Effect of dura mater on enhancement of cranial osteogenesis in rats / 中国组织工程研究

BACKGROUND:The dura mater and skull are physically and functionally related,although there have been few investigations on primary extraction of dura mater and cranial cells,as well as co-culture of the two.The use of primary cells to investigate the influence of the dura mater on the skull is novel,and it is hoped that it may give a theoretical foundation for therapeutic therapy. OBJECTIVE:Rat dura mater and cranial bone cells were retrieved in situ to observe the influence of dura mater on cranial bone proliferation and differentiation,as well as to get a basic knowledge of the involvement of Twist1 in this process. METHODS:The enzyme digestion method was used in conjunction with the tissue block method to extract dural cells and cranial osteoblasts from rats within three days of birth.Immunofluorescence staining was used to identify the extracted cells,and alizarin red staining was used to identify and evaluate cranial osteoblasts and their mineralization ability.After co-culturing dural cells and cranial osteoblasts,real-time PCR was utilized to identify the expression of genes associated to cranial osteoblast proliferation and osteogenesis,as well as Twist1. RESULTS AND CONCLUSION:(1)Morphology:The retrieved dural cells had morphological traits similar to fibroblasts,while the osteoblasts were spindle-shaped.(2)Cell identification:immunofluorescence staining revealed that extracted dural cells expressed high levels of vimentin and cranial osteoblasts expressed high levels of alkaline phosphatase;cranial osteoblasts were stained with alizarin red 28 days after osteogenic induction,and obvious mineralized nodules were observed.(3)Real time PCR detection showed that the co-culture group had higher levels of PCNA,alkaline phosphatase,and RUNX2 mRNA expression than the control group(P＜0.01);however,Twist1 mRNA expression was lower(P＜0.01).(4)The findings showed that the primary extracted cranial osteoblasts had a high mineralization capacity,and that the dura mater was a key factor in promoting cranial growth and development and osteogenic differentiation,with Twist1 playing a key role in this process.

Mechanism of TFF3-dependent TWIST1 Upregulating TRIB3 to Promote Colorectal Cancer Metastasis / 肿瘤防治研究

Objective To investigate the effects of TFF3 overexpression on the proliferation, migration, and invasion ability of colorectal cancer HT29 cells and the mechanisms involved in cancer metastasis. Methods HT29 cells were transfected with pIRES2-TFF3, and the expression levels of mRNAs and proteins related to TFF3 gene, TWIST1/TRIB3 signaling pathway, and epithelial-mesenchymal transition (EMT) were detected by qRT-PCR and Western blot. The proliferation, migration, and invasion ability of HT29 cells were detected by the CCK-8, cell scratch, and Transwell assays. Changes in cell morphology after TFF3 overexpression were observed through transmission electron microscopy. Results After the HT29 cells were transfected with pIRES2-TFF3, the expression levels of TFF3 mRNA and protein significantly increased (P<0.01); cell proliferation, migration, and invasion were significantly enhanced (P<0.01); and the expression of related genes, such as TWIST1, TRIB3, Vimentin, and Snail were significantly upregulated. By contrast, the expression of E-cadherin significantly decreased (P<0.05). Various changes in cell morphology were observed after TFF3 overexpression, such as decrease in cell junctions, loss of cilia, formation of pseudopodia, and increase in fusiform cells. Conclusion TFF3 overexpression may promote EMT in colorectal cancer cells through the Twist1/TRIB3 signaling pathway, increase their metastatic potential, and accelerate the malignant progression of colorectal cancer.

