. Taken together, GDF15 in inflammatory microenvironment induces colon cancer invasion and metastasis by regulating EMT genes by activating c-Fos, which might be a potential therapeutic target for metastatic colon cancer.

Silence of TGF-β1 gene expression reduces prrsv replication and potentiates immunity of immune cells of tibetan pig

Transforming growth factor beta 1 (TGF-β1) was of importance in the pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV). To determine whether knockdown of TGF-β1 gene expression could facilitate the control of PRRSV infection, specific sequences for expressing shRNA targeted to porcine TGF-β1 gene were synthesized and cloned into pSilencer 3.1-H1 neovector. Then they were used to transfect peripheral blood mononuclear cells of Tibetan pig (Tp-PBMCs) followed by PRRSV inoculation. The positive recombinant plasmids were screened for inhibition of TGF-β1 gene expression by real-time quantitative RT-PCR. Conversely, the mRNA level of PRRSV in shRNA treated Tp-PBMCs dramatically decreased, and there were significant increases of the transcription of immune genes, such as interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), toll-like receptor 3 (TLR3), toll-like receptor 7 (TLR7), Myeloid differentiation primary response gene (88) (MyD88), and interleukin-27p28 (IL-27p28). However, the expressions of IL-8 and IL-10 genes significantly reduced in comparison to the control infected cells. In addition, transfection with the shRNA plasmids significantly elevated the viability of immune cells. Therefore the knockdown of TGF-β1 gene expression by shRNA not only inhibits the replication of PRRSV but also improves immune responsiveness following viral infection, suggesting a novel way to facilitate the control of PRRSV infection in pigs.

Circ-ITCH correlates with less advanced tumor features as well as prolonged survival, and it inhibits cells proliferation but promotes apoptosis in non-small cell lung cancer

BackgroundThis study was to investigate the correlation of circ-ITCH expression with tumor features, disease free survival (DFS) and overall survival (OS) in non-small cell lung cancer (NSCLC) patients, and the effect of its dysregulation on cells proliferation and apoptosis of NSCLC cells.MethodsTumor tissue and paired adjacent normal tissue samples from 190 surgical NSCLC patients were collected and used for detecting circ-ITCH expression by qPCR. Circ-ITCH expression was also measured in normal pulmonary epithelial cell line and 4 NSCLC cell lines by qPCR. Patients’ baseline characteristics were recorded and DFS as well as OS were calculated. Blank, circ-ITCH overexpression, blank shRNA, circ-ITCH shRNA plasmids were transferred into NSCLC cell line (A549 cells) as NC(+), Circ-ITCH(+), NC(−) and Circ-ITCH(−) groups. Cells proliferation was measured using CCK-8 and cells apoptosis rate was measured using annexin V (AV)/propidium iodide (PI).ResultsCirc-ITCH expression was decreased in tumor tissue compared with paired adjacent tissue, and was negatively correlated with tumor size, lymph node metastasis and TNM stage. And circ-ITCH high expression was associated with favorable DFS and OS. In vitro experiments revealed that compared with normal pulmonary epithelial cell line BEAS-2B, circ-ITCH expression was reduced in NSCLC cell lines HCC827, A549, NCI-H1299 and NCI-H460. And cells proliferation was decreased in Circ-ITCH(+) group and elevated in Circ-ITCH(−) group at 72 h. Cells apoptosis rate was increased in Circ-ITCH(+) group and decreased in Circ-ITCH(−) group at 72 h.ConclusionsCirc-ITCH associates with less advanced tumor features and prolonged survival, and it inhibits cells proliferation while promotes cells apoptosis in NSCLC.

SALL4 promotes the tumorigenicity of cervical cancer cells through activation of the Wnt/β-catenin pathway via CTNNB1.

