SRY-related High-mobility Group Box 9 Research Articles

Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression

BackgroundCalcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms.MethodsSmall RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves.ResultsThe data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition.ConclusionmiR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

SRY-related high-mobility group box 9 (SOX9) alleviates cigarette smoke extract (CSE)-induced inflammatory injury in human bronchial epithelial cells by suppressing stromal interaction molecule 1 (STIM1) expression.

Chronic obstructive pulmonary disease (COPD) is a chronic airway disease with airflow limitation and abnormal inflammatory response. It has been verified that SOX9 plays a key role in lung function of various lung diseases and SOX9 is closely associated with COPD. Additionally, literature has reported that STIM1 is involved in lung injury and is highly expressed in neutrophils from COPD patients. This study aimed to characterize the biological roles of SOX9 and STIM1 in the pathogenesis of COPD and to elucidate the regulatory mechanism. Human bronchial epithelial cells (BEAS-2B) were treated with CSE to construct in vitro COPD model. The levels of SOX9 and STIM1 in CSE-treated BEAS-2B cells were detected by western blot and RT-qPCR assay. Then, JASPAR datasets were utilized to analyze SOX9 binding sites in the promoter region of STIM1. Besides, luciferase reporter assay and ChIP assay were employed to validate the binding sites in STIM1 promoter region to SOX9. In addition, viability and apoptosis of BEAS-2B cells were assessed by utilizing MTT assay and TUNEL staining. ELISA kits and corresponding commercial kits were applied to measure the levels of TNF-α, IL-6, IL-1β, SOD, GSH-Px and MDA. CSE treatment dose- and time-dependently reduced SOX9 expression in BEAS-2B cells. SOX9 overexpression enhanced the viability and suppressed the apoptosis of CSE-treated BEAS-2B cells as well as attenuated CSE-induced inflammation and oxidative stress. Then, it was validated that SOX9 bound to the promoter region of STIM1. Moreover, SOX9 overexpression-mediated impacts on cell viability, cell apoptosis, inflammation and oxidative stress in CSE-treated BEAS-2B cells were partially abolished by upregulation of STIM1. To sum up, results here suggested that overexpression of SOX9 could mitigate inflammatory injury in CSE-treated bronchial epithelial cells by suppressing STIM1.

Adipose mesenchymal stem cell-derived exosomes accelerate skin wound healing via the lncRNA H19/miR-19b/SOX9 axis

It has been reported that adipose mesenchymal stem cells (ADSCs) accelerate wound healing. Moreover, exosomes, which serve as paracrine factors, play a vital role in wound healing. However, the mechanism remains unclear. This research aimed to determine the roles of exosomes derived from ADSCs (ADSC-Exos) in wound skin tissue repair. Flow cytometry and electron microscopy were carried out to identify ADSCs and ADSC-Exos, respectively; RT-qPCR was performed to assess the lncRNA H19 (H19), microRNA19b (miR-19b) and SRY-related high-mobility-group box 9 (SOX9) levels; Western blotting was carried out to evaluate collagen and β-catenin expression; CCK-8, scratch and transwell assays were conducted to evaluate human skin fibroblast (HSF) cell proliferation, migration and invasion, respectively; the potential binding sites between H19 and miR-19b, miR-19b and SOX9 were detected by dual-luciferase reporter gene assay and RIP assay; and H&E staining was conducted to observe skin wound tissues. ADSC-Exos accelerated the proliferation, migration and invasion of HSF cells via H19. H19 acts as a molecular sponge towards miR-19b, which targets SOX9. ADSC-Exos inhibited miR-19b expression via H19, resulting in accelerated HSF proliferation, migration and invasion. ADSC-Exos upregulated SOX9 to activate the Wnt/β-catenin pathway, resulting in accelerated HSF cell proliferation, migration and invasion, and ADSC-Exos promoted skin wound healing via H19 in mice.The high expression of H19 in ADSC-Exos may upregulate SOX9 expression via miR-19b to accelerate wound healing of skin tissues. Our study may provide novel perspectives for therapy to accelerate skin wound healing.This study reveals that the long non-coding RNA H19 is highly expressed in exosomes derived from adipose mesenchymal stem cells and accelerates the proliferation, migration and invasion of human skin fibroblasts by upregulation of SOX9 and activation of the Wnt/β-catenin pathway The authors show that H19 affects SOX9 expression via the microRNA miR-19b to promote wound healing in injured skin.