Influence of compound Huangbai liquid on wound healing in rats with skin ulcer by activating Notch1/Twist1 axis / 中国免疫学杂志

Objective:To explore the influence and possible mechanism of compound Huangbai liquid on wound healing in rats with skin ulcer.Methods:A rat model of skin ulcer was constructed and randomly separated into model group,compound Huang-bai liquid low-dose,medium-dose and high-dose groups(compound Huangbai liquid L,M,H groups)and erythromycin ointment group,10 rats/group;another 10 rats were randomly collected as the normal group.Compound Huangbai liquid L,M,H groups were given 2.5 ml,5 ml and 10 ml of compound Huangbai liquid,respectively;the erythromycin ointment group was given erythromycin ointment to spread evenly on the wound surface using cotton swabs;the model group and normal group were not given drug treatment.At 0 d,3 d,7 d and 14 d of administration,the wound healing of the rats were recorded by taking pictures,and the wound healing rate were calculated.On the 14th day of administration,the pathological changes and collagen deposition in the wounds of rats were determined by HE and Masson staining methods;the levels of IL-1β,TNF-α,nitric oxide(NO),transforming growth factor-β(TGF-β)and hydroxyproline in wound tissue were determined by kits;and the expressions of Notch1 and Twist1 proteins in wound tissue were determined by Western blot.Results:On the 7th and 14th day of administration,compared with model group,the wound healing rate of the rats in the compound Huangbai liquid L,M,H groups and the erythromycin ointment group were significantly improved(P<0.05),and the inflammatory cell infiltration and other phenomena in rats were significantly improved on 14 days of administration,and the deposition of collagen in the wound surface of rats was also significantly increased.On the 14th day of administration,com-pared with the normal group,the levels of IL-1β,TNF-α,NO,hydroxyproline and TGF-β in the wound tissues of the model group were significantly increased(P<0.05),the protein expressions of Notch1 and Twist1 were significantly decreased(P<0.05);com-pared with model group,the levels of IL-1β,TNF-α and NO in the compound Huangbai liquid L,M,H groups and erythromycin oint-ment group were significantly decreased(P<0.05),the levels of hydroxyproline and TGF-β and the protein expressions of Notch1 and Twist1 were significantly increased(P<0.05).Conclusion:Compound Huangbai liquid can promote wound healing in rats with skin ulcers by activating the Notch1/Twist1 axis.

The mechanism of FAK/Twist1 signal pathway in the closure of cranial suture / 安徽医科大学学报

Objective@#To investigate the function of FAK / Twist1 signaling pathway during craniosynostosis closure.@*Methods@#Ten days old rats were divided into a control group (n = 50) and a rotation group (n = 50) . Both of them were made a approximately 0. 5 cm circle bone window at the midpoint of the lambdoid suture of the rat.The bone flaps were left free without damaging the dura mater. The bone flaps in the control group were repositioned in situ , and the bone flaps in the rotation group were rotated 180 ° and repositioned 3 weeks later. Then the experiments were performance as followed :open field test , measurement of body weight , head circumference , bone flap area , and thickness of bone flap in the two groups , observation of cranial suture closure by microscopy and HE staining , FAK / Twist1 expression determined by Western blot , real⁃time PCR , and immunohistochemical staining in the bone flap and dure , respectively. @*Results@#The cranial sutures was completely closed in the rotation groupand that was open in the control group through detecting by microscopic examination and HE staining. The thickness of the bone flap in the derotation group was greater than that in the control group , with statistical significance (P < 0. 01) . There were no significant differences between two groups in head circumference , weight , bone flap area , and operative area. The results of behavioral test showed that after the closure of cranial suture , the acsion of FAK was significantly increased in the calvaria and dura as well as Twsit1 was significantly decreased in the dura in rotating group measuring by Western blot , real⁃time PCR , and immunohistochemical staining (P < 0. 05) .@*Conclusion@#FAK/Twist1 may play an important role in craniosynostosis after rotation.

