SOX2 Protein Expression Research Articles

The anti-tumor effect of taxifolin on lung cancer via suppressing stemness and epithelial-mesenchymal transition in vitro and oncogenesis in nude mice.

BackgroundTaxifolin is a natural flavonoid with anti-oxidant and anti-proliferative properties. In this study, we investigated the stemness-related inhibitory effects of taxifolin in two lung cancer cell lines, A549 and H1975, as well as in A549 xenografts.MethodsA549 and H1975 cells, as well as A549 xenograft BALB/c mice were treated with taxifolin. Cell viability, stemness, mobility and protein expression were tested with Cell counting kit-8 (CCK-8), Colony formation assay, Flow cytometry, Transwell, Western blot and Immunohistochemistry, respectively.ResultsCCK-8 exhibited an obvious toxicity of taxifolin to both cell lines at higher dose. Then taxifolin of 0, 25, 50, and 100 µM/L were subsequently used. Taxifolin exhibited inhibitory effects on stemness and sphere formation, reduced protein expression of SOX2 and OCT4, and reduced CD133-positive cells. Furthermore, taxifolin decreased invasive cells, reduced N-cadherin and vimentin while increased E-cadherin expression, indicating that epithelial-mesenchymal transition (EMT) was inhibited. All of the effects observed were exhibited in a dose-dependent manner, and A549 cells proved to be more sensitive to taxifolin than H1975 cells. Taxifolin inactivated PI3K and TCF4 protein phosphorylation; however, taxifolin was not observed to have an effect on NF-κB P65 or STAT3. Taxifolin also suppressed tumor growth in A549 xenograft BALB/c mice, with decreased SOX2 and OCT4 expression and inhibited PI3K and TCF4.ConclusionsIn summary, taxifolin inhibited stemness and EMT in lung cancer cells possibly via the inactivation of PI3K and OCT4. Taxifolin could be a potential prodrug or serve as an adjuvant in lung cancer treatment.

Neofunctionalized gene RTl3 and SOX9 collectively modulate COL2A1 expression in human articular chondrocytes

Purpose: Retrotransposon-derived DNA is a known source of genetic variation, but the biological significance of these molecules in chondrocytes, the resident cell type present in cartilage, is still unknown. A mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 was found to be differentially expressed in human OA cartilage vs normal samples but the function of RTL3 in cartilage remained unexplored. The goal of our study was to determine the functional role of the mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 in human chondrocytes. Methods: These studies were approved by the institutional review boards (IRBs) of Northeast Ohio Medical University and Summa Health Systems (Akron, OH) prior to the initiation of the studies. Chondrocytes were isolated by sequential enzymatic digestion of discarded and de-identified cartilage samples from patients undergoing total joint arthroplasty. Total RNA was isolated using Trizol. mRNA expression was assessed via Taqman assays and protein expression by immunoblotting using validated antibodies. Human RTL3 and SOX-9 expression was depleted using validated siRNAs (GE Dharmacon ONTARGETplus) and X-tremeGENE siRNA Transfection Reagent (SigmaAldrich). For ectopic expression, human chondrocytes were transfected with a RTL3 expression plasmid (Origene) using X-tremeGENE HP DNA Transfection Reagent (SigmaAldrich). COL2A1 promoter reporter assays employed a pCOL2A1-luciferase reporter (GeneCopoeia) and the luciferase activity normalized with a Renilla reporter (Promega). Statistical analyses were conducted using student t-tests or one-way analysis of variation (ANOVA) with Tukey’s post-hoc tests. Results: RTL3 contains a nucleic acid binding domain so we first determined if RTL3 was directly involved in regulating the expression of chondrocyte signature genes expression through gain of function and loss of function analyses. Upon RTL3 depletion, COL2A1 mRNA and protein levels were significantly reduced and activity of the COL2A1 promoter reporter was also significantly decreased. Transfection with an RTL3 expression plasmid under the control of the CMV promoter significantly increased the COL2A1 mRNA and protein levels and enhanced COL2A1 promoter reporter activity was observed in a dose-dependent manner. Together,these results indicated that RTL3 has a direct role in regulating chondrocyte expression of COL2A1. To determine a possible mode of action, we determined if RTL3 modulates COL2A1 expression via known COL2A1 regulator SOX-9. SOX-9 depletion reduced expression of COL2A1 as well as COL2A1 promoter reporter activity. When RTL3 was depleted, constitutive expression of SOX-9 mRNA and protein were significantly reduced compared to controls and the activity of the COL2A1 promoter reporter was also inhibited. Conversely, over-expression of RTL3 was able to significantly enhance mRNA and protein levels of SOX-9 in chondrocytes and increase COL2A1 promoter reporter activity in vitro. Interestingly, in chondrocytes with SOX-9 knockdown and RTL3 overexpression, COL2A1 promoter activity was half that of cells expressing endogenous SOX-9. These results indicate RTL3 or SOX9 alone are not sufficient for optimal expression of COL2A1 but both act in concert to to regulate COL2A1 expression in chondrocytes. Conclusions: The mammalian retrotransposon RTL3 has lost the ability to retrotranspose but has retained an open reading frame suggestive of new cellular functions. Here, we utilized loss-of-function and gain-of-function approaches to demonstrate a previously unknown role of the mammalian retrotransposon transcript RTL3 in the regulation of COL2A1 in human chondrocytes in vitro. siRNA-depletion of RTL3 reduced constitutive SOX-9 expression, pCOL2A1 promoter reporter activity and COL2A1 transcription. Forced expression of RTL3 rescued these effects. SOX-9 siRNA-depletion and RTL3 over expression experiments elucidated that expression of both RTL3 and SOX-9 are important for COL2A1 regulation in chondrocytes. The details of this previously unknown mechanism of regulation warrants further study.

