BACKGROUND AND AIMS:The type X collagen gene (Col10a1), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. Col10a1 expression is known to be influenced by many regulators. In this study, we aim to investigate how DEAD-box helicase 5 (DDX5), a potential binding factor for Col10a1 enhancer, may play a role in Col10a1 expression and chondrocyte hypertrophic differentiation in vitro.METHODS:The potential binding factors of the 150-bp Col10a1 cis-enhancer were identified with the hTFtarget database. The expression of DDX5 and COL10A1 was detected by quantitative real-time PCR (qRT-PCR) and Western blot in chondrogenic ATDC5 and MCT cell models with or without Ddx5 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the Col10a1 enhancer. The effect and mechanism of DDX5 on chondrocyte differentiation and maturation was evaluated by alcian blue, alkaline phosphatase (ALP), and alizarin red staining in ATDC5 cell lines with stable knockdown of Ddx5.RESULTS:DDX5 was identified as a potential binding factor for the Col10a1 enhancer. The expression of DDX5 in hypertrophic chondrocytes was higher than that in proliferative chondrocytes. Knockdown of Ddx5 decreased, while overexpression of Ddx5 slightly increased COL10A1 expression. DDX5 promotes the enhancer activity of Col10a1 as demonstrated by dual-luciferase reporter assay, and the ChIP experiment suggests a direct interaction between DDX5 and the Col10a1 enhancer. Compared to the control (NC) group, we observed weaker alcian blue and ALP staining intensity in the Ddx5 knockdown group of ATDC5 cells cultured both for 7 and 14 days. Whereas weaker alizarin red staining intensity was only found in the Ddx5 knockdown group of cells cultured for 7 days. Meanwhile, knockdown of Ddx5 significantly reduced the level of runt-related transcription factor 2 (RUNX2) in related ATDC5 cells examined.CONCLUSIONS:Our results suggest that DDX5 acts as a positive regulator for Col10a1 expression and may cooperate with RUNX2 together to control Col10a1 expression and promote the proliferation and maturation of chondrocytes.

Objectives: Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation. Methods: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation. Results: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1. Conclusions: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.

URI, a prefoldin family member, has been implicated roles in cancer development. We have previously shown that URI can attenuate DNA damage in gastric cancer cells treated with potassium dichromate. The aim of this study was to investigate how URI involves cisplatin-induced DNA damage response (DDR) in gastric cancer cells and its possible mechanism relating to the ATM/CHK2 pathway. Here, MGC-803 and SGC-7901 gastric cancer cells were treated with different concentrations of cisplatin. Comet assay was used to detect DNA damage and the results confirmed the dose-effect of cisplatin-induced DNA damage in gastric cancer cells. URI knockdown cell lines were established with siRNA transfection. Cell viability and proliferation were detected by counting kit 8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays respectively. Apoptosis and cell cycle were analyzed by flow cytometry. The results indicated that URI knockdown increased the sensitivity of cells to cisplatin by inhibiting proliferation and promoting apoptosis. The levels of P-ATM, P-CHK2 and γH2AX were detected by Western blot. Increased levels of P-ATM, P-CHK2, and γH2AX were observed in cisplatin treated cells, indicating that cisplatin induced a DNA damage response (DDR). URI knockdown in cisplatin-treated cells significantly decreased the levels of P-ATM and P-CHK2 at 12 hours, but not at 0 and 6 hours after drug withdrawal, while significantly increased γH2AX levels were detected at 6 hours, but not at 0 and 12 hours after drug withdrawal compared with the control cells. However, the levels of γH2AX were significantly increased in URI knockdown cells after cisplatin treatment for 12 hours. The cell cycle analysis showed that the number of cells entering S phase was significantly reduced and the cells were arrested in the G1 phase in URI-silenced cisplatin-exposed cells, indicating that cell cycle progression was inhibited. In conclusion, our results suggest that URI is involved in the cisplatin-induced DNA damage response via the ATM/CHK2 pathway, and silencing URI can increase cisplatin-induced DNA damage and enhance drug sensitivity in gastric cancer cells.

