Sox9 mRNA Expression Research Articles

The effect of SOX9 on islet β cells in high glucose environment through regulation of ERK/P38 signaling pathway.

Islet beta cells are involved in insulin secretion. SRY-related high mobility group 9 (SX-S9) is involved in the progression of various diseases, but the role of SOX9 in islet β cells remains unclear. The islet β cell MIN6 cells were cultured in vitro and randomly divided into control group, high glucose group, and SOX9 siRNA group followed by analysis of SOX9 mRNA and protein expression by real-time PCR and Western blot, respectively, cell proliferation by MTT assay, cell apoptosis by flow cytometry, secretion of inflammatory factors TNF-α and IL-2 by ELISA, insulin secretion levels by spectrophotometer, myeloperoxidase (MPO), and superoxide dismutase (SOD) activities, as well as ERK/P38 signaling protein expression by Western blot. Under high glucose environment, SOX9 mRNA and protein expression were significantly increased, MIN6 cell proliferation was inhibited, apoptosis rate and secretion of TNF-α and IL-2 were increased, along with decreased insulin secretion, increased MPO content, decreased SOD activity and phosphorylation of ERK/P38, compared with control group (p < 0.05). However, transfection of SOX9 siRNA reduced SOX9 expression, promoted proliferation of MIN6 cells, decreased apoptotic rate and secretion of TNF-α and IL-2, increased insulin secretion, decreased MPO content, increased SOD and ERK/P38 protein phosphorylation. Compared with high glucose group, the differences were statistically significant (p < 0.05). The expression of SOX9 is increased under high glucose environment. Down-regulation of SOX9 expression can inhibit islet cell apoptosis, oxidative stress and inflammation, and promote islet cell proliferation and insulin secretion by regulating ERK/P38 signaling pathway.

Herpes Simplex Virus Type-1 Infection Impairs Adult Hippocampal Neurogenesis via Amyloid-β Protein Accumulation

We previously reported that Herpes simplex virus type-1 (HSV-1) infection of cultured neurons triggered intracellular accumulation of amyloid-β protein (Aβ) markedly impinging on neuronal functions. Here, we demonstrated that HSV-1 affects in vitro and in vivo adult hippocampal neurogenesis by reducing neural stem/progenitor cell (NSC) proliferation and their neuronal differentiation via intracellular Aβ accumulation. Specifically, cultured NSCs were more permissive for HSV-1 replication than mature neurons and, once infected, they exhibited reduced proliferation (assessed by 5'-bromo-deoxyuridine incorporation, Ki67 immunoreactivity, and Sox2 mRNA expression) and impaired neuronal differentiation in favor of glial phenotype (evaluated by immunoreactivity for the neuronal marker MAP2, the glial marker glial fibrillary astrocyte protein, and the expression of the proneuronal genes Mash1 and NeuroD1). Similarly, impaired adult neurogenesis was observed in the subgranular zone of hippocampal dentate gyrus of an in vivo model of recurrent HSV-1 infections, that we recently set up and characterized, with respect to mock-infected mice. The effects of HSV-1 on neurogenesis did not depend on cell death and were due to Aβ accumulation in infected NSCs. Indeed, they were: (a) reverted, in vitro, by the presence of either β/γ-secretase inhibitors preventing Aβ production or the specific 4G8 antibody counteracting the action of intracellular Aβ; (b) not detectable, in vivo, in HSV-1-infected amyloid precursor protein knockout mice, unable to produce and accumulate Aβ. Given the critical role played by adult neurogenesis in hippocampal-dependent memory and learning, our results suggest that multiple virus reactivations in the brain may contribute to Alzheimer's disease phenotype by also targeting NSCs. Stem Cells 2019;37:1467-1480.

Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor β-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, β-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of β-glycerophosphate on SOCE. Moreover, the β-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

Gene expression profile of Sox2 and Nestin both were regulated by histone deacetylase in C17.2 neural stem cells

Histone deacetylation is a key modulator involved in proliferation, apoptosis and mRNA transcription . However, the effects of histone deacetylation in C17.2 neural stem cells (NSCs) are still not fully understood. In this study, C17.2 NSCs were used to evaluate the effects of nicotinamide and trichostatin A (TSA) on the transcription of several important genes. Here, we confirmed that the mRNA expression of sox1, sox2, p53 and bax in E14.5 NSCs were significantly higher than in C17.2 NSCs. There was no difference between E14.5 and C17.2 NSCs in the transcription of nestin. The transcription levels of p53 and nestin were significantly higher in C17.2 NSCs than in cerebral cortex, hippocampus, cerebellar cortex and olfactory bulb. The expression levels of sox1, sox2 and bax were upregulated in olfactory bulb as compared to the other three brain tissues. To further assess the effects of histone deacetylation in C17.2 NSCs, we used two histone deacetylase (HDAC) inhibitors, nicotinamide and TSA, to explore the differential changes in the expression of genes. The results indicated that the mRNA transcription of sox2, p53, nestin and bax was generally downregulated after exposure to nicotinamide and TSA, but the difference was statistically significant only for sox2 and nestin. In addition, there was no transcription of sox1. Thus our studies demonstration that C17.2 NSCs have different mRNA expression profiles compared to E14.5 NSCs and adult mouse brain tissues. Furthermore, HDAC inhibitors nicotinamide and TSA regulate gene expression through different histone acetylation.

Impact of Maternal Malnutrition on Gut Barrier Defense: Implications for Pregnancy Health and Fetal Development.

Small intestinal Paneth cells, enteric glial cells (EGC), and goblet cells maintain gut mucosal integrity, homeostasis, and influence host physiology locally and through the gut-brain axis. Little is known about their roles during pregnancy, or how maternal malnutrition impacts these cells and their development. Pregnant mice were fed a control diet (CON), undernourished by 30% vs. control (UN), or fed a high fat diet (HF). At day 18.5 (term = 19), gut integrity and function were assessed by immunohistochemistry and qPCR. UN mothers displayed reduced mRNA expression of Paneth cell antimicrobial peptides (AMP; Lyz2, Reg3g) and an accumulation of villi goblet cells, while HF had reduced Reg3g and mucin (Muc2) mRNA and increased lysozyme protein. UN fetuses had increased mRNA expression of gut transcription factor Sox9, associated with reduced expression of maturation markers (Cdx2, Muc2), and increased expression of tight junctions (TJ; Cldn-7). HF fetuses had increased mRNA expression of EGC markers (S100b, Bfabp, Plp1), AMP (Lyz1, Defa1, Reg3g), and TJ (Cldn-3, Cldn-7), and reduced expression of an AMP-activator (Tlr4). Maternal malnutrition altered expression of genes that maintain maternal gut homeostasis, and altered fetal gut permeability, function, and development. This may have long-term implications for host-microbe interactions, immunity, and offspring gut-brain axis function.

RETRACTED ARTICLE: Down-regulated lncRNA DLX6-AS1 inhibits tumorigenesis through STAT3 signaling pathway by suppressing CADM1 promoter methylation in liver cancer stem cells