SOX2 is a positive regulator of osteoclast differentiation

Elucidating the mechanism underlying osteoclast differentiation is important to improve our understanding of the pathophysiologies related to skeletal diseases and osteolytic metastasis in cancer. Sex-determining region Y-box containing gene 2 (SOX2), a stemness marker, is known to affect osteoblast differentiation and cancer metastasis. However, its role in osteoclastogenesis has not been investigated to date. Here, we report that SOX2 protein and mRNA expression was upregulated during osteoclast differentiation. The overexpression or knockdown of SOX2 in osteoclast precursor cells enhanced or suppressed, respectively, receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and migration, and nuclear factor of activated T-cell c1 (NFATc1) and factor-associated suicide ligand (FASL) expression. In addition, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) activation were regulated by SOX2 expression; both EGFR and ERK inhibitors abrogated the SOX2 overexpression-induced increase in osteoclast differentiation and NFATc1 expression under RANKL stimulation. Overall, these results suggest SOX2 as a positive regulatory factor during osteoclast differentiation partly through the EGFR and ERK signaling pathways, highlighting a new potential target for restoring abnormal osteoclast activation.

Abstract WP161: Sovateltide Promotes Regeneration and Functional Recovery of Ischemic Brain by Improving Mitochondrial Function

Mitochondrial dysfunction is associated with cerebral ischemic stroke. Sovateltide (IRL-1620; PMZ-1620) undergoing clinical trials (NCT04047563; NCT04046484) has been demonstrated, in preclinical studies, to provide recovery of brain functions when administered following acute cerebral ischemic stroke. We hypothesize that sovateltide preserves mitochondrial activity and provides neural protection and regeneration. We investigated the effect of sovateltide on mitochondrial morphology and function following acute cerebral ischemic stroke. Rats underwent permanent middle cerebral artery occlusion (MCAO) and then received three intravenous injections of vehicle or sovateltide (5 μg/kg) at 4, 6, and 8 h and sacrificed at 24 h post MCAO. Another set of rats were treated similarly on day 0, 3 and 6 and sacrificed on day 7. Western blot and/or immunofluorescence were used to evaluate protein expression of NeuroD1, DoubleCortin, HUC+HUD, NeuN, Sox2, Oct4, DRP1 and MFN2. Mitochondrial biogenesis was evaluated using in situ PCR and transmission electron microscopy (TEM). Sovateltide produced (p<0.001) improvement in neurological deficit and motor function compared to vehicle at 24 h and day 7 post MCAO. Expression of NeuroD1 (p=0.0003), HuC-HuD (p=0.0373) and DoubleCortin (p=0.013) was higher in sovateltide compared to vehicle at 24 h but not at day 7. No change in Oct4 and Sox2 was observed. Immunofluorescence of cultured NPCs showed elevated expression of NeuroD1 and NeuN in sovateltide compared to vehicle. Sovateltide significantly decreased fission marker, DRP1 (p<0.001) and increased fusion marker MFN2 (p<0.0001) at 24 h and day 7 compared to vehicle. A significantly increased mitochondrial DNA (MT-ATP8, mitochondria specific gene) fluorescence in sovateltide group compared to vehicle (p=0.0418) and sham (p=0.0085) was observed. TEM analysis showed significant increase in mitochondrial cross-sectional area x number and % mitochondrial/tissue area in sovateltide group at 24 hours compared to vehicle. Mitochondrial number increased (p=0.032) at day 7 compared to 24 h in vehicle group. Sovateltide preserves mitochondrial activity and provides neural protection and regeneration in rat model of acute cerebral ischemic stroke.