BACKGROUND:The type X collagen gene (Col10a1) is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by in silico analysis as potential Col10al gene regulators.OBJECTIVES:In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation in vitro.METHODS:First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes in situ. Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp Col10a1 cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a.RESULTS:The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes in situ. Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (Runx2), increased SRY-box transcription factor 9 (Sox9), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls.CONCLUSIONS:In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.

CircCRIM1 (hsa_circ_0002346) is a circular RNA derived from gene CRIM1 (the cysteine rich transmembrane BMP regulator 1 circRNAs) by back-splicing. Recent studies have suggested the diverse function of CircCRIM1 in the tumorigenesis of multiple malignancies, including osteosarcoma (OS). Here, we investigated the role and mechanism of circCRIM1 during OS progression. Differentially expressed circRNAs (including circCRIM1) in OS and human osteoblast (hFOB1.19) cell lines were selected by searching the circRNA expression microarray dataset of GSE96964. The expression levels of circCRIM1 and its sponging miRNAs and target genes were examined by RT-qPCR. The effects of circCRIM1 on the proliferation, migration, and invasion of OS cells were investigated by in vitro gain of function experiments. The in vivo function of circCRIM1 on OS was evaluated by measuring the subcutaneous and in situ tumor growth in nude mice. In addition, dual-luciferase reporter assay and in situ hybridization (FISH) were performed to explore the underlying mechanisms of circCRIM1 and its sponging miRNAs and target genes in OS. CircCRIM1 is downregulated in human OS cell lines and predominantly presents in the cytoplasm as demonstrated by RT-qPCR and FISH assays. Overexpression of circCRIM1 suppressed the migration, invasion, proliferation of OS cells in vitro and OS tumor growth in vivo. Mechanistically, we identified miR146a-5p as a sponge miRNA of circCRIM1 through bioinformatic prediction and confirmed their interaction and colocalization via reporter gene assay and FISH analysis. This interaction leads to increase expression of the downstream target gene NUMB, which will cause inhibition of the Notch signal pathway. We further demonstrated that miR146a-5p overexpression could reverse the antitumor effect induced by circCRIM1 in OS cells. Our results support that circCRIM1 acts as a tumor suppressor in OS by sponging miR146a-5p and its downstream target NUMB.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an essential role in embryo development and carcinogenesis, circNRIP1 was recently identified to promote development of multiple human cancers. This study investigated the role of circNRIP1 in osteosarcoma (OS) cells and the potential mechanisms relating to the sponging miRNAs and their target genes. OS cell lines and normal human osteoblasts were grown for qRT-PCR analysis of circNRIP1 expression and functions of circNRIP1 expression in OS cell proliferation, migration, and invasion in vitro. Bioinformatics analysis was then performed to predict the sponge miRNA of circNRIP1 and the target gene, which was confirmed by using the dual-luciferase reporter assay. The in vivo functions of circNRIP1 was evaluated in OS cell xenograft models, while levels of relevant marker genes were examined using immunohistochemistry. CircNRIP1 was mainly localized in OS cell cytoplasm and significantly lower in OS cell lines than in normal human osteoblasts. CircNRIP1 overexpression significantly inhibited OS cell proliferation, migration, and invasion in vitro. miR-1200 was predicted as the sponge miRNA of circNRIP1 and directly interacted with circNRIP1 confirmed by the dual-luciferase reporter assay. Moreover, miR-1200 overexpression significantly alleviated the inhibitory effect of circNRIP1 on OS cells. A protein-coding gene MIA2 was identified as the miR-1200 targeting gene and reversely associated with miR-1200 expression in OS cells. Increase in MIA2 expression in a murine OS cell xenograft model was associated with circNRIP1 expression in inhibition of OS cell xenograft growth in vivo. These data support the circNRIP1 OS-suppressive role by sponge of miR-1200 expression and in turn to upregulate MIA2 expression.