SALL4 is overexpressed in many cancers and is found to be involved in tumorigenesis and tumor progression. However, the function of SALL4 in cervical cancer remains unknown. Here, we showed that the expression of SALL4 was gradually increased from normal cervical tissue to high‐grade squamous intraepithelial lesions and then to squamous cervical carcinoma. SALL4 was upregulated or downregulated in cervical cancer cells by stably transfecting a SALL4‐expressing plasmid or a shRNA plasmid targeting SALL4, respectively. In vitro, cell growth curves and MTT (3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assays showed that SALL4 promoted the cell proliferation of cervical cancer cells. In vivo, xenograft experiments verified that SALL4 enhanced the tumor formation of cervical cancer cells in female BALB/c Nude mice. Cell cycle analysis by fluorescence‐activated cell sorting found that SALL4 accelerates cell cycle transition from the G0/G1 phase to the S phase. TOP/FOP‐Flash reporter assay revealed that SALL4 significantly upregulates the activity of Wnt/β‐catenin pathway. Western blotting showed that the expression levels of β‐catenin and important downstream genes, including c‐Myc and cyclin D1, were increased by SALL4 in cervical cancer cells. Furthermore, dual‐luciferase reporter and chromatin immunoprecipitation assays confirmed that SALL4 transcriptionally activated CTNNB1 by physically interacting with its promoters. Taken together, The results of this study demonstrated that SALL4 may promote cell proliferation and tumor formation of cervical cancer cells by upregulating the activity of the Wnt/β‐catenin signaling pathway by directly binding to the CTNNB1 promoter and trans‐activating CTNNB1.

Integrin α7 high expression correlates with deteriorative tumor features and worse overall survival, and its knockdown inhibits cell proliferation and invasion but increases apoptosis in breast cancer.

BackgroundThis study aimed to investigate the correlation of integrin α7 (ITGA7) expression with clinical/pathological characteristics and overall survival (OS), and its knockdown on inhibiting cell activities in breast cancer.MethodsA total of 191 breast cancer patients underwent surgery were retrospectively reviewed, and ITGA7 expression in tumor tissues was determined by immunofluorescence and real‐time quantitative polymerase chain reaction. Patients’ clinical/pathological data were recorded, and OS was calculated. In vitro, control shRNA and ITGA7 shRNA plasmids were transfected into MCF7 cells to evaluate the influence of ITGA7 knockdown on cell proliferation, apoptosis, and invasion.ResultsNinety‐two (48.2%) patients presented with ITGA7 high expression, and 99 patients (51.8%) presented with ITGA7 low expression. ITGA7 expression was positively correlated with T stage, tumor‐node metastasis (TNM) stage, and pathological grade. Kaplan‐Meier curves showed that ITGA7 high expression was associated with shorter OS, and multivariate Cox's proportional hazards regression displayed that ITGA7 high expression was an independent predictive factor for poor OS. Moreover, in vitro experiments disclosed that cell proliferation (by Cell Counting Kit‐8 assay) and cell invasion (by Matrigel invasion assay) were reduced, while cell apoptosis rate (by Annexin V/propidium iodide assay) was enhanced by ITGA7 knockdown in MCF‐7 cells.ConclusionIntegrin α7 high expression correlates with increased T stage, TNM stage, and pathological grade as well as worse OS, and its knockdown enhances cell apoptosis but inhibits cell proliferation and invasion in breast cancer.

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

Regulation mechanism of aquaporin 9 gene on inflammatory response and cardiac function in rats with myocardial infarction through extracellular signal-regulated kinase1/2 pathway.

The aim of this study was to investigate the regulation mechanism of aquaporin 9 (AQP9) gene on inflammatory response and cardiac function in rats with myocardial infarction (MI) through extracellular signal-regulated kinase1/2 (ERK1/2) pathway. The constructed rats models of MI were randomly divided into 6 groups: control group (sham operation group, MI modeling sham operation), model group (MI modeling), NC group (MI modeling, tail vein injection of AQP9 negative control sequence vector), AQP9 shRNA group (MI modeling, tail vein injection of AQP9 shRNA plasmid vector), U0126 group (MI modeling, tail vein injection of ERK signaling pathway inhibitor), and AQP9 shRNA + U0126 group. The hemodynamics and cardiac function of rats in each group were detected on the seventh day of modeling. The levels of AQP9 and inflammatory factors [tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10)] in peripheral blood of rats were detected by ELISA method. qRT-PCR and western blot were used to detect the mRNA and protein expression of AQP9, ERK1/2, B-cell lymphoma-2 (Bcl-2), Bcl-associated x (Bax) in the myocardial tissue of rats. TTC and TUNEL staining were used to observe myocardial infarct size and apoptosis of myocardial cells in each group. Compared with control group, the levels of heart rate, left ventricular end-diastolic pressure, TNF-α, and IL-6 were increased in each group of rats with MI (all p < 0.05), while the levels of systolic blood pressure, diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, and IL-10 were significantly decreased (all p < 0.05). The mRNA and protein expression levels of AQP9, ERK1/2 phosphorylation and Bax were significantly increased, as well as the myocardial infarct size, apoptosis index of myocardial tissue (all p < 0.05), the mRNA and protein expression levels of Bcl-2 were significantly decreased (all p < 0.05). The AQP9 gene knock-down or exogenous administration of the ERK1/2 inhibitor U0126 could improve the above indexes. However, the combination of AQP9 gene knock-down and U0126 showed no further effect. Silencing AQP9 gene can inhibit the activation of ERK1/2 signaling pathway, attenuate the inflammatory response in rats with MI, inhibit apoptosis of myocardial cells, and improve cardiac function.