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

BackgroundContinuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS.MethodsHuman bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting.ResultscLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2.ConclusionsCollectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

MiR-145-5p attenuates paclitaxel resistance and suppresses the progression in drug-resistant breast cancer cell lines.

miR-145-5p has been identified as a tumor suppressor involved in a wide variety of human cancers. Herein, we aimed to further explore the functional role and molecular mechanism of miR-145-5p on breast cancer (BC) progression and chemoresistance. The expressions of miR-145-5p and sex determining region Y-box2 (SOX-2) mRNA were assessed by qRT-PCR assay. SOX2 protein expression was measured using western blot. CCK-8 assays were used to determine the IC50 values of paclitaxel (PTX) and cell proliferation. Transwell assays were performed to detect cell migration and invasion. The target of miR-145-5p was verified by dual-luciferase reporter assays. Xenograft model was used to observe the role of miR-145-5p in vivo. The results showed that miR-145-5p was downregulated in BC tissues and cells and associated with PTX resistance of BC cells. Overexpression of miR-145-5p or SOX2 knockdown repressed the proliferation, migration, invasion, and attenuated PTX resistance in PTX-resistant BC cells. Mechanistically, miR-145-5p negatively regulated SOX2 expression by targeting SOX2. The inhibitory effects of miR-145-5p on the proliferation, migration, invasion, and PTX resistance were antagonized by SOX2 level restoration in PTX-resistant BC cells. Additionally, miR-145-5p repressed tumor growth in vivo. In conclusion, our study suggested that miR-145-5p reduced PTX resistance and repressed the progression at least partly by targeting SOX2 in PTX-resistant BC cells, highlighting miR-145-5p as a promising biomarker for BC treatment.

Differential expression of Sox9 protein and proteoglycans in the epiphyseal cartilage of bromocriptine-treated pregnant and lactating rats.

Several investigations have shown that pregnancy and lactation are able to induce elongation of long bone by altering epiphyseal cartilage function in a prolactin-dependent manner. Since the transcription factor Sox9 is of utmost importance for chondrocyte proliferation and differentiation and since bromocriptine, a dopaminergic D2 agonist widely used to suppress milk production, is known to disrupt the production and release of prolactin, we herein aimed to investigate whether pregnancy and lactation as well as bromocriptine could alter the expression of Sox9. Our immunohistochemical analysis showed that the Sox9 expression levels were markedly upregulated in the tibial proliferative zone of day 21 pregnant rats. In day 8 (early) and day 14 (mid) lactating rats, the Sox9 expression was enhanced only in the proliferative zone, but not in the resting and hypertrophic zones. There was no change in Sox9 expression in day 21 (late) lactating rats. Postweaning rats manifested a decreased Sox9 expression in the hypertrophic zone. Bromocriptine had no effect on Sox9 expression in the proliferative zone of day 21 pregnant rats; however, it completely prevented the Sox9 upregulation in those of early and mid-lactating rats. A differential response was observed in the proliferative and hypertrophic zones of late lactating rats, in which bromocriptine enhanced Sox9 expression. Further investigation of cartilaginous matrix revealed no change in proteoglycans accumulation in lactating rats. In conclusion, the upregulated Sox9 expression predominantly occurred in the proliferative zone during late pregnancy and early and mid-lactation, while the bromocriptine effects depended on the periods and epiphyseal zones.

Increased Expression of Gankyrin and Stemness Factor Oct-4 are Associated with Unfavorable Clinical Outcomes and Poor Benefit of Tamoxifen in Breast Carcinoma Patients.