BackgroundLiver cancer stem cells (LCSCs) are a small subset of cells characterized by unlimited self-renewal, cell differentiation, and uncontrollable cellular growth. LCSCs are also resistant to conventional therapies and are thus believed to be held responsible for causing treatment failure of hepatocellular carcinoma (HCC). It has been recently found that long non-coding RNAs (lncRNAs) are important regulators in HCC. This present study aims to explore the underlying mechanism of how lncRNA DLX6-AS1 influences the development of LCSCs and HCC.MethodsA microarray-based analysis was performed to initially screen differentially expressed lncRNAs associated with HCC. We then analyzed the lncRNA DLX6-AS1 levels as well as CADM1 promoter methylation. The mRNA and protein expression of CADM1, STAT3, CD133, CD13, OCT-4, SOX2, and Nanog were then detected. We quantified our results by evaluating the spheroid formation, proliferation, and tumor formation abilities, as well as the proportion of tumor stem cells, and the recruitment of DNA methyltransferase (DNMT) in LCSCs when lncRNA DLX6-AS1 was either overexpressed or silenced.ResultsLncRNA DLX6-AS1 was upregulated in HCC. The silencing of lncRNA DLX6-AS1 was shown to reduce and inhibit spheroid formation, colony formation, proliferation, and tumor formation abilities, as well as attenuate CD133, CD13, OCT-4, SOX2, and Nanog expression in LCSCs. Furthermore, downregulation of lncRNA DLX6-AS1 contributed to a reduction in CADM1 promoter methylation via suppression of DNMT1, DNMT3a, and DNMT3b in LCSCs and inactivating the STAT3 signaling pathway.ConclusionThis study demonstrated that down-regulated lncRNA DLX6-AS1 may inhibit the stem cell properties of LCSCs through upregulation of CADM1 by suppressing the methylation of the CADM1 promoter and inactivation of the STAT3 signaling pathway.

Hepatic progenitor cell activation is induced by the depletion of the gut microbiome in mice.

The homeostasis of the gut microbiome is crucial for human health and for liver function. However, it has not been established whether the gut microbiome influence hepatic progenitor cells (HPCs). HPCs are capable of self‐renewal and differentiate into hepatocytes and cholangiocytes; however, HPCs are normally quiescent and are rare in adults. After sustained liver damage, a ductular reaction occurs, and the number of HPCs is substantially increased. Here, we administered five broad‐spectrum antibiotics for 14 days to deplete the gut microbiomes of male C57BL/6 mice, and we measured the plasma aminotransferases and other biochemical indices. The expression levels of two HPC markers, SRY‐related high mobility group‐box gene 9 (Sox9) and cytokeratin (CK), were also measured. The plasma aminotransferase activities were not affected, but the triglyceride, lactate dehydrogenase, low‐density lipoprotein, and high‐density lipoprotein concentrations were significantly altered; this suggests that liver function is affected by the composition of the gut microbiome. The mRNA expression of Sox9 was significantly higher in the treated mice than it was in the control mice (p < 0.0001), and a substantial expression of Sox9 and CK was observed around the bile ducts. The mRNA expression levels of proinflammatory factors (interleukin [IL]‐1β, IL‐6, tumor necrosis factor [TNF]‐α, and TNF‐like weak inducer of apoptosis [Tweak]) were also significantly higher in the antibiotic‐treated mice than the levels in the control mice. These data imply that the depletion of the gut microbiome leads to liver damage, negatively impacts the hepatic metabolism and function, and activates HPCs. However, the underlying mechanisms remain to be determined.

MicroRNA-30e inhibits proliferation and invasion of non-small cell lung cancer via targeting SOX9.

Previous studies have reported that microRNA-30e (miR-30e) is dysregulated in multiple human cancers. However, the expression, functions and molecular mechanism of miR-30e in NSCLC remain unknown. In this study, we found that miR-30e was expressed at a low level in NSCLC tissues and cell lines. In NSCLC cell lines, enforced expression of miR-30e could inhibit cell proliferation and invasion in vitro. In addition, miR-30e negatively regulated SOX9 expression through directly binding to the 3'UTR of SOX9, and an inverse correlation was found between miR-30e and SOX9 mRNA expression in NSCLC tissues. Moreover, knockdown of SOX9 led to decreased proliferation and invasion of NSCLC cells. Taken together, miR-30e acts as a tumor suppressor in NSCLC, and inhibits cell proliferation and invasion possibly by directly targeting SOX9. These findings might provide novel therapeutic targets for NSCLC.

Activation of Estrogen Receptor Alpha by Decitabine Inhibits Osteosarcoma Growth and Metastasis.