Long Noncoding RNAs and Messenger RNAs Expression Profiles Potentially Regulated by ZBTB7A in Nasopharyngeal Carcinoma.

Our previous studies showed that ZBTB7A played an important role in promoting nasopharyngeal carcinoma (NPC) progression. However, molecular mechanisms of different levels of ZBTB7A are still unclear. It is necessary to search molecular markers which are closely connected with ZBTB7A. We selected NPC sublines CNE2 with stably transfecting empty plasmid (negative control, NC) and short hair RNA (shRNA) plasmid targeting ZBTB7A as research objectives. Microarray was used to screen differentially expressed long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) via shRNA-CNE2 versus NC-CNE2. Quantitative PCR (qPCR) was used to validate lncRNAs and mRNAs from the sublines, chronic rhinitis, and NPC tissues. Bioinformatics was used to analyze regulatory pathways which were connected with ZBTB7A. The 1501 lncRNAs (long noncoding RNAs) and 1275 differentially expressed mRNAs were upregulated or downregulated over 2-fold. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the upregulated or downregulated carbohydrate and lipid metabolisms probably involved in carcinogenicity of shRNA-CNE2 (P-value cut-off was 0.05). In order to find the molecular mechanisms of ZBTB7A, we validated 12 differentially expressed lncRNAs and their nearby mRNAs by qPCR. Most of the differentially expressed mRNAs are closely connected with carbohydrate and lipid metabolisms in multiply cancers. Furthermore, part of them were validated in NPC and rhinitis tissues by qPCR. As a result, NR_047538, ENST00000442852, and fatty acid synthase (FASN) were closely associated with NPC. ZBTB7A had a positive association with NR_047538 and negative associations with ENST00000442852 and FASN. The results probably provide novel candidate biomarkers for NPC progression with different levels of ZBTB7A.

Circular RNA ABCB10 promotes tumor progression and correlates with pejorative prognosis in clear cell renal cell carcinoma.

Our study aimed to evaluate the effect of circular RNA ABCB10 (circ-ABCB10) on proliferation and apoptosis of clear cell renal cell carcinoma (ccRCC) cells, and its prognostic value in patients with ccRCC. Circ-ABCB10 expression in five ccRCC cell lines and normal kidney epithelial cell line was measured by quantitative polymerase chain reaction (qPCR). Empty overexpression, circ-ABCB10 overexpression, empty shRNA, and circ-ABCB10 shRNA plasmids were transfected into A498 cells as negative control for circ-ABCB10 over expression {NC (+)}, Circ-ABCB10(+), negative control (-){NC(-)}, and Circ-ABCB10(-) groups, then cell proliferation and apoptosis were evaluated by Cell Counting Kit-8 and annexin V/propidium iodide. Meanwhile, apoptotic markers were measured by western blot. Subsequently, circ-ABCB10 expression in tumor tissues and paired adjacent tissues from 120 ccRCC patients was measured by qPCR. Circ-ABCB10 expression was elevated in all the ccRCC cell lines compared with the normal kidney cells line. A498 cell proliferation was enhanced in the Circ-ABCB10(+) group compared with the NC(+) group, while it was inhibited in the Circ-ABCB10(-) group compared with the NC (-) group; and A498 cell apoptosis was repressed in the Circ-ABCB10(+) group than the NC(+) group, but was promoted in the Circ-ABCB10(-) group compared with the NC(-) group. In addition, circ-ABCB10 was up-regulated in tumor tissues compared with paired adjacent tissues, and its high expression correlated with the advanced pathological grade and the tumor node metastasis stage as well as independently predicting worse overall survival in ccRCC patients. Circ-ABCB10 promotes tumor progression and correlates with pejorative prognosis in ccRCC.