The distal-less (dlx) homeobox transcription factors have been implicated roles in bone development. DLX5, in particular, was shown to play essential roles in osteoblast differentiation by targeting RUNX2, a master transcription factor for bone development. Interestingly, DLX5 has also been shown to play an oncogenic role in lung and other cancers, possibly via regulation of MYC expression. Given its dual roles in bone and cancer, this study aimed to investigate the effect of DLX5 on progression of osteosarcoma (OS), the primary bone cancer that is characterized by abnormal bone formation and osteoblast activity. Expression of DLX5 in OS cell lines was detected by quantitative real-time PCR (qRT-PCR) and western blot (WB). In vitro and in vivo assays were performed to investigate the oncogenic function of DLX5 in OS cells and xenograft models. Luciferase reporter assay was performed to determine the underlying mechanism of DLX5-mediated OS aggressiveness. The results showed that DLX5 was differentially expressed in OS cell lines, with significantly upregulated levels in HOS and MG-63 and relatively low levels in U2OS and 143B cell lines, compared with the normal bone cell line. DLX5 knockdown in HOS and MG-63 cell lines by siRNA inhibited OS cell growth and progression, and induced cell apoptosis and cell cycle changes both in vitro and in vivo. Meanwhile, DLX5 overexpression had the opposite effect on U2OS and 143B cell lines. Notably, a positive correlation between the expression patterns of NOTCH1 and DLX5 was also observed. The expression levels of NICD (NOTCH1 intracellular domain) and HES1 (classical target of NOTCH) were closely associated with DLX5 expression. Whereas knockdown of DLX5 in OS cells resulted in decreased expression of NOTCH1 and reduced cell proliferation and migration, which were rescued by overexpression of NOTCH1. We further analyzed DLX5 and NOTCH1 genes using JASPAR software and found two potential DLX5 binding sites within the NOTCH1 promoter. Dual-luciferase assay demonstrated that DLX5 specifically activates the NOTCH1 promoter and controls its expression. Taken together, our results support that DLX5 plays an oncogenic role in OS development, which can at least partially, be attributed to activation of the NOTCH signaling pathway.

Hypertrophic chondrocytes and their specific marker, the type X collagen gene (Col10a1), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse Col10a1 gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp Col10a1 cis-enhancer. However, the roles of these candidate TFs in Col10a1 expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp Col10a1 enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of Col10a1 expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, Tbx5 was downregulated upon Col10a1 upregulation, overexpression of Tbx5 decreased Col10a1 expression, and knock-down of Tbx5 increased Col10a1 expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of Col10a1. We further generated a stable Tbx5-overexpressing ATDC5 cell line and ColX-Tbx5 transgenic mice driven by Col10a1-specific enhancers and promoters. Tbx5 overexpression decreased Col10a1 expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate Col10a1 transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.

Abstract Gastric cancer is the most common gastrointestinal malignant tumor and also the second leading cause of cancer-related mortality worldwide. Given its high rate of non- symptoms at early stage, gastric cancer is usually diagnosed at middle or advanced stage with metastasis. Therefore, chemotherapy, including Cisplatin, becomes a main treatment option, in addition to surgery. However, it is not uncommon for patients to develop cisplatin resistance which limits its clinical applications. URI, a prefoldin family member and also an oncoprotein, has been shown to promote multiple cancer development, including gastric cancer. The aim of this study was to investigate how URI may participate in the DNA damage repair of gastric cancer cells induced by Cisplatin and its possible mechanism via the ATM/CHK2 pathway. URI knockdown cell lines from gastric cancer cells MGC-803 and SGC-7901 were established and treated with different concentrations of Cisplatin. Cell viability, proliferation and apoptosis were analyzed by CCK-8, EDU assay, and flow cytometry respectively. DNA injury marker H2AX were examined by qRT-PCR and Western blot analysis. Comet assay was used to detect the DNA damage. The results showed that URI knockdown reduces cell viability and proliferation, as well as the Cisplatin-induced apoptosis of gastric cancer cells. Cisplatin dose responsively induced DNA damage in gastric cancer cells and URI knockdown enhanced this damage as indicated by comet assay and increased γH2AX expression. We have also detected increased levels of P-ATM and P-CHK2 in gastric cancer cells treated with Cisplatin, while URI knockdown decreased the P-ATM and P-CHK2 expression after the cells were treated with cisplatin for 12 hours. Taken together, our results support that URI reduces DNA damage and promotes DNA repair of gastric cancer cells induced by cisplatin and therefore, enhances its resistance possibly via the ATM/CHK2 pathway Citation Format: Huiqin Bian, Yaojuan Lu, Yu Gu, Xiaojing Zhang, Lichun Sun, Junxia Gu, Qiping Zheng. URI participates in cisplatin-induced DNA damage repair of gastric cancer cells via the ATM/CHK2 pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5914.

Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63γ. To investigate the role of TAp63γ in chondrocyte differentiation and maturation, we developed stable TAp63γ expressing ATDC5 cells. Compared to control cells, TAp63γ cells showed significant upregulation of Col10a1 after 4 and 7 days in culture. Moreover, alkaline phosphatase, Alizarin red, and Alcian blue staining were stronger in TAp63γ cells, suggesting that TAp63γ promotes chondrocyte proliferation, hypertrophic differentiation, and possibly matrix mineralization. To investigate the in vivo function of TAp63γ during endochondral bone formation, we established transgenic mice that express flag-tagged TAp63γ driven by Col10a1 regulatory elements. Skeletal staining of transgenic mice at postnatal day 1 showed accelerated ossification in long bone, tail, and digit bones compared to wild-type littermates. Furthermore, Sox9 expression was reduced and Runx2 expression was increased in the proliferative and/or hypertrophic zones of these mice. Altogether, these results suggest that TAp63γ promotes endochondral ossification and skeletal development, at least partially via controlling chondrocyte differentiation and maturation.

The type X collagen gene (COL10A1) is specifically expressed in chondrocytes undergoing hypertrophy, which is an essential late stage of endochondral ossification during the development of long bones. We have previously localized multiple murine Col10a1 promoter-enhancer elements and used these elements for transgenic studies with LacZ reporter gene or genes of interest. Here, we report two additional transgenic mouse lines in which Cre was driven by the 10 kb Col10a1 promoter/intron and the 300-bp enhancer elements respectively. Cre activity was assessed by breeding the transgenic founders onto the RosA26R genetic background and to examine its β-gal activity (blue staining) via Cre/Lox P recombination. Our results showed that, in addition to the Cre activity in hypertrophic chondrocytes, we also observed blue staining of the bone marrow and the surrounding digits when the 10 kb Col10a1 promoter/intron element was used, whereas the 300-bp enhancer element could drive Cre expression exclusively within the hypertrophic zone as demonstrated by the blue staining pattern. This is intriguing, as the 10 kb promoter covers the 300-bp enhancer element. We then further reanalyzed the LacZ transgenic mice. We did observe non-specific blue staining in 10 kb-LacZ mice but not the mice with the 300-bp enhancer. In addition, the Cre reporter construct was on a coat-color vector backbone, which enables direct visual genotyping of the transgenic mice in the FVB/N albino background. Together, our results support that the 300 bp Col10a1 enhancer provides a more efficient genetic tool to target the hypertrophic zone for studies of skeletal development and disease.