Estudo da imunoexpressão de proteínas envolvidas na transição epitélio-mesênquima em tumores de glândula salivar / Study of the immunoexpression of proteins involved in the epithelial-mesenchymal transition in salivary gland tumors

Os tumores de glândula salivar (TGS) apresentam notável complexidade clínica e biológica, razão para a qual muitos estudos investigam os eventos envolvidos na sua progressão. Uma das dinâmicas envolvidas na invasão tumoral de diversos tipos de carcinomas é a transição epitélio-mesênquima (TEM). Neste processo, as células epiteliais sofrem transição para um estado mesenquimal móvel, favorecendo a invasão e metástase. Sendo assim, esta pesquisa analisou a expressão imuno-histoquímica de E-caderina, Twist1, Snail1, α-SMA, metaloproteinases de matriz 9 (MMP-9) e Vimentina (VM) em 90 casos de TGS, correlacionando-os entre si e com parâmetros clinicopatológicos. Foram selecionados 20 casos de Adenoma pleomórfico (AP), 20 casos de Carcinoma mucoepidermoide (CME), 20 casos de Carcinoma adenoide cístico (CAC), 10 casos de Adenocarcinoma polimorfo (ACP), 10 casos de Carcinoma epitelial-mioepitelial (CEME) e 10 casos de Carcinoma ex-adenoma pleomórfico (CexAP). A análise de E-caderina, Twist1, Snail1 foi realizada em parênquima tumoral sendo observado o percentual de células positivas (PP), com escores variando de 0 a 4, e a intensidade de expressão (IE), cujos escores variaram de 0 a 3. A avaliação de MMP-9 foi realizada em parênquima e estroma tumoral, também avaliando-se a PP e a IE, ambos baseados em escores que variaram de 0 a 3. A marcação para α-SMA e VM foi analisada em região de estroma tumoral. Células positivas para α-SMA foram contabilizadas em 10 campos, obtendo-se, então a média. A VM foi avaliada de forma qualitativa, utilizando-se 4 escores de acordo com a IE e se a marcação é difusa ou focal. Os dados obtidos foram analisados no software Statistical Package for Social Science, GraphPad Prism e STATA. O nível de significância de 5% foi adotado para os testes estatísticos. Foi verificada menor imunomarcação de E-caderina nos APs em relação às neoplasias malignas de glândula salivar (NMGS). Observou-se baixa imunoexpressão de Twist1 e Snail1 em APs. Em relação a expressão nuclear do Twist1, constatou-se maior expressão nas neoplasias malignas quando comparadas aos APs. Ainda, Twist1 em núcleo foi correlacionado à expressão citoplasmática de E-caderina nas NMGS. No que concerne aos parâmetros clinicopatológicos, esta proteína se relacionou estatisticamente com maiores chances de óbito. Foi evidenciada baixa imunoexpressão de Snail1 entre as NMGS. No entanto, na análise dos CACs, foi verificada maior expressão nuclear na variante sólida em relação às demais. A expressão de MMP-9 em parênquima demonstrou correlação positiva com Twist1 citoplasmático e Snail1nuclear nas NMGS. A MMP-9 também apresentou correlação positiva na comparação da sua imunoexpressão em região de parênquima e de estroma. A VM se apresentou como um biomarcador a ser considerado na avaliação clínica dos pacientes, já que esta apresentou relação significativa com tamanho do tumor (T3-T4) e maior frequência de óbito. Ademais, a alta expressão desta proteína se apresentou como um fator preditivo independente para piores taxas de sobrevida global (SG). A avaliação dos demais fatores clinicopatológicos apresentou estágios clínicos avançados como indicador de valor prognóstico independente para menores taxas de SG, enquanto que para a sobrevida livre da doença, estes foram a localização em glândula salivar menor e presença de metástase à distância. Os resultados deste estudo sugerem que o processo de TEM pode estar relacionado ao estágio de diferenciação celular em APs e à progressão tumoral nas NMGS. Ressalta-se, também, maior participação de Twist1 e MMP-9 no cenário da TEM em tumores malignos de glândula salivar, além da possibilidade de utilização da VM como indicador de valor prognóstico (AU).