Tamoxifen is the most important treatment component in estrogen receptor positive (ER+) breast carcinoma patients. Tamoxifen resistance incidence presents an important obstacle in clinical treatment. Mechanisms underlying tamoxifen refractory are not completely understood. Although elevated expression of Gankyrin (P28GANK) and stem cell markers Nanog, Oct-4 and Sox-2 have been reported in breast carcinoma, their role in tamoxifen resistance progression has not been explored. In the present study, P28GANK and stem cell markers Nanog, Oct-4 and Sox-2 expression were evaluated using quantitative RT-PCR and immunohistochemical technology in 72 breast carcinoma patients who received tamoxifen as adjuvant anti-hormone treatment. Expression data were correlated with the clinical outcome and survival of patients. Data analysis showed that P28GANK, Oct-4 and Sox-2 transcripts were significantly overexpressed in tamoxifen resistance patients. Immunohistochemical staining indicated that protein expression of P28GANK and Oct-4 were also significantly higher in tamoxifen resistance patients. We have shown a positive correlation between mRNA and protein expression of P28GANK, Oct-4 and Sox-2. Multivariate logistic regression analysis indicated that P28GANK (P = 0.002) and Oct-4 (P = 0.013) overexpression could be negative independent factors of disease outcome. Additionally, in the whole study group, multivariate Cox regression analysis revealed that high expression of P28GANK and Oct-4 remained significant and unfavorable predictive factors for patients' survival. These findings suggest that Gankyrin and Oct-4 overexpression could promote tamoxifen refractory in breast cancer patients. More studies are warranted to clarify the predictive role of these potential biomarkers for patients who don't benefit from tamoxifen treatment and their possible application as prognostic markers in ER+ tamoxifen-treated breast carcinoma patients.

Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst

Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to a posteriori externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.

Glioma SOX2 expression decreased after adjuvant therapy

BackgroundSOX2 is regarded as an important marker in stem cell. The change of SOX2 expression after adjuvant therapy in high grade glioma (HGG) remains unknown so far. Few patients with recurrent glioma have opportunity to undergo operation once again, so the recurrent glioma samples are scarce. This study tries to analyze SOX2 expression in paired primary and recurrent HGG, aims to better understand the transformation law of SOX2 after adjuvant therapy in HGG.MethodsTwenty-four recurrent HGG patients who undergone a second resection were included. 16 patients received adjuvant therapy, the remaining 8 patients didn’t receive any adjuvant therapy at all. The protein expression of SOX2 in paired primary and recurrent HGG was tested by immunohistochemistry. The statistical analysis was conducted by IBM SPSS Statistics 19.0.ResultsIn primary HGG, SOX2 expression of 3 + , 2 + , 1+ and 0+ were seen in 20 (83.3%), 1 (4.2%), 1 (4.2%) and 2 cases (8.3%), respectively. The expression of SOX2 was decreased in recurrent HGG compared to the paired primary sample (p = 0.001). The decrease of SOX2 was often seen in patients received chemotherapy, radiotherapy or both (p = 0.003). Patients with SOX2 high expression in primary glioma had a longer median PFS than those with SOX2 low expression with marginal statistic significance (12.7 vs. 5.4 months, p = 0.083). For cases with SOX2 high expression in the primary glioma, those had SOX2 low expression after recurrence seemed to have worse prognosis as compared to patients with stable SOX2 high expression (PFS: 10.4 vs. 14.9 months, p = 0.036; OS: 27.0 vs 49.5 months, p = 0.005).ConclusionsThis is the first study comparing the protein expression of SOX2 in recurrent HGG and its paired primary tumor. SOX2 high expression is common in brain HGG, a tendency of decreased SOX2 expression in recurrent gliomas was evidenced. Lower SOX2 expression was seen in those patients who received adjuvant chemotherapy and/or radiotherapy. Patients with low SOX2 expression in primary HGG usually have poorer prognosis, those with SOX2 expression decreased in recurrent HGG had worse outcome.

MiR-590-3p and Its Downstream Target Genes in HCC Cell Lines.

miRNAs are small non-coding RNA sequences of 18-25 nucleotides. They can regulate different cellular pathways by acting on tumor suppressors, oncogenes, or both. miRNAs are mostly tissue-specific, and their expression varies depending on the cancer or the tissue in which they are found. hsa-miR-590-3p was found to be involved in several types of cancers. In this study, we identified potential downstream target genes of hsa-miR-590-3p computationally. Several bioinformatics tools and more than one approach were used to identify potential downstream target genes of hsa-miR-590-3p. CX3CL1, SOX2, N-cadherin, E-cadherin, and FOXA2 were utilized as potential downstream target genes of hsa-miR-590-3p. SNU449 and HepG2, hepatocellular carcinoma cell lines, were used to carry out various molecular techniques to further validate our in silico results. mRNA and protein expression levels of these genes were detected using RT-PCR and western blotting, respectively. Co-localization of hsa-miR-590-3p and its candidate downstream target gene, SOX2, was carried out using a miRNA in situ hybridization combined with immunohistochemistry staining through anti-SOX2. The results show that there is an inverse correlation between hsa-miR-590-3p expression and SOX2 protein expression in SNU449. Subsequently, we suggest that SOX2 can be a direct downstream target of has-miR-590-3p indicating that it may have a role in the self-renewal and self-maintenance of cancer cells. We also suggest that CX3CL1, E-cadherin, N-cadherin, and FOXA2 show a lot of potential as downstream target genes of hsa-miR-590-3p signifying its role in epithelial-mesenchymal transition. Studying the expression of hsa-miR-590-3p downstream targets can enrich our understanding of the cancer pathogenesis and how it can be used as a therapeutic tool.