Osteosarcoma is a malignant tumor in the bone, which originates from normal osteoblasts or osteoblast precursors. Normal osteoblasts express estrogen receptor alpha (ERα); however, osteosarcomas do not express ERα due to promoter DNA methylation. Here we show that treatment of 143B osteosarcoma cells with decitabine (DAC, 5-Aza-2'-deoxycytidine) induces expression of ERα and leads to decreased proliferation and concurrent induction of osteoblast differentiation. DAC exposure reduced protein expression of metastasis-associated markers VIMENTIN, SLUG, ZEB1, and MMP9, with a concurrent decrease in mRNA expression of known stem cell markers SOX2, OCT4, and NANOG. Treatment with 17β-estradiol (E2) synergized with DAC to reduce proliferation. Overexpression of ERα inhibited proliferation and induced osteoblast differentiation, whereas knockout of ERα by CRISPR/Cas9 prevented the effects of DAC. In an orthotopic model of osteosarcoma, DAC inhibited tumor growth and metastasis of 143B cells injected into the tibia of NOD SCID gamma mice. Furthermore, ERα overexpression reduced tumor growth and metastasis, and ERα knockout prevented the effects of DAC in vivo. Together, these experiments provide preclinical evidence that the FDA-approved DNA methylation inhibitor DAC may be repurposed to treat patients with osteosarcoma based on its efficacy to decrease proliferation, to induce osteoblast differentiation, and to reduce metastasis to visceral organs.Significance: These findings describe the effects of DNA methyltransferase inhibition on ERα and its potential role as a tumor suppressor in osteosarcoma.See related commentary by Roberts, p. 1034 See related article by El Ayachi and colleagues; Cancer Res 79(5);982-93.

Simvastatin promotes restoration of chondrocyte morphology and phenotype

In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol—conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.

Epigenetic mechanisms are behind the regulation of the key genes associated with the osteoblastic differentiation of the mesenchymal stem cells: The role of zoledronic acid on tuning the epigenetic changes.

Mesenchymal stem cells (MSCs) are multipotent stem cells and show distinct features such as capability for self-renewal and differentiation into several lineages of cells including osteoblasts, chondrocytes, and adipocytes. In this study, the methylation status of the promoter region of zinc finger and BTB domain containing 16 (ZBTB16), twist-related protein 1(Twist1), de novo DNA methyltransferases 3A (DNMT3A), SRY-box 9 (Sox9), osteocalcin (OCN), and peroxisome proliferator-activated receptor γ2 (PPARγ2) genes and their messenger RNA (mRNA) expression levels were evaluated during the osteoblastic differentiation of MSCs (ODMSCs). We planned two experimental groups including zoledronic acid (ZA)-treated and nontreated cells (negative control) which both were differentiated into the osteoblasts. Methylation level of DNA in the promoter regions was assayed by methylation-specific-quantitative polymerase chain reaction (MS-qPCR), and mRNA levels of the target inhibitory/stimulatory genes during osteoblastic differentiation of MSCs were measured using real-time PCR. During the experimental induction of ODMSCs, the mRNA expression of the OCN gene was upregulated and methylation level of its promoter region was decreased. Moreover, Sox9 and PPARγ2 mRNA levels were attenuated and their promoter regions methylation levels were significantly augmented. However, the mRNA expression of the DNMT3A was not affected during the ODMSCs though its methylation rate was increased. In addition, ZA could enhance the expression of the ZBTB16 and decrease its promoter regions methylation and on the opposite side, it diminished mRNA expression of Sox9, Twist1, and PPARγ2 genes and increased their methylation rates. Intriguingly, ZA did not show a significant impact on gene expression and methylation levels the OCN and DNMT3A. We found that methylation of the promoter regions of Sox9, OCN, and PPARγ2 genes might be one of the main mechanisms adjusting the genes expression during the ODMSCs. Furthermore, we noticed that ZA can accelerate the MSCs differentiation to the osteoblast cells via two regulatory processes; suppression of osteoblastic differentiation inhibitor genes including Sox9, Twist1, and PPARγ2, and through promotion of the ZBTB16 expression.