URI, a member of the prefoldin family of molecular chaperones, functions in the regulation of nutrient-sensitive, mTOR-dependent transcription signaling pathways. Previous studies of several tumor types demonstrated that URI exhibits characteristics similar to those of an oncoprotein. URI has been shown as a mitochondrial substrate of S6 kinase 1 (S6K1), which acts to integrate nutrient and growth factor signals to promote cell growth and survival. Notably, the Akt/mTOR/p70S6K signaling pathway constitutes major negative regulatory mechanism of autophagy. However, the role of URI in autophagy has not been explored. Here, we investigated the involvement of URI in autophagy by manipulating its expression in MGC-803 and HGC-27 cells using siRNA and transfection approaches. GFP-LC3 punctum aggregation was assessed by confocal microscopy, whereas formation of autophagic vesicles was assessed using transmission electron microscopy. NH4Cl was used to inhibit autophagosome-lysosome fusion and to monitor autophagic flux. Expression of LC3-I, LC3-II, beclin1, total and phosphorylated mTOR, and p70S6k was assessed by Western blotting. The results showed that knockdown of URI induced significant autophagic flux in gastric cancer cells. URI regulates the expression of beclin1, which is essential for initiation of conventional autophagy. Levels of p-mTOR (Ser2448) and p-p70S6K (Thr389) increased in URI-overexpressing cells treated with the mTOR inhibitor rapamycin but decreased in URI-silenced cells. The inhibitory effect of URI silencing on mTOR and p70S6K phosphorylation was antagonized by the autophagy inhibitor 3-methyladenine. These results suggest that URI knockdown-induced autophagy is associated with the mTOR/p70S6K signaling pathway, indicating the potential existence of a novel autophagy regulatory mechanism mediated by URI.

目的:探讨癌蛋白非经典前折叠素RPB5相互作用因子(unconventional prefoldin RPB5 interactor,URI)在胃癌组织中的表达及其临床意义,分析URI对顺铂诱导的胃癌细胞凋亡的影响.方法:采用免疫组化辣根过氧化物酶法检测胃癌组织芯片中URI的表达,并分析其与临床特征的相关性;体外培养胃癌MGC-803细胞和SGC-7901细胞,运用Hiperfect转染试剂分别在两类胃癌细胞株中转染URI siRNA片段构建URI基因敲低细胞株,并用荧光定量PCR和蛋白质印迹法验证敲低效果;将细胞分为空白组、siRNA URI干扰组以及顺铂诱导下的未转染组、无关序列组和siRNA URI干扰组,采用流式细胞术检测各组细胞凋亡情况.结果:胃癌组织芯片检测结果显示,胃癌组织胞质中URI的表达量显著高于癌旁组织(P<0.05),胞核中URI的表达量虽比癌旁组织增高,但无显著性差异.URI的表达量与胃癌患者生存期、临床分期等无显著相关性.URI基因敲低后两类细胞株顺铂诱导的凋亡率均显著增加(P<0.01).结论:URI可能作为一种凋亡抑制因子在胃癌的发生中起作用.

The T-box gene family refers to a group of transcription factors that share a highly conserved, sequence-specific DNA-binding domain (T-box) containing around 180-amino acids. According to HUGO gene nomenclature committee (HGNC), there are 18 T-box family members. These T-box genes have been implicated essential roles during embryogenesis and cardiac development, given their specific expression pattern in developing mammalian heart for several T-box genes, including TBX5. TBX5 is consisted of three transcriptional variants which cover 9 exons and encode two distinct isoforms that differ in N-terminus. TBX5 is probably the most frequently studied T-box gene over the past decade due to the typical cardiac defects observed in Holt-Oram syndrome (HOS), which is caused by TBX5 mutation. Most of the mutations are within exons 3-7 where locate sequence coding for the T-box domain. Notably, a variety of cardiac defects, as well as abnormalities in limb and other organs have been seen in HOS syndrome with different kinds of TBX5 mutations, suggesting a heterogeneous disease mechanism. We have performed a meta-analysis of TBX5 and found a significant correlation between its single nucleotide polymorphism (SNP) rs3825214 (A to G), and risk of atrial fibrillation and its subtypes, supporting TBX5 as a master transcription factor for cardiac development. In addition, bioinformatics analysis of this SNP identified several TFs that may be affected for their binding affinity with TBX5. Identification and characterization of more TBX5 mutations and SNPs hold promise for therapeutic strategy targeting TBX5 associated developmental abnormalities and diseases.