SOX2 Expression Is an Independent Predictor of Oral Cancer Progression.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.

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.

Decreased expression of microRNA-145 promotes the biological functions of fibroblasts in hypertrophic scar tissues by upregulating the expression of transcription factor SOX-9.

The present study aimed to determine the expression of microRNA (miRNA or miR)-145 in hypertrophic scars at the tissue and cellular levels, and to investigate its biological functions and mechanism of action. A total of 36 patients who were diagnosed with hypertrophic scar were included in the present study. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of miR-145 in tissues and fibroblasts. Primary fibroblasts were transfected with negative control miRNA, miR-145 mimics or inhibitor. A Cell Counting Kit-8 assay was performed to determine the level of proliferation of fibroblasts. Flow cytometry was employed for cell cycles determination and apoptosis in fibroblasts. A Matrigel assay was used to evaluate the invasion ability of fibroblasts. Western blotting was used to determine the expression of the transcription factor SOX-9 (SOX-9) protein in fibroblasts. Rescue experiments were performed to examine the effect of SOX-9 on the regulation of fibroblasts by miR-145. The dual luciferase reporter assay was performed to identify the direct interaction between SOX-9 and miR-145. The expression of miR-145 was reduced in hypertrophic tissues and fibroblasts. Overexpression of miR-145 inhibited the proliferation, G1/S phase transition and invasion of fibroblasts, and promoted the apoptosis of fibroblasts. In addition, overexpression of miR-145 inhibited SOX-9 protein expression. By contrast, the expression of SOX-9 reversed the effects of miR-145 on the proliferation, cell cycle, apoptosis and invasion of fibroblasts. The miR-145 seed region was able to bind with the 3′-untranslated region of the SOX-9 mRNA to regulate its expression. The present study demonstrated that miR-145 expression is reduced in hypertrophic scar tissues and negatively associated with SOX-9 expression. In addition, miR-145 inhibits the proliferation, cell cycle and invasion, and promotes the apoptosis of fibroblasts by down-regulating the expression of SOX-9. The current study provides a potential target for the clinical diagnosis and treatment of hypertrophic scars.

Evaluation of SOX2 and podoplanin expression in oral epithelial dysplasia and its correlation with malignant transformation.

Oral carcinogenesis cascade is a complex process, characterized by variable numbers of genetic and epigenetic alterations of various genes with manifold roles that could serve as biological hallmarks. This study was undertaken to assess the protein expression of SOX2 and podoplanin in oral epithelial dysplasia and correlate the expression with clinicopathological parameters and risk of malignant transformation. SOX2 and podoplanin expression were analyzed in 60 cases of oral epithelial dysplasia. The association between SOX2 and podoplanin expression with various clinicopathological parameters and transformation to oral cancer was analyzed. A higher Histoscore was seen in 55% of moderate and 30% of severe dysplasia. 25% of the cases showed a negative podoplanin expression and 30% of patients had higher podoplanin expression (score 2 and 3). Though there was significant association of both SOX2 and podoplanin expression with the degree of dysplasia, the association of their expression with transformation to oral squamous cell carcinoma did not reach statistical significance. Alteration in SOX2 and podoplanin is likely an important event in head and neck carcinogenesis; however, their expression may be valuable only in a few cases of oral epithelial dysplasia to assess the risk of malignant transformation.