Mechanical Stimulation Promotes the Proliferation and the Cartilage Phenotype of Mesenchymal Stem Cells and Chondrocytes Co-Cultured &lt;i&gt;in vitro&lt;/i&gt;

Mesenchymal stem cells and chondrocytes are an important source of the cells for cartilage tissue engineering. Therefore, the culture and expansion methods of these cells need to be improved to overcome the aging of chondrocytes and induced chondrogenic differentiation of mesenchymal stem cells. The aim of this study was to expand the cells for cartilage tissue engineering by combining the advantages of growing cells in co-culture and under a mechanically-stimulated environment. Rabbit chondrocytes and co-cultured cells (bone mesenchymal stem cells and chondrocytes) were subjected to cyclic sinusoidal dynamic tensile mechanical stimulationusing the FX-4000 tension system. Chondrocyte proliferation was assayed by flow cytometry and CFSE labeling. The cell cartilage phenotype was determined by detecting GAG, collagen II and TGFβ1 protein expression by ELISA and the Col2α1, TGF-β1 and Sox9 mRNA expression by RT-PCR. The results show that the co-culture improved both the proliferation ability of chondrocytes and the cartilage phenotype of co-cultured cells. A proper cyclic sinusoidal dynamic tensile mechanical stimulation improved the proliferation ability and cartilage phenotype of chondrocytes and co-cultured cells. These results suggest that the coculture of mesenchymal stem cells with chondrocytes and proper mechanical stimulation may be an appropriate way to rapidly expand the cells that have an improved cartilage phenotype for cartilage tissue engineering. Funding Statement: This study was supported by the National Natural Science Foundation of China (11802209), the Natural Science Foundation of Shandong Province China (ZR2017PA011), the Medical and Health Technology Development Project of Shandong Province China (2016WS0669) and the Visiting Scholar Program of Weifang Medical University. Declaration of Interests: The authors have no proprietary interest in any of the materials discussed in this article. Ethics Approval Statement: The experimental protocols were approved by the Animal Care and Protection Committee of Weifang Medical University. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.

Correlation of SOX9 and NM23 genes with the incidence and prognosis of prostate cancer.

Correlation of sex determining region Y-box (SOX)9 and NM23 genes with the incidence and prognosis of prostate cancer (PC) was investigated. SOX9-small interfering ribonucleic acid (siRNA) and NM23-siRNA were constructed and transfected into PC-3 cells. The expression levels of SOX9 and NM23 messenger RNAs (mRNAs) and proteins in PC-3 cells were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. MTT assay was adopted to examine the proliferation ability of the transfected cells, and Transwell assay was applied to detect the migration ability of the transfected cells. Sixty-three patients with PC and 56 patients with benign prostatic hyperplasia who were treated in Huadu District People's Hospital were enrolled. Correlation analyses were conducted for the relative expression levels of SOX9 and NM23 and the Gleason grade; and the survival curves of the patient SOX9/NM23 (S/N) Cq values were plotted. The proliferation and migration abilities of PC-3 cells were remarkably reduced after low expression of SOX9 (P<0.01), while those of PC-3 cells were significantly improved after low expression of NM23 (P<0.01). Compared with that in tissues of PC patients, the relative expression of SOX9 mRNA in patients with benign prostatic hyperplasia was obviously decreased (P<0.01), while that of NM23 mRNA was significantly elevated (P<0.01). The larger the S/N was, the shorter the patient's survival time would be (P<0.05). The low expression of SOX9 gene can significantly reduce the proliferation and migration abilities of PC cells, which is negatively associated with the incidence and prognosis of patients. The low expression of NM23 gene can markedly enhance the proliferation and migration abilities of PC cells, and it is positively related to the incidence and prognosis of patients.