AIM2 promotes non-small-cell lung cancer cell growth through inflammasome-dependent pathway.

The human absent in melanoma 2 (AIM2) is considered as a DNA recognizer. AIM2 has been described as a tumor suppressor gene in the early years. But recent studies suggested that it functions as an oncogene in several cancers. However, its roles in non-small-cell lung cancer (NSCLC) remain unclear. Here we reported that AIM2 highly expressed in NSCLC cells and exhibited a tumor-promoting property both in vitro and in vivo. Besides, AIM2 short hairpin RNA (shRNA)-mediated suppression of cell proliferation was triggered by the accumulation of cells at the G2/M phase. Knockdown of AIM2 reduced the inflammasome formation, while overexpression of AIM2 or stimulation by poly(dA:dT) induced the inflammasome formation. Interestingly, blockade of the inflammasome by caspase-1 inhibitor VX-765 or ASC small interfering RNA (siRNA) abolished the effects brought by AIM2 shRNA and AIM2 plasmid. In summary, our results revealed that AIM2 functioned as an oncogene in NSCLC in an inflammasome-dependent way.

PKA- and Ca2+-dependent p38 MAPK/CREB activation protects against manganese-mediated neuronal apoptosis

Although manganese (Mn) is an essential trace element, its excessive consumption may lead to neuronal death and neurodegenerative disorders. Human cells launch adaptive responses to attenuate Mn-induced neurotoxicity. However, the regulation of the responsive proteins and their function during Mn-stimulated neurotoxicity remain largely unknown. We report the role of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in Mn-induced neuronal apoptosis. Mn increased CREB phosphorylation and cellular apoptosis in both PC12 cells and mouse brain tissue. Furthermore, downregulation of CREB with shRNA plasmid transfection significantly worsened the PC12 cell apoptosis by decreasing mRNA and protein expression of brain-derived neurotrophic factor (BDNF). Moreover, Mn enhanced protein kinase A (PKA) activation and activation of the p38 MAPK and JNK pathways. Inhibition of p38 MAPK rather than JNK effectively reduced the CREB phosphorylation. Subsequent analysis showed that a PKA inhibitor blocked p38 MAPK and CREB phosphorylation. Moreover, the intracellular Ca2+ chelator BAPTA-AM decreased the phosphorylation of p38 MAPK and CREB but failed to reduce PKA activation. In summary, p38 MAPK/CREB activation via PKA activation and increased cellular Ca2+ helped to alleviate Mn-induced neuronal apoptosis via BDNF regulation. These findings improve our understanding of Mn-induced neurotoxicity and the molecular targets to antagonise it.

HIF‐prolyl Hydroxylase‐3 as the downstream pathway of TRPC6 Mediates Hypertensioninduced Renal Injury in 5/6 Ablation/Infarction Model