NF-YA transcriptionally activates the expression of SOX2 in cervical cancer stem cells

Roles for SOX2 have been extensively studied in several types of cancer, including colorectal cancer, glioblastoma and breast cancer, with particular emphasis placed on the roles of SOX2 in cancer stem cell. Our previous study identified SOX2 as a marker in cervical cancer stem cells driven by a full promoter element of SOX2 EGFP reporter. Here, dual-luciferase reporter and mutagenesis analyses were employed, identifying key cis-elements in the SOX2 promoter, including binding sites for SOX2, OCT4 and NF-YA factors in SOX2 promoter. Mutagenesis analysis provided additional evidence to show that one high affinity-binding domain CCAAT box was precisely recognized and bound by the transcription factor NF-YA. Furthermore, overexpression of NF-YA in primitive cervical cancer cells SiHa and C33A significantly activated the transcription and the protein expression of SOX2. Collectively, our data identified NF-YA box CCAAT as a key cis-element in the SOX2 promoter, suggesting that NF-YA is a potent cellular regulator in the maintenance of SOX2-positive cervical cancer stem cell by specific transcriptional activation of SOX2.

Abstract 785: The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein

Abstract Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that SOX2 is downregulated by ChlA-F at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3’-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients. Citation Format: Xiaohui Hua, Jiheng Xu, Chuanshu Huang, Jingxia Li, Zhongxian Tian, Haishan Huang. The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 785.

In Vivo Study on the Effects of Xiaoaiping on the Stemness of Hepatocellular Carcinoma Cells.

Aims The aim of this study was to examine the effects of Xiaoaiping on the stemness of hepatocellular carcinoma (HCC) cells in vivo and to investigate the underlying molecular mechanism. Methods A subcutaneous xenograft nude mouse model was established using Hep3B-derived HCC cells. The mice were randomly assigned to the 100 mg/kg Xiaoaiping or 100 μL/20 g normal saline (control) groups (n =3/sex/group) for daily intragastric administration for 14 days. The tumor size was closely monitored during the dosing phase. After the treatment period, the tumor tissues were weighed and harvested for mRNA and protein isolation. qPCR and Western blotting were used to evaluate the expression of cancer stemness markers (epithelial cell adhesion molecule [EpCAM], cluster of differentiation [CD13], CD90, aldehyde dehydrogenase 1 [ALDH1], CD44, and CD45), totipotency factors (sex determining region Y-box 2 [Sox2], Nanog, and octamer-binding transcription factor 4 [Oct4]), and genes involved in the Notch, Wnt/β-catenin, Hedgehog, and Hippo signaling pathways. Key Findings The tumor size and weight were significantly reduced in the nude mice treated with 100 mg/kg Xiaoaiping when compared with the controls. The Xiaoaiping effects on the stemness markers and totipotency factors included decreased expression of EpCAM, CD24, CD47, Sox2, Oct4, and sal-like protein 4 (SALL4), as well as increased expression of CD13 and ALDH1. In addition, Xiaoaiping inhibited the Hippo, Wnt, and Hedgehog signaling pathways. Conclusion Xiaoaiping significantly inhibited the growth of HCC xenograft in nude mice. These antitumor effects may be mediated by modulating the expression of multiple stemness markers and totipotency factors and inhibition of the Hippo, Wnt, and Hedgehog signaling pathways.

Expression of Gp78/Autocrine Motility Factor Receptor and Endocytosis of Autocrine Motility Factor in Human Thyroid Cancer Cells.

Gp78/autocrine motility factor receptor (Gp78/AMFR) is a cancer-associated endoplasmic reticulum-localized E3 ubiquitin ligase and also the cell surface receptor for autocrine motility factor (AMF). The study objective was to determine the association between Gp78/AMFR and AMF endocytosis in thyroid cancer cells. Gp78/AMFR expression and AMF internalization were measured in differentiated thyroid cancer (DTC) and anaplastic thyroid cancer (ATC) cell lines and in freshly resected human papillary thyroid cancers (PTC) relative to benign thyroid tissue. Spheroid-like aggregates generated from explants of cancer, goiter, and collateral thyroid tissue were assessed for expression of cancer stem cell markers, surface Gp78/AMFR and AMF endocytosis. DTC cell lines showed elevated total and surface Gp78/AMFR and AMF internalization relative to ATC lines. Gp78/AMFR, Oct-4 and Sox-2 protein expression, Gp78/AMFR surface expression and AMF internalization were elevated in PTC-derived aggregates relative to fibroblasts. Elevated levels of Gp78/AMFR expression and AMF internalization in PTC were associated with expression of cancer stem cell markers. Gp78/AMFR expression and AMF uptake are more closely associated with DTC compared to benign thyroid lesions or ATC and with PTC-derived cancer stem-like cells.

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.