Sox2 is associated with cancer stem-like properties in colorectal cancer

Sox2 is known as the undifferentiated cell marker. Recent studies have shown that Sox2 may also be involved in the maintenance of cancer stem cells (CSCs) in skin and bladder cancers. In this study, we aimed to clarify the role of Sox2 in colorectal CSCs. Sox2 expression was measured in colon cancer cells and colorectal clinical samples by qRT-PCR and western blot analysis. To visualize the active Sox2 mRNA production, we generated a Sox2 promoter-dependent DsRed fluorescence emission system. Colon cancer cell lines and colorectal tumor tissues generally expressed the Sox2 protein. Knockdown of Sox2 by siRNA led to increased proliferative activity in Caco2 cells. Kaplan-Meier survival curves showed that the group with high Sox2 mRNA expression had a worse prognosis for relapse-free survival (RFS) than the low expression group (P = 0.045, median follow-up 60.0 months). Time-lapse image analysis revealed that most DsRed+ cells exhibited typical asymmetric cell division and had higher CSC marker expressions. The DsRed+ cells exhibited chemoresistance and they grew slower in vitro, yet they established rather larger tumors in vivo. Our data suggest that Sox2 may be a potential biomarker for colorectal CSCs.

Retraction: MicroRNA-371-5p targets SOX2 in gastric cancer.

[This retracts the article DOI: 10.18632/oncotarget.8289.].

Potential Effect of Simvastatin as an Anti-Cancer Agent on SOX7 and SOX9 Expression in Prostate Cancer Cell Lines

BackgroundProstate cancer (PCa) is the second leading cause of cancer death in men, worldwide. Geranylgeranylation and farnesylation have a main role in the carcinogenic process, which can be prevented via statins as HMGCOA reductase enzyme inhibitors in cholesterol biosynthesis. These effects might be controlled by several transcription factors such as SOX7 and SOX9, which have been involved in PCa initiation and progression. To the best of our knowledge, no study has demonstrated the association of simvastatin and SOX status in PCa. Therefore, this study is an attempt to evaluate whether simvastatin induces anti-neoplastic effects via the SOX9 and SOX7 transcription factors.MethodsProstate cancer cell lines LNCaP and PC3 were used to evaluate the expression of SOX7 and SOX9 using quantitative RT-PCR.ResultsOur data was analyzed by applying one-way ANOVA and Tukey's test and determined that 0.07 µM of simvastatin after 24 h was sufficient to upregulate SOX7 mRNA expression ratio by 3.58 fold in LNCaP. In addition, the level of SOX9 mRNA expression was increased by 12.18 fold at 0.07 µM after 24 h, 8.67 fold at 0.001 µM after 24 h, and 6.33 fold at 0.07 µM after 12 h in LNCaP and in PC3 cell line. The level of SOX9 mRNA expression was increased by 2.64 fold at 0.5 µM after 24 h and 2.78 fold at 0.1 µM after 12 h, however, it decreased by 0.67 fold at 0.1 µM after 24 h.ConclusionsOur findings suggest that simvastatin can induce the anti-cancer properties via manipulating the expression of SOX7 in LNCaP, as the androgen-dependent cell.

Different murine-derived feeder cells alter the definitive endoderm differentiation of human induced pluripotent stem cells.

The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.

VEGFC/VEGFR3 axis mediates TGFβ1-induced epithelial-to-mesenchymal transition in non-small cell lung cancer cells.