The activation of transient receptor potential canonical 6 (TRPC6) channel participates in the glomerulosclerosis. The TRPC6 also participates in the mechanical or pressure sensation in various cell types, including podocyte. Thus, TRPC6 may be the upstream mediator that transmits the pressure stress into downstream molecular pathways to cause hypertension‐induced renal injury. We previously showed that TRPC6 shRNA blocked the hypertension‐induced renal injury in rats with 5/6 renal ablation/infarction (A/I), a model in which the renal injury is closely associated with blood pressure, suggesting that TRPC6 is the mediator of hypertension‐induced renal injury. However, the signaling pathways downstream of TRPC6 remain unclear. It has been shown that TRPC6 signaling inhibits the activity of HIF‐proly hydroxylase to stabilize hypoxia‐inducible factor (HIF)‐1α, while renal HIF‐1α activation is known to produce chronic renal injury. The present study tested the hypothesis that inhibition of proly hydroxylase (PHD) mediates the injurious effect of activated TRPC6 in the kidney of 5/6 A/I model. Adults male Sprague Dawley rats were divided into shame (S), 5/6 A/I control (C), 5/6 A/I TRPC6 shRNA (T), and 5/6 A/I TRPC6 shRNA+PHD3 shRNA (T+P) groups. The control and shRNA plasmids were transfected via intra‐renal arterial injection using the ultrasound‐microbubble technique. The results showed that the levels of TRPC6 in isolated glomeruli were increased in C group compared with S group and that the increase of TRPC6 was blocked in T and T+P groups, the relative protein levels of TRPC6 by Western blot analysis were 1.0±0.16, 3.7±0.26, 1.3±0.14 and 1.5±0.19 in S, C, T and T+P groups, respectively*. The levels of hydroxylated HIF‐1α (HO‐HIF‐1α), the indicator of PHD activity, were significantly reduced in C group compared with S group; TRPC6 shRNA recovered the levels of HO‐HIF‐1α, whereas PHD3 shRNA blocked the increase of HO‐HIF‐1α by TRPC6 shRNA; the relative protein levels of HO‐HIF‐1α were 1.0±0.06, 0.47±0.03, 0.83±0.09 and 0.31±0.09 in S, C, T and T+P groups, respectively*, suggesting that activated TRPC6 inhibites PHD activities in 5/6 A/I model and that PHD3 shRNA mimics the inhibitory effect of TRPC6 on PHD activity. The shRNA plasmids had no effect on hypertension in 5/6 A/I rats (mean arterial pressure: 153.9 ± 8.1 mmHg in C group vs. 154.7 ± 8.9 and 153.6±14 in T and T+P groups). However, the TRPC6 shRNA‐induced attenuation of albuminuria was eliminated by PHD3 shRNA. The levels of urinary albumin were 0.7±0.11 mg/Kg/24h in S, 35.6±6.7 in C, 4.9±1.5 in T, and 57.9±13.5 in T+P groups, respectively*. The regression slops between the blood pressure and the % of glomerulosclerosis were significantly steeper in C and T+P groups than in T group*, indicating that PHD3 shRNA abolished the protective effect of TRPC6 shRNA in hypertension‐induced glomerular injuries. These data suggested that hypertension‐induced activation of TRPC6 may be the molecular sensor of pressure stress to produce kidney damage associated with hypertension and that PHD3 is the downstream pathway of TRPC6 mediates the damage. * P&lt;0.05Support or Funding InformationNIH grants DK107991 to NL, DK102539 to JR and DK54927 to PLThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LncRNA SNHG1 correlates with higher T stage and worse overall survival, and promotes cell proliferation while reduces cell apoptosis in breast cancer.

BackgroundThe aim of this study was to investigate the correlation of long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) with the prognosis in breast cancer patients, and its effect on breast cancer cell proliferation and apoptosis.MethodsA total of 178 breast cancer patients were consecutively recruited, then tumor tissue and the paired adjacent tissue were obtained during surgery for lncRNA SNHG1 determination by quantitative polymerase chain reaction (qPCR). LncRNA SNHG1 expression was also measured in breast cancer cell lines and normal breast epithelial cell line. Subsequently, negative control (NC) overexpression plasmids, lncRNA SNHG1 overexpression plasmids, NC short hairpin RNA (shRNA) plasmids and lncRNA SNHG1 shRNA plasmids were transfected into MDA-MB-453 cells as well as MCF7 cells, and cell proliferation and apoptosis were measured afterward.ResultsLncRNA SNHG1 expression in tumor tissue was increased compared with paired adjacent tissue, and it correlated with higher T stage and worse overall survival (OS) in breast cancer patients. LncRNA SNHG1 expression was also elevated in breast cancer cell lines compared with normal breast epithelial cell line. Cell Counting Kit-8 (CCK8) assay revealed that lncRNA SNHG1 overexpression promoted while lncRNA SNHG1 shRNA reduced cell proliferation, and Annexin V-fluorescein isothiocyanate/propidium iodide staining (AV/PI) assay illustrated that lncRNA SNHG1 overexpression decreased while lncRNA SNHG1 shRNA increased cell apoptosis rate. In addition, Western Blot assay disclosed that lncRNA SNHG1 overexpression downregulated while lncRNA SNHG1 shRNA upregulated pro-apoptotic marker (C-Caspase3) expression, and lncRNA SNHG1 overexpression increased while lncRNA SNHG1 shRNA decreased anti-apoptotic marker (p-P38) expression.ConclusionsLncRNA SNHG1 is upregulated in tumor tissue and correlates with higher T stage and worse OS, and it promotes cell proliferation but inhibits cell apoptosis in breast cancer.