URI is known to act as an oncoprotein in several tumors. Our previous studies have shown that URI is associated with the migration process in cervical and gastric cancer cells, but the mechanisms remain to be determined. Given the fact that URI positively regulates vimentin expression, we therefore investigated how URI regulated vimentin expression affects the migration and invasion of cells from two human cervical cancer cell lines HeLa and C33A, which differentially express URI. We have shown that knock-down of URI in HeLa cells using URI siRNA caused decreased vimentin mRNA and protein levels along with attenuated cell motility. Meanwhile, overexpression of URI by transfection of PCMV6-URI in C33A cells resulted in increased vimentin expression and enhanced cell migration and invasion. We have also used TGF-β to induce vimentin expression, which enhanced the cell migration and invasion abilities affected by URI, while inhibition of vimentin by siRNA attenuated URI's effect on cell migration and invasion. In addition, we have performed luciferase reporter and ChIP assays, and the results support that URI indirectly enhances the activity of vimentin promoter. Taken together, our results suggest that URI plays essential roles in the migration and invasion of human cervical cancer cells, possibly via targeting vimentin expression.

Runx2, a member of the Runt domain family, is a well-known master transcription factor for osteoblast differentiation. Runx2 has also been shown to play essential roles during chondrocyte hypertrophy, an important late stage of endochondral ossification linking both bone and cartilage development. To identify the co-factors that may interact with Runx2 together to regulate this critical process, we have performed yeast two-hybrid (Y2H) screening using Runx2 as a bait to screen a cDNA library of hypertrophic chondrocytes. The bait expressing cassette was constructed by fusing Runx2 with the pGBKT7 vector containing the Gal4 DNA binding domain (BD). The Mate & Plate libraries were constructed using pGADT7-Rec and cDNAs derived from hypertrophic chondrocytes enriched limb tissues or hypertrophic MCT cells. After co-transformation of pGBKT7-Runx2 and the cDNA libraries, colonies that grew in nutrition deficient medium were selected and subjected to PCR and sequencing analysis. We successfully identified more than 30 candidate genes, including Lectin-1 (Lgals1), Col1a2, Edf1 and Timp-2. We have performed literature review and bioinformatics analysis of these genes using GenePaint. Most of them show ubiquitous expression with Lgals1 show enhanced expression in hypertrophic chondrocytes. We further performed preliminary expression analysis by quantitative PCR and detected differential expression of these candidate genes in proliferative and hypertrophic MCT cells, with Timp-2 significantly (around 3-fold) and Lgals1 moderately (around 1.5 fold) upregulated in hypertrophic MCT cells. Our results suggest that, candidate gene Timp-2 is very likely to interact with Runx2 and together to play essential function during cartilage development, and possibly its homeostasis.

Cyclooxygenase 2 (Cox-2) has been implicated an essential role during bone repair, but the mechanisms remain elusive. Bone repair healing is known to include processes similar to endochondral ossification. In this study, we investigated the in vitro effect of Cox-2 on Col10a1 expression and chondrocyte hypertrophy, two critical components of endochondral ossification. Using quantitative RT-PCR, we detected increased mRNA levels of Cox-2 and Col10a1 in hypertrophic MCT cells, while cells treated with Cox-2 inhibitor, NS398, showed decreased mRNA and protein levels of Cox-2 and Col10a1. Increased Cox-2 also correlated with significantly upregulated Col10a1 in hypertrophic ATDC5 cells, whereas inhibition of Cox-2 significantly decreased Col10a1 expression. We further generated a Cox-2-expressing ATDC5 stable cell line. Compared with the controls, Cox-2 over-expression significantly increased Col10a1 as early as day 7 of continuous culturing, but not at days 14 and 21. Enhanced Alp staining was also observed in day 7 stable cell line. Correspondingly, we detected significantly increased levels of Runx2, Alp, Bcl-2, Bax, Col1a1, Osterix, and Bsp in day 7 stable line. Most of these genes have been associated with chondrocyte maturation and apoptosis. Together, our results support that Cox-2 promotes Col10a1 expression and chondrocyte hypertrophy in vitro, possibly through upregulation of Runx2 and other relevant transcription factors.