Downregulation of SOX9 suppresses breast cancer cell proliferation and migration by regulating apoptosis and cell cycle arrest.

SRY-related high-mobility group box 9 (SOX9) is an important transcriptional factor that regulates diverse genes involved in development and stemness. Dysregulation of SOX9 encourages carcinogenesis in various types of cancer, including breast cancer. The present study aimed to explore the role of SOX9 in triple-negative breast cancer (TNBC). SOX9 expression was significantly upregulated in the TNBC MDA-MB-231, MDA-MB-436 and MDA-MB-468 cell lines compared with that in BT-549 cells. Based on a lentivirus assay, SOX9 inhibition in MDA-MB-231 and MDA-MB-436 cells suppressed cell proliferation and colony formation. Apoptosis was increased and the cell cycle was arrested at the G0/G1 phase in SOX9-knockdown cells. Transwell and wound-healing assays demonstrated that SOX9 inhibition decreased the migration and invasion of MDA-MB-231 and MDA-MB-436 cells. RNA sequencing identified that numerous genes were regulated by SOX9, including nucleophosmin, thioredoxin reductase 1, succinate dehydrogenase complex subunit D, nuclear receptor binding SET domain protein 2, eukaryotic translation initiation factor 4γ1 and glycogen phosphorylase L. Overall, the current study suggested that SOX9 acted as an oncogene in TNBC.

SOX9 is associated with advanced T-stages of clinical stage II colon cancer in young Mexican patients.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and includes colon cancer (CC) and rectal cancer (RC). Regarding CC, the development of novel molecular biomarkers for the accurate diagnosis and prognosis, as well as the identification of novel targets for therapeutic intervention, are urgently needed. SRY-related high-mobility group box 9 (SOX9), a transcription factor, is involved in development, and has been associated with the progression of human cancer. However, its underlying clinical and functional effects in CRC have not been fully understood. Therefore, the present study aimed to evaluate the clinical and functional relevance of SOX9 expression in CC. The expression of SOX9 in tumor tissues was evaluated in 97 biopsies from Mexican patients with CC with early-stage I and II disease by immunohistochemistry (IHC). In addition, SOX9 silencing in the HCT116 cell line was performed using specific small interfering RNAs, while downregulation efficiency was verified by reverse transcription-quantitative PCR and immunofluorescence. Spheroid-formation assay was carried out using ultra-low attachment plates. The IHC results showed that SOX9 was upregulated in patients with stage II (91%) and advanced T3 stage (67%) CC. Interestingly, higher SOX9 expression was associated with clinical stage, tumor size and tumor location. Furthermore, increased SOX9 expression was found in relapsed cases with local tumors; however, it was not associated with increased survival probability. Additionally, functional analysis indicated that SOX9 silencing significantly attenuated the sphere-formation capability of HCT116 cells. The present study was the first to evaluate the expression levels of SOX9 in Mexican patients diagnosed with early-stage CC. The aforementioned findings indicated that high SOX9 expression could play an important role in tumorigenesis and be associated with advanced T-stages of clinical-stage II patients, but not with relapse-free survival.

RETRACTED: H3K27 demethylase KDM6B aggravates ischemic brain injury through demethylation of IRF4 and Notch2-dependent SOX9 activation

Lysine demethylase 6B (KDM6B) is a histone H3 lysine 27 (H3K27) demethylase that serves as a key mediator of gene transcription. Although KDM6B has been reported to modulate neuroinflammation after ischemic stroke, its role in ischemic brain injury is yet to be well elucidated. Therefore, this study aimed to thoroughly demonstrate the molecular mechanism underlying the effect of KDM6B on neurological function and astrocyte response in post-ischemic brain injury. Middle cerebral artery occlusion/reperfusion (MCAO) mouse models were constructed, while the oxygen-glucose deprivation/reperfusion (OGD/R) model was developed in astrocytes to mimic injury conditions. KDM6B was upregulated post-MCAO in mice and in astrocytes following the induction of OGD/R. Silencing of KDM6B resulted in suppressed neurological deficit, reduced cerebral infarction volume, attenuated neuronal cell apoptosis, and disrupted inflammation. Dual-luciferase reporter gene and chromatin immunoprecipitation-quantitative polymerase chain reaction assays revealed that KDM6B inhibited H3K27 trimethylation in the interferon regulatory factor 4 (IRF4) promoter region, resulting in the upregulation of IRF4 expression, which in turn bound to the Notch2 promoter region to induce its downstream factor SRY-related high-mobility group box 9 (SOX9). SOX9 knockdown reversed the effects of KDM6B overexpression on ischemia-triggered brain damage. Based on these findings, we concluded that KDM6B-mediated demethylation of IRF4 contributes to aggravation of ischemic brain injury through SOX9 activation.

LncRNA-CRNDE regulates BMSC chondrogenic differentiation and promotes cartilage repair in osteoarthritis through SIRT1/SOX9.

Osteoarthritis (OA) is the most common chronic and degenerative joint disease. Although traditional OA medications can partially relieve pain, these medications cannot completely cure OA. Therefore, it is particularly important to find an effective treatment for OA. This study explored the function of long non-coding RNA (lncRNA)-colorectal neoplasia differentially expressed gene (CRNDE) in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the underlying molecular mechanism, aiming to develop a new treatment method for osteoarthritis. BMSCs were isolated from rat bone marrow using the gradient centrifugation method. And BMSC chondrogenic differentiation was induced with chondrogenic medium. The expression of lncRNA-CRNDE was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Silent information regulator factor 2-related enzyme 1(SIRT1) and cartilage marker genes Aggrecan and collagen 2 (α1) protein expression were researched using western blot. Alcian blue staining was employed to examine the content of cartilage matrix proteoglycan glycosaminoglycan (GAG). The interaction between lncRNA-CRNDE and SIRT1 was detected by RNA pull-down and RNA immunoprecipitation (RIP) assay. Ubiquitination experiments were performed to measure the ubiquitination level of SIRT1. The combination between SMAD ubiquitination regulatory factor 2 (SMURF2) and SIRT1, as well as SRY-related high-mobility-group box 9 (SOX9) and collagen 2 (α1) promoter, was detected by Co-immunoprecipitation or ChIP. With the prolongation of induction time, the expression of lncRNA-CRNDE, SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) in BMSC osteogenic differentiation was gradually increased. Also, the content of cartilage matrix proteoglycan GAG was gradually elevated with the extension of the induction time. Further increase in the expression of SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) by overexpression of lncRNA-CRNDE also indicated elevated GAG content. RNA pull-down and RIP assay confirmed the binding between lncRNA-CRNDE and SIRT1. qRT-PCR and western blot showed that interference with lncRNA-CRNDE significantly inhibited the protein expression of SIRT1. BMSCs transfected with si-CRNDE increased ubiquitination levels of SIRT1 mediated by the E3 ligase SMURF2, leading to the reduced protein stability of SIRT1. However, overexpression of lncRNA-CRNDE increased the binding ability of SOX9 and collagen 2 (α1) promoter, which was reversed by the simultaneous transfection ofCRNDE overexpression (pcDNA-CRNDE) and SIRT1small interferingRNA (si-SIRT1). lncRNA-CRNDE regulates BMSC chondrogenic differentiation to promote cartilage repair in osteoarthritis through SIRT1/SOX9.

Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells by regulating the lncRNA H19/miR-29b-3p/SOX9 axis.

Platelet lysate (PL) has been shown to induce chondrogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs). However, the underlying mechanism is still not clear. The aim of this study was to investigate whether long noncoding RNA H19 is involved in this process. hUCMSCs were isolated, identified and cultured in 5% PL-supplemented chondrogenic differentiation medium. Chondrogenic differentiation was assessed by Alcian blue staining. The expressions of H19, miR-29b-3p, SRY-related high-mobility-group box 9 (SOX9), collagen II and aggrecan were determined by quantitative real-time PCR and western blot. The interaction between miR-29b-3p and H19 or SOX9 was analyzed by luciferase reporter assay. During PL-induced chondrogenic differentiation of hUCMSCs, expressions of H19 and SOX9 were increased, whereas miR-29b-3p expression was decreased. H19 overexpression promoted, whereas H19 silencing attenuated the PL-induced chondrogenic differentiation of hUCMSCs. SOX9 was identified as a direct target of miR-29b-3p, and H19 was observed to act as a sponge of miR-29b-3p to up-regulate SOX9 expression. The chondrogenic differentiation-promoting effect of H19 overexpression was negated when miR-29b-3p expression was up-regulated by Lenti-miR-29b-3p infection. In conclusion, PL induced chondrogenic differentiation of hUCMSCs by regulating the H19/miR-29b-3p/SOX9 axis.

SOX9 represses hepatitis B virus replication through binding to HBV EnhII/Cp and inhibiting the promoter activity

Hepatitis B virus (HBV) infection affects 364 million people worldwide and causes a serious global public health problem. The SRY-related high mobility group-box 9 (SOX9) is a risk of developing cirrhosis in patients with chronic hepatitis B and a cancer stem cell marker. However, the role of SOX9 in HBV replication has not been reported. This study revealed a distinct mechanism underling the regulation of HBV replication mediated by SOX9. HBV induces SOX9 mRNA and protein expression in human hepatoma cells, including HepG2.2.15, HepG2, Huh7, and HepG2-NTCP cells. Further study demonstrated that HBV activates SOX9 expression at the transcriptional level through inducing SOX9 promoter activity and HBc could induce the activity of SOX9 promoter. Interestingly, SOX9 in turn represses HBV replication in human hepatoma cells. More importantly, SOX9 inhibits HBV infection in HepG2-NTCP cells and C57/BL6 mice. Detailed study revealed that SOX9 suppresses HBV replication through directly binding to HBV EnhII/Cp (HBV 1667–1672 nt) to inhibit EnhII/Cp activation. Results from deletion mutant analysis, ChIP assay, nuclear and cytoplasmic extraction analysis, and immunofluorescence demonstrated that SOX9 high mobility group (HMG) domain is required for SOX9 anti-HBV activity. Moreover, we demonstrated that SOX9 and hepatocyte nuclear factor 4 alpha (HNF4α) can bind to HBV EnhII/Cp (HBV 1667–1672 nt) individually and simultaneously to regulate the promoter activity. Collectively, the results revealed a distinct negative feedback mechanism underlying HBV replication and SOX9 expression, and identified SOX9 as a new host restriction factor in HBV replication and infection. ImportanceHBV infection is a global public health problem by causing serious liver diseases, but the mechanisms underlying HBV pathogenesis remain largely unknown. SOX9 is a risk of developing cirrhosis and a cancer stem cell marker, however, the role of SOX9 in HBV infection has not been reported. The authors revealed a distinct mechanism underling the regulation of HBV replication and SOX9 expression. On the one hand, HBV induces SOX9 expression in human hepatoma cells through activating SOX9 promoter. On the other hand, SOX9 in turn represses HBV replication in human hepatoma cells by binding to and inhibiting HBV EnhII/Cp through its HMG domain. More importantly, SOX9 inhibits HBV infection in HepG2-NTCP cells and C57/BL6 mice. Therefore, this study identifies SOX9 as a novel and potential therapeutic reagent for the prevention and treatment of HBV-associated diseases.

Down-regulation of MiR-138-5p Protects Chondrocytes ATDC5 and CHON-001 from IL-1 β-induced Inflammation Via Up-regulating SOX9.

Osteoarthritis (OA) pertains to a chronic disease of degenerative joints distinguished by articular cartilage destruction, subchondral bone remodeling, osteophyte formation, and inflammatory changes. Chondrocyte apoptosis is inextricably linked to cartilage degeneration. SRY-related high-mobility-group-box 9 (SOX9) is a well-acknowledged transcription factor in the chondrogenesis. Nevertheless, the detailed function of miR-138-5p/SOX9 in OA remains to be fully clarified. qRT-PCR was performed to measure the expressions of miR-138-5p and SOX9 mRNA in OA and normal cartilage tissues and cells. Human chondrocyte cell lines, CHON-001 and ATDC5, were treated with different doses of interleukin-1β (IL-1β) to simulate the inflammatory response environment of OA. miR-138-5p mimics, miR-138-5p inhibitors, and SOX9 small interfering RNA (siRNA) were constructed and transfected into CHON-001 and ATDC5 cells. CCK-8 was conducted to determine the cell viability and transwell assay was used to monitor the migration of cells. Western blot was carried out to detect the expressions of apoptosis- related factors. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the contents of inflammatory factors. TargetScan predicted SOX9 was a target gene of miR-138-5p, which was then verified by luciferase assay. miR-138-5p expression was down-regulated in OA and regulated SOX9 expression. The downregulation of miR-138-5p facilitated the proliferation and migration of CHON-001 and ATDC5 cells, while impeded their apoptosis and inflammatory response. Besides, down-regulated SOX9 can counteract the promoting effect of down-regulated miR-138-5p on the proliferation and migration of chondrocytes. miR-138-5p can arrest the proliferation and migration of CHON-001 and ATDC5 via restraining SOX9, and facilitate the apoptosis and inflammation. This study revealed the protective effect of down-regulated miR-138-5p on the inflammatory injury of chondrocytes caused by IL-1β.

SOX9 promotes nasopharyngeal carcinoma cell proliferation, migration and invasion through BMP2 and mTOR signaling

SRY-related high-mobility-group box 9 (SOX9) is a member of the SOX family of transcription factors. Accumulating evidence has shown that SOX9 plays a significant role in various malignancies. However, the role of SOX9 in nasopharyngeal carcinoma (NPC) remains unknown. In the present study, up-regulation of SOX9 was observed in both NPC tissues and different NPC cells. Overexpression of SOX9 promoted NPC cell proliferation, migration and invasion. Conversely, knock down of SOX9 inhibited NPC proliferation, colony formation, migration and invasion. Mechanistically, SOX9 bound directly to the promoter region of BMP2 and increased BMP2 expression. In addition, overexpression of SOX9 activated the mTOR pathway partly through BMP2. Collectively, these results identify a novel role for SOX9 as a potential therapeutic marker for the prevention and treatment of NPC.

Honokiol improved chondrogenesis and suppressed inflammation in human umbilical cord derived mesenchymal stem cells via blocking nuclear factor-\u03baB pathway

BackgroundCartilage degradation is the significant pathological process in osteoarthritis (OA). Inflammatory cytokines, such as interleukin-1β (IL-1β), activate various downstream mediators contributing to OA pathology. Recently, stem cell-based cartilage repair emerges as a potential therapeutic strategy that being widely studied, whereas, the outcome is still far from clinical application. In this study, we focused on an anti-inflammatory agent, honokiol, which is isolated from an herb, investigated the potential effects on human umbilical cord derived mesenchymal stem cells (hUC-MSCs) in IL-1β stimulation.MethodsSecond passage hUC-MSCs were cultured for multi-differentiation. Flow cytometry, qRT-PCR, von Kossa stain, alcian blue stain and oil red O stain were used for characterization and multi-differentiation determination. Honokiol (5, 10, 25, 50 μM) and IL-1β (10 ng/ml) were applied in hUC-MSCs during chondrogenesis. Analysis was performed by MTT, cell apoptosis evaluation, ELISA assay, qRT-PCR and western blot.ResultshUC-MSC was positive for CD73, CD90 and CD105, but lack of CD34 and CD45. Remarkable osteogenesis, chondrogenesis and adipogenesis were detected in hUC-MSCs. IL-1β enhanced cell apoptosis and necrosis and activated the expression of caspase-3, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-1, −9, 13 in hUC-MSCs. Moreover, the expression of SRY-related high-mobility group box 9 (SOX-9), aggrecan and col2α1 was suppressed. Honokiol relieved these negative impacts induced by IL-1β and suppressed Nuclear factor-κB (NF-κB) pathway by downregulating expression of p-IKKα/β, p-IκBα and p-p65 in dose-dependent and time-dependent manner.ConclusionsHonokiol improved cell survival and chondrogenesis of hUC-MSCs and inhibited IL-1β-induced inflammatory response, which suggested that combination of anti-inflammation and stem cell can be a novel strategy for better cartilage repair.

H-rev107 regulates prostaglandin D2 synthase-mediated suppression of cellular invasion in testicular cancer cells

BackgroundH-rev107 is a member of the HREV107 type II tumor suppressor gene family which includes H-REV107, RIG1, and HRASLS. H-REV107 has been shown to express at high levels in differentiated tissues of post-meiotic testicular germ cells. Prostaglandin D2 (PGD2) is conjectured to induce SRY-related high-mobility group box 9 (SOX9) expression and subsequent Sertoli cell differentiation. To date, the function of H-rev107 in differentiated testicular cells has not been well defined.ResultsIn the study, we found that H-rev107 was co-localized with prostaglandin D2 synthase (PTGDS) and enhanced the activity of PTGDS, resulting in increase of PGD2 production in testis cells. Furthermore, when H-rev107 was expressed in human NT2/D1 testicular cancer cells, cell migration and invasion were inhibited. Also, silencing of PTGDS would reduce H-rev107-mediated increase in PGD2, cAMP, and SOX9. Silencing of PTGDS or SOX9 also alleviated H-rev107-mediated suppression of cell migration and invasion.ConclusionsThese results revealed that H-rev107, through PTGDS, suppressed cell migration and invasion. Our data suggest that the PGD2-cAMP-SOX9 signal pathway might play an important role in H-rev107-mediated cancer cell invasion in testes.

Abstract 5384: Hrev107 enhances prostaglandin D2 synthase-mediated cancer cells suppression in the testis.

Abstract Hrev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family which includes HREV107, Retinoid-inducible gene 1 (RIG1), and HRASLS. The protein in this family contains an NC domain, with unknown function at the N-terminus, and a hydrophobic membrane-anchoring domain at the C-terminus. HREV107 has shown that expressed at high levels in differentiated tissues of post-meiotic testicular germ cells but not in human testicular germ cell tumors. Prostaglandin D2 (PGD2) has shown that can induce SOX-9 nuclear translocation and subsequent Sertoli cell differentiation. In addition, expression of the Prostaglandin D2 synthase (PTGDS) has been detected in the cellular lineage. This gives rise to the Sertoli cells, and the Sertoli cells appear to express the PTGDS only during the VI-VIII stages of the spermatogenic cycle after spermiation. These results suggested that PGD2 may play an important role in the testis differentiation. Our previous result showed that RIG1 (as RIG1 is expressed in the human species only) can interact with PTGDS. We further examined the effect of mouse Hrev107 on the growth of mouse testicular cells. When Hrev107 was expressed in TM3 or TM4 mouse testicular cancer cells, the cell migration and invasion were inhibited. Further, we found PTGDS was co-localized with Hrev107 in TM4 testis cells. In Hrev107-expressing NT2/D1 cells, elevated levels of PGD2, cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This result indicated that Hrev107 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased h-REV107-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated Hrev107-mediated suppression of migration and invasion. These results suggest that Hrev107 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, Hrev107 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Citation Format: Fu-Ming Tsai, Chang-Chieh Wu, Rong-Yaun Shyu, Shun-Yuan Jiang. Hrev107 enhances prostaglandin D2 synthase-mediated cancer cells suppression in the testis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5384. doi:10.1158/1538-7445.AM2013-5384

Analysis of androgen receptor and anti-Müllerian hormone pathways in human granulosa cells under luteinizing hormone treatment

BackgroundThe objective of this study was to determine the gene expression profiles of the androgen/androgen receptor (AR) and anti-Müllerian hormone (AMH)/ Sry-related high-mobility group box 9 (SOX9) pathways in granulosa-luteal cells from patients undergoing standard in vitro fertilization (IVF) with or without recombinant luteinizing hormone (rLH) therapy.MethodsLevels of reproductive hormones in the pre-ovulatory follicular fluid and the expression levels of LHR (luteinizing hormone receptor), AR, SOX9, AMH, AR-associated protein 54(ARA54)and ARA70 were determined in granulosa-luteal cells by real-time reverse-transcription PCR. The effects of androgen and rLH treatments on AR and AMH expression levels were also tested in vitro using HO23 cells.ResultsWe collected 35 an 70 granulosa cell samples from patients cycled with and without rLH supplementation, respectively. The clinical outcomes were similar in patients who received rLH therapy and those who did not, though the pre-ovulatory follicular fluid levels of androstenedione, testosterone, and estradiol were significantly higher and progesterone was lower in the rLH supplementation group. Moreover, granulosa-luteal cell mRNA levels of LHR, AR, AMH, and SOX9 were significantly higher in the rLH supplementation group relative to the group that did not receive rLH supplementation. In addition, we observed significant correlations between LHR and AR mRNA expression and among AR, AMH, and SOX9 mRNA expression in granulosa-luteal cells from patients undergoing standard IVF treatment.ConclusionsIncreased expression of LHR, AR, AMH, and SOX9 is characteristic of granulosa-luteal cells from IVF/ intracytoplasmic sperm injection (ICSI) patients receiving rLH supplementation.

Involvement of the prostaglandin D2 signal pathway in retinoid-inducible gene 1 (RIG1)-mediated suppression of cell invasion in testis cancer cells

Retinoid-inducible gene 1 (RIG1), also called tazarotene-induced gene 3, belongs to the HREV107 gene family, which contains five members in humans. RIG1 is expressed in high levels in well-differentiated tissues, but its expression is decreased in cancer tissues and cancer cell lines. We found RIG1 to be highly expressed in testicular cells. When RIG1 was expressed in NT2/D1 testicular cancer cells, neither cell death nor cell viability was affected. However, RIG1 significantly inhibited cell migration and invasion in NT2/D1 cells. We found that prostaglandin D2 synthase (PTGDS) interacted with RIG1 using yeast two-hybrid screens. Further, we found PTGDS to be co-localized with RIG1 in NT2/D1 testis cells. In RIG1-expressing cells, elevated levels of prostaglandin D2 (PGD2), cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This indicated that RIG1 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated RIG1-mediated suppression of migration and invasion. These results suggest that RIG1 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Our study suggests that RIG1-PGD2 signaling might play an important role in cancer cell suppression in the testis.

Possible Risk Factors Associated with Relapse in Patients Treated with Neoadjuvant Chemohormonal Therapy for High Risk Prostate Cancer

Objectives: To perform a pilot hypothesis generating study of neoadjuvant docetaxel, estramustine, and androgen deprivation therapy for high-risk patients prior to radical prostatectomy. Patients and Methods: Twenty-eight patients received 4 cycles of docetaxel and estramustine administered on an every three week schedule in combination with androgen deprivation therapy (LHRH analog and bicalutamide) prior to radical prostatectomy. Following the prostatectomy, androgen deprivation therapy was continued for 8 months. End-points were pathological complete response (pCR), time-to-relapse (TTR), feasibility and tolerability, and biochemical or clinical correlates of relapse. Results: With a median of 80 months (6.6 years) of follow-up, 18/28 patients have relapsed and one patient died from unrelated causes while in remission, with median TTR of 44 months (3.6 years). Tumor downstaging, perhaps as a result of neoadjuvant therapy, was associated with a decreased risk of relapse (P=0.0002). Consistent with this result, positive margin status was associated with an increased risk of relapse, which was not affected by adjuvant radiation therapy. Increased expression of a possible tumor stem cell marker, (Sry-related high mobility group box-9) Sox-9, both at the time of prostatectomy (P=0.005) and in the pre-treatment tumor biopsy cores was associated with an increased risk of relapse (P=0.03). Conclusions: The neoadjuvant chemohormonal regimen may have benefited some patients, especially those who exhibited pathologic downstaging. Sox-9 expression in prostate cancer specimens warrants prospective validation in both pre-prostatectomy tissue as well as patients undergoing an ongoing randomized trial using a similar neoadjuvant regimen.

Early step in mammalian sex determination

Since the cloning of the Y-located testis-determining factor, sex determining region Y ( SRY), several other genes have been implicated in the process of mammalian sex determination. Mutations of an SRY-related gene, SRY-related high-mobility group box 9 ( SOX9), result in autosomal sex reversal and campomelic dysplasia. The genes Steroidogenic factor 1 ( SF1) and Wilm's tumour 1 ( WT1) are required for early gonadal development as well as for the formation of adrenals and kidneys respectively. The gene responsible for adrenal hypoplasia congenita, DAX1, is a candidate for the X-linked dosage sensitive sex reversal gene ( DSS).