SLC5A1 promotes growth and proliferation of pancreatic carcinoma via glucose-dependent AMPK/mTOR signaling.

Background: Accumulating studies have reported that aberrant expression of SLC5A1 is a negative prognostic factor to various cancer patients.Purpose: Pancreatic cancer tissue has also shown to harbor higher expression of SLC5A1, however how SLC5A1 mediates pancreatic cancer cells growth remains unclear.Methods: In this study, we examined the mRNA and protein expressions of SLC5A1 in human pancreatic tissue and various cell lines. The in vitro and in vivo roles of SLC5A1 in pancreatic cancer were investigated through stably transfected pancreatic cells with shRNA plasmid targeting SLC5A1.Results: Our results observed SLC5A1 was over-expressed in human pancreatic cancer tissues as well as most pancreatic cancer cell lines. Both in vitro and in vivo inhibition of SLC5A1 retarded pancreatic cancer cell growth and progression. The SLC5A1 knockdown mediated growth suppression is mainly regulated by reduced cellular glucose uptake by pancreatic cancer cells. Our further mechanistic observation showed that inhibition of SLC5A1 induced AMPK-dependent mTOR suppression and pharmacological inhibition of AMPK rescued the effect of SLC5A1 blockade. Further protein-protein interaction analysis showed association of SLC5A1 with EGFR and knockdown of EGFR also showed decreased cellular survival and glucose uptake by pancreatic cancer cells.Conclusion: Our findings postulated SLC5A1/EGFR as the potential therapeutic target of pancreatic cancer patients.

The molecular mechanisms of Aloin induce gastric cancer cells apoptosis by targeting High Mobility Group Box 1.

Purpose: Aloin (ALO), a bioactive ingredient extracted from aloe vera, has anti-tumor effects. High Mobility Group Box 1 (HMGB1), a highly conserved nuclear DNA-binding protein, has been implicated in various cancer types. Highly expressed HMGB1 is closely associated with tumor cells apoptosis, proliferation and migration. We investigated the specific molecular mechanisms by which ALO-induced apoptosis by targeting HMGB1 in gastric cancer cells.Materials and methods: Human gastric cancer HGC-27 cells were treated with different doses of ALO (100, 200 and 400 µg/ml) for 24 h, after which DAPI staining was used to observe the nuclear morphology, Annexin V/PI double staining assay was used to determine the rate of apoptosis; Western blotting was used to detect the levels of PARP, pro-caspase3, HMGB1 and RAGE; nuclear translocation of HMGB1 was determined by conducting a nucleoplasm separation experiment. The Enzyme linked immunosorbent assay (ELISA) assay was used to detect release of HMGB1. The HGC-27 cells, transfected with HMGB1 shRNA plasmids, were stimulated with ALO for 24 h, after which a flow cytometry assay was used to detect the rate of apoptosis. HGC-27 cells were pre-treated with or without ALO and then stimulated with rhHMGB1, the phosphorylation of Akt, mTOR, P70S6K, S6, 4EBP1, ERK, P90RSK, cAMP regulatory element binding (CREB) were detected by Western blotting.Results: After different doses of ALO treatment, the nuclei showed morphological changes characteristic of apoptosis. Apoptotic rates were enhanced in a dose dependent manner. The level of cleaved PARP was enhanced and pro-caspase3, HMGB1 and RAGE levels were reduced, HMGB1 nuclear translocation and release were inhibited. The activation of rhHMGB1-induced Akt-mTOR-P70S6K and ERK-CREB signalling pathways was inhibited by ALO. Blocking these signalling pathways by special inhibitors and HMGB1 knockdown could enhance ALO-induced HGC-27 cell apoptosis.Conclusion: ALO- induced HGC-27 cell apoptosis by down-regulating expressions of HMGB1 and RAGE, inhibiting HMGB1 release and then suppressing rhHMGB1-induced activation of Akt-mTOR-P70S6K and ERK-P90RSK-CREB signalling pathways.