Salivary gland tumors (SGTs) present remarkable clinical and biological complexity; therefore, many studies investigate the events involved in their progression. One of the dynamics involved in the tumor invasion of different types of carcinomas is the epithelial-mesenchymal transition (EMT). In this process, epithelial cells undergo a transition to a mobile mesenchymal state, favoring invasion and metastasis. Therefore, this research analyzed the immunohistochemical expression of E-cadherin, Twist1, Snail1, α-SMA, vimentin (VM) and matrix metalloproteinase 9 (MMP-9) in 90 SGTs cases; correlations among the biomarkers, as well as between the biomarkers and clinicopathological parameters were made. We selected 20 cases of pleomorphic adenoma (PA), 20 cases of mucoepidermoid carcinoma (MEC), 20 cases of adenoid cystic carcinoma (ACC), 10 cases of polymorphous adenocarcinoma (PAC), 10 cases of epithelial-myoepithelial carcinoma (EMC) and 10 cases of carcinoma ex-pleomorphic adenoma (CXPA). E-cadherin, Twist1, and Snail1 were analyzed in tumor parenchyma, observing the percentage of positive cells (PP) using scores ranging from 0 to 4, and the expression intensity (EI), whose scores were ranged from 0 to 3. The evaluation of MMP-9 was performed in tumor parenchyma and stroma, also evaluating PP and IE, both based on scores that ranged from 0 to 3. The labeling for α-SMA and VM was analyzed in stromal cells. Positive cells for α-SMA were counted in 10 fields and the mean was calculated. VM was evaluated qualitatively, using 4 scores according to EI and whether the labeling was diffuse or focal. Obtained data were analyzed using Statistical Package for Social Science, GraphPad Prism, and STATA software. The significance level of 5% was adopted for the statistical tests. Patients were mostly female, with a mean age of 49.8 years; the major salivary glands were the most affected anatomical site, mainly the parotid gland. A lower E-cadherin immunostaining was verified in PAs in comparison to malignant neoplasms of salivary glands (MNSGs). Low immunoexpression of Twist1 and Snail1 was observed in PAs. Regarding the nuclear expression of Twist1, it was found greater expression in malignant neoplasms than in PAs. Furthermore, Twist1 in the nucleus was correlated with cytoplasmic expression of E-cadherin in MNSGs. Regarding clinicopathological parameters, this protein was statistically related to higher chances of death. Low immunoexpression of Snail1 was evidenced among the MNSGs. However, in the analysis of CACs, greater nuclear expression was observed in the solid variant compared to the others. Expression of MMP-9 in parenchyma showed a positive correlation with cytoplasmic Twist1 and Snail1nuclear in MNSGs. MMP-9 also showed a positive correlation when comparing its immunoexpression in the parenchyma and the stroma. VM was presented as a biomarker to be considered in the clinical evaluation of patients since it showed a significant correlation between greater tumor size and a higher frequency of death. Furthermore, the high expression of this protein appeared as an independent predictive factor for worse overall survival (OS) rates. The evaluation of the rest of the clinicopathological factors showed advanced clinical stages as an indicator of independent prognostic value for lower rates of OS. For disease-free survival, these indicators were the location in the minor salivary gland and the presence of distant metastasis. Our results suggest that the EMT may be related to myoepithelial differentiation in PAs and tumor progression in MNSGs. Also, Twist1 and MMP-9 appear to play a greater role in the scenario of EMT in MNSGs; finally, VM might be used as a prognostic value indicator (AU).

Síndrome de Saethre-Chotzen: a propósito de un caso / Saethre-Chotzen syndrome: a case report

El síndrome de Saethre-Chotzen es un síndrome malformativo craneofacial caracterizado por una sinostosis de las suturas coronales y alteraciones de extremidades. Tiene una prevalencia de 1 de cada 25 000-50 000 recién nacidos vivos. Se presenta el caso de un neonato sin antecedentes de interés con alteraciones craneofaciales al nacer. Ante los rasgos fenotípicos del paciente, se realizó una tomografía axial computada craneal, que mostró la fusión parcial de la sutura coronal y evidenció la presencia de huesos wormianos en localización metópica y lambdoidea derecha. Con la sospecha clínica de síndrome malformativo craneofacial, se solicitó análisis del exoma dirigido, que confirmó que el paciente era portador heterocigoto de la variante patogénica c.415C>A, que inducía un cambio de prolina a treonina en la posición 139 del gen TWIST1, responsable del síndrome. La presencia de huesos wormianos, hallazgo no descrito hasta ahora en la literatura, amplía la variabilidad fenotípica conocida de este síndrome.

The Saethre-Chotzen syndrome is a craniofacial malformation syndrome characterized by synostosis of coronal sutures and limb anomalies. The estimated prevalence of this syndrome is 1 in 25 000-50 000 live births. We present a case report of a neonate, without relevant family history, who presented craniofacial alterations at birth. Given the phenotypic features, a cranial computed tomography scan was performed, showing partial fusion of the coronal suture, evidencing the presence of wormian bones in the metopic and right lambdoid location. With the clinical suspicion of craniofacial malformation syndrome, an analysis of the directed exome was requested confirming that the patient is a heterozygous carrier of the pathogenic variant c.415C>A, which induces a change of proline to threonine at position 139 of the TWIST1 gene, responsible for Saethre-Chotzen syndrome.The presence of wormian bones, a finding not described so far in the literature, extends the well-known phenotypic variability of this syndrome.

Expression of Twist1 and Twist2 in hypopharyngeal carcinoma and its relationship with clinicopathological features / 中国医师杂志

Objective:To investigate the relationship between the expression of Twist1 and Twist2 protein and with clinicopathological features in hypopharyngeal carcinoma.Methods:The clinical and pathological data of 58 patients with hypopharyngeal carcinoma who underwent surgery in Liaoyang Central Hospital from May 2014 to September 2019 were retrospectively analyzed. The general demographic and pathological data of the patients were collected, and the expression of Twist1 and Twist2 protein in the hypopharyngeal cancer tissues and adjacent tissues were detected by immunohistochemistry. The patients were followed up to record their survival. The relationship between protein expression of Twist1 and Twist2 and clinical pathological stages and prognosis was analyzed.Results:Immunohistochemistry showed that the positive expression rates of Twist1 and Twist2 protein in 58 patients with hypopharyngeal cancer were significantly higher than those in adjacent tissues ( P<0.05). The positive expression rates of Twist1 and Twist2 in patients with T stage T 3+ T 4 and clinical stage Ⅲ+ Ⅳ were significantly higher than those in patients with T 1+ T 2 and Ⅰ+ Ⅱ ( P<0.05). The positive expression rates of Twist1 and Twist2 in patients with lymph node metastasis were significantly higher than those without lymph node metastasis, and the positive expression rates of Twist1 and Twist2 in low differentiation were significantly higher than those in high differentiation ( P<0.05). 52 patients were followed up postoperatively, the follow-up rate was 89.67%, among which 28 patients recurred (53.85%), and the 1-, 3-, and 5-year survival rates were 80.77%(42/52), 53.85%(28/52), and 21.15%(11/52), respectively. The survival rate of patients with positive expression of Twist1 and Twist2 protein was significantly lower than that of patients with negative expression ( P<0.05). Multiple analysis showed that recurrence, tumor stage, positive expression of Twist1 and Twist2 protein were independent risk factors for survival ( P<0.05). Conclusions:The expression of Twist1 and Twist2 protein in hypopharyngeal carcinoma is stronger than that in normal tissues. The expression of Twist1 and Twist2 protein is related to T stage, clinical stage, pathological tissue type and lymph node metastasis. Meanwhile, the high expression of Twist1 and Twist2 proteins is an independent factor affecting the prognosis of patients. They may be potential indicators to judge the prognosis of patients with hypopharyngeal cancer.

Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway / 中国中药杂志

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P<0.05 or P<0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P<0.05 or P<0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P<0.05 or P<0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P<0.05 or P<0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P<0.05 or P<0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

The combination of mammaglobin A and TWIST-1 increases detection of circulating tumor cells in breast cancer / A combinação de mamaglobina A e TWIST-1 aumenta a detecção de células tumorais circulantes no câncer de mama

ABSTRACT Objectives: Breast cancer cells that are released into the bloodstream are called circulating tumor cells (CTCs). CTCs can express different genes, like TWIST-1 and mammaglobin A (MGA). The aims of this study were to analyze the expression of TWIST-1 and MGA in the blood of breast cancer patients to detect CTCs and to assess the association between the presence of CTCs and prognostic parameters of breast cancer. Methods: Prospective study. Total ribonucleic acid (RNA) from blood mononucleated cells was obtained from breast cancer patients (n = 36; age: 51.5 ± 12.5 years) and healthy donors (n = 14; age: 49.4 ± 9.4 years). Real-time polymerase chain reaction (RT-PCR) was performed to analyze the expression of TWIST-1 and MGA. Results: Patient carcinomas - ductal (86.7%), other types (13.3%). MGA gene expression was not detected in the donors' samples, while it was detected in 14% of the patient samples. Overexpression of TWIST-1 gene was observed in 17% of the patient samples. The combined analysis of both markers allowed the detection of CTCs in 27.8% of the samples, resulting in a significant (p < 0.05) sensitivity increase of detection. No significant associations (p > 0.05) were found between expression of the analyzed genes and the breast cancer prognostic factors. Conclusion: Combined analysis of TWIST-1 and MGA increased the sensitivity of CTCs detection compared to the single analysis of each gene. The detection of CTCs was not associated with known prognostic factors, suggesting that it is able to provide clinical information in addition to routine breast cancer clinicopathological parameters.

RESUMEN Objetivos: Las células de cáncer de mama liberadas al torrente sanguíneo se llaman células tumorales circulantes (CTCs). Las CTCs pueden expresar diferentes genes, como TWIST-1 y mamaglobina A (MGA). Los objetivos de este estudio fueron analizar la expresión de TWIST-1 y MGA en la sangre de pacientes con cáncer de mama (CM) para detectar CTCs y evaluar la asociación entre la presencia de CTCs y los parámetros pronósticos del CM. Métodos: Estudio prospectivo. Se obtuvo el ácido ribonucleico (ARN) de las células mononucleadas en la sangre de pacientes con CM (n = 36, edad: 51,5 ± 12,5 años) y donantes sanas (n = 14; edad: 49,4 ± 9,4 años). Se realizó reacción en cadena de la polimerasa en tiempo real (RT-PCR) para analizar la expresión de TWIST-1 y MGA. Resultados: Carcinoma ductal (86,7%), otros tipos (13,3%). No se detectó la expresión del gen MGA en las muestras de las donantes, pero en el 14% de las muestras de las pacientes. Se observó elevada expresión de TWIST-1 en el 17% de las muestras de pacientes con CM. El análisis combinado de ambos marcadores permitió detección de CTCs en el 27,8% de las muestras, resultando en un aumento significativo (p < 0,05) en la sensibilidad de detección. No se encontraron asociaciones significativas (p > 0,05) entre la expresión de los genes y los factores pronósticos. Conclusión: El análisis combinado de TWIST-1 y MGA aumentó la sensibilidad de detección de CTCs en comparación con el análisis de cada gen. La detección de CTCs no se asoció a factores pronósticos conocidos, sugiriendo que podría ofrecer informaciones clínicas adicionales a los parámetros clínico-patológicos de rutina del CM.

RESUMO Objetivos: As células cancerígenas da mama liberadas na corrente sanguínea são chamadas de células tumorais circulantes (CTCs). As CTCs podem expressar diferentes genes, como TWIST-1 e mamaglobina A (MGA). Os objetivos deste estudo foram analisar a expressão de TWIST-1 e MGA no sangue de pacientes com câncer da mama (CM) para detectar CTCs e avaliar a associação entre a presença de CTCs e os parâmetros prognósticos do CM. Métodos: Estudo prospectivo. O ácido ribonucleico (RNA) das células mononucleadas no sangue foi obtido de pacientes com CM (n = 36, idade: 51,5 ± 12,5 anos) e doadoras saudáveis (n = 14; idade: 49,4 ± 9,4 anos). Reação da cadeia da polimerase em tempo real (RT-PCR) foi realizada para analisar a expressão de TWIST-1 e MGA. Resultados: Carcinoma ductal (86,7%), outros tipos (13,3%). A expressão do gene MGA não foi detectada nas amostras das doadoras, mas foi observada em 14% das amostras das pacientes. Superexpressão de TWIST-1 foi observada em 17% das amostras dos indivíduos com CM. A análise combinada de ambos os marcadores permitiu a detecção de CTCs em 27,8% das amostras, resultando em um aumento significativo (p < 0,05) na sensibilidade da detecção. Associações significativas (p > 0,05) entre a expressão dos genes e os fatores prognósticos não foram encontradas. Conclusão: A análise combinada de TWIST-1 e MGA aumentou a sensibilidade da detecção de CTCs em comparação com a análise de cada gene. A detecção de CTCs não foi associada a fatores prognósticos conhecidos, sugerindo que ela pode fornecer informações clínicas adicionais aos parâmetros clinicopatológicos de rotina do CM.

Experimental study on the effect of curcumin on endometriosis in rats / 中国临床药理学与治疗学

AIM: To analyze the effect of curcumin on the expression of transcription factors Twist1, Twist2, E-cadherin and Syndecan-1 in rats with endometriosis. METHODS: Thirty SD rats were randomly divided into five groups: model group, low dose curcumin group (25 mg•kg-1•d-1), medium dose curcumin group (50 mg•kg-1•d-1), high dose curcumin group (100 mg•kg-1•d-1) and gestrinone group (0.5 mg•kg-1•d-1). There were 6 rats in each group, and 5 rats in sham operation group were set up as blank control group. The expression of Twist1, Twist2, E-cadherin and Syndecan-1 was detected by immunohistochemistry. RESULTS: Compared with the model group, the ectopic tissue volume of curcumin group and gestrinone group was significantly lower, the difference was statistically significant (P0.05). CONCLUSION: Curcumin can reduce the expression of Twist1, Twist2 and Syndecan-1, increase the expression of E-cadherin, and inhibit the growth of endometrium in the model rats, so as to achieve the purpose of treatment.

Acquisition of multidrug resistance by overexpression of Twist1 in colorectal cancer cells SW620 and its mechanism / 中国免疫学杂志

Objective: To investigate the effect of overexpression of Twist1 on multidrug resistance( MDR) in colorectal cancer SW620 cells and its possible mechanism. Methods: The colorectal cancer SW620 cells were transfected with Twist1 overexpressing lentivirus and negative control virus, which were called experimental group ( SW620-Twist1) and control group ( SW620-NC) respectively. The protein and mRNA expression of Twist1、ABCB1 and ABCG2 in two groups were detected by Western blot and RT-qPCR, respectively. The growth inhibitory rate of 5-Fluorouracil(5-FU) and Oxaliplatin( OXA) at 24 ,48 and 72 h in two groups were determined by CCK8 assay. Cancer stem cell markers CD133,CD44 expression levels were detected by Flow cytometry. Results: The experimental group was compared with the control group, the expressions of Twsit1 gene mRNA and protein increased significantly( P<0. 01) ,Twist1 gene overexpressing SW620 cell line was successfully constructed. The inhibition rates of cell proliferation of the three chemotherapeutic drugs at 48 h and 72 h were significantly decreased ( 48 h, P<0. 05 ; 72 h, P<0. 01) . The mRNA and protein expression levels of ABCB1,ABCG2 were significantly increased( P<0. 01). The expression of CSCs markers CD133 and CD44 increased significantly (P<0. 01). Conclusion: Overexpression of Twist1 can promote the expression of ABCB1 and ABCG2 genes in SW620 cells and make the tumor cells acquire MDR. This phenomenon may be related to the promotion of stemness.

Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells / 中国结合医学杂志

OBJECTIVE@#To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms.@*METHODS@#The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively.@*RESULTS@#COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells.@*CONCLUSIONS@#COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.

Effect of Twist1 gene on migration and invasion of esophageal squamous cell carcinoma / 中国医师进修杂志

Objective To investigate the expression and clinicopathological significance of Twist1 gene in esophageal squamous cell carcinoma(ESCC),and its effect on migration and invasion of ESCC.Methods The ESCC tissues and adjacent normal tissues of 30 ESCC patients having underwent surgery were selected. The expression levels of Twist1 mRNA and protein were detected by real time fluorescence quantitative polymerase chain reaction (RT- qPCR) and Western blot methods. The expression level of Twist1 mRNA in adjacent normal tissues was used as control,the relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70,K140 and EC109 were detected,and the relationship between the expression level and clinicopathological features was analyzed. ESCC cell line EC109 was selected for cell scratch test and Transwell chamber invasion assay respectively.Twist1 small interfering RNA and control double stranded nonsense RNA were transfected respectively, and the changes were compared.Results The expression level of Twist1 mRNA in ESCC tissue was significantly higher than that in adjacent normal tissue(1.91 ± 0.93 vs.0.54 ± 0.26),and there was statistical difference(P<0.01). The relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70, K140 and EC109 were significantly higher than those in adjacent normal tissue (2.30 ± 0.34, 1.78 ± 0.28 and 4.37 ± 0.25 vs. 1.00 ± 0.48), and there were statistical differences (P<0.05). The expression level of Twist1 protein in ESCC tissue was significantly higher than that in adjacent normal tissue (1.35 ± 0.66 vs. 0.25 ± 0.05), and there was statistical difference (P<0.05). The relative expression level of Twist1 mRNA in ESCC tissue had correlation with lymph node metastasis and invasion degree(P<0.05).The expression levels of Twist1 mRNA in ESCC cell lines EC109 after transfected Twist1 small interfering RNA and double stranded nonsense RNA were 0.45 ± 0.16 and 1.00 ± 0.20, the express levels of Twist1 protein were 0.24 ± 0.09 and 0.58 ± 0.10,and there were statistical differences(P<0.05).The cell scratch test result showed that the migration rates of 36 and 72 h of ESCC cell line EC109 were significantly lower than those of the control double stranded nonsense RNA,after Twist1 expression was down regulated by RNA interference:(32.6 ± 4.7)% vs.(16.2 ± 6.0)% and(71.9 ± 4.7)% vs.(53.2 ± 6.6)%,there were statistical differences(P<0.05).The Transwell chamber invasion assay result showed that the total number of cells in the bottom of Twist1 through the small interfering RNA of EC109 was lower than that of the transfected control double stranded nonsense RNA (92.5 ± 8.1 vs. 32.7 ± 7.5), and there was statistical difference (P<0.05). Conclusions Twist1 gene may be highly abnormally expressed in ESCC, which has some correlation with invasion and lymph node metastasis,and may have some influences on the migration and invasion of ESCC.

Inhibition of in vitro carcinogenicity by 8-bromo-7-methoxychrysin mediated by modulating miR-519d/TWIST1 axis in liver cancer stem-like cells from SMMC-7721 cell line / 中国药理学通报

Aim To determine whether 8-bromo-7-me-thoxychrysin ( BrMC) inhibits in vitro carcinogenicity via up-regulating miR-519d expression and down-regu-lating Twist1 expression in liver cancer stem-like cells ( LCSLCs) derived from SMMC-7721 cell line. Meth-ods The second generation spheroids derived from SMMC-7721 cell line were obtained by sphere-forming assay and were considered as LCSLCs . Then LCSLCs were treated with various concentrations ( 1.0, 3.0, 10.0 μmol·L-1) of BrMC. The expression level of miR-519d was detected using real-time PCR. And in