In the tumor progression, transforming growth factor β1 (TGFβ1) plays a critical role in tumorigenesis as well as metastasis. It is known that high plasma level of TGFβ1 in patients with advanced non-small cell lung cancer (NSCLC) is correlated with poor prognostics. In addition, the generation of cancer stem-like cells is associated with metastasis, drug resistance, and tumor recurrence, which also lead to poor outcomes in NSCLC patients. However, it remains unclear how TGFβ1 promotes NSCLC cells to acquire stem-like properties and accelerate tumor metastasis. In our study, we found that short term TGFβ1 treatment resulted in a significant epithelial-mesenchymal transition (EMT) morphological change in TGFβ1–sensitive NSCLC cells but not in insensitive cells. Western blotting confirmed increased Vimentin and reduced E-Cadherin protein expression after TGFβ1 treatment in A549, NCI-H1993, and NCI-H358 cells. TGFβ1 incubation dramatically decreased in vitro cell proliferation and increased cell invasion in TGFβ1–sensitive NSCLC cells but not in NCI-H1975, NCI-H1650, and HCC827 cells. Moreover, TGFβ1 was able to enhance the mRNA expression of Oct4, Nanog and Sox2 and drastically increased anchorage-independent colony formation in TGFβ1–sensitive NSCLC cells, suggesting the acquisition of cancer stem-like properties. Interestingly, we found that vascular endothelial growth factor receptor 3 (VEGFR3) mRNA expression was significantly elevated in TGFβ1–sensitive NSCLC cells compared to insensitive cells. And TGFβ1 was capable of inducing VEGF-C gene expression. Pharmacological blocking TGFβ type I receptor kinase (ALK5) significantly inhibited TGFβ1-induced VEGF-C expression. Silencing of ALK5 by siRNA also dramatically reduced TGFβ1-induced VEGF-C expression in TGFβ1–sensitive NSCLC cells. Therefore, TGFβ1 contributes for NSCLC metastasis through promoting EMT, generation of high invasive cancer cells with stem-like properties, and increasing VEGF-C expression. Blocking TGFβ pathway is a potential therapeutic target in human non-small cell lung cancer.

Expression and correlation of MALAT1 and SOX9 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths in the world. MALAT1 and SOX9 have important roles in tumour formation and development in several types of cancers. However, little is known about the function and co-relationship of these 2 factors in NSCLC in vivo. To explore the role of MALAT1 and SOX9 expression relationship, their clinical pathological characteristics and OS on NSCLC patients. Paired of primary lung cancer tissues and the matched tumour adjacent tissues were collected in 121 NSCLC patients. MALAT1 and SOX9 mRNA expression was measured by SYBR green q RT-PCR assay. SOX-9 protein expression was measured by streptavidin-peroxidase (SP) staining method. MALAT1and SOX9 expression was higher in NSCLC tissues than the adjacent tissues, and they have positive correlation. Moreover, SOX9 protein expression was higher in NSCLC tissues, especially in MALAT1 mRNA higher expressed NSCLC tissues. MALAT1 and SOX9 mRNA expression were associated with age (x2 =11.474, P = .009), tumour size (x2 =26.839, P = .000), TNM stage (x2 =8.010, P = .046) and LEL. (x2 =53.908, P = .000). NSCLC patients with higher MALAT1 and SOX9 mRNA expression had poorer OS rates. MALAT1 and SOX9 could be used as prognostic co-biomarker in NSCLC.

Synthesis of photo-reactive poly (vinyl alcohol) and construction of scaffold-free cartilage like pellets in vitro.

Photo-reactive poly(vinyl alcohol) (PRPVA) was synthesized by introduction of phenyl azido groups into poly(vinyl alcohol) (PVA) and applied for surface modification. PRPVA was grafted onto cell culture plate surface homogeneously or in a micropattern. Human mesenchymal stem cells (hMSCs) cultured on cell culture plate surface and PVA-modified surface showed different behaviors. Cells adhered and spread well on cell culture plate surface, while they did not adhere on PVA-grafted surface at all. When hMSCs were cultured on PVA-micropatterned surface, they formed a cell micropattern. Cells formed pellets after cultured on PVA homogeneously modified surface in chondrogenic induction medium for 2 weeks. The pellets were positively stained by hematoxylin/eosin, safranin-O/fast green and toluidin blue, and they were also stained brown by Type II collagen and proteoglycan immunohistological staining. Real-time PCR analysis was conducted to investigate the expression of colI, colII, colX, aggrecan and sox9 mRNA. Results of gene expression were in agreement with those of histological and immunohistological observations. These results indicated that hMSCs cultured on PVA-modified surface performed chondrogenic differentiation, and it was possible to construct scaffold-free cartilage like pellets with PVA-modified surface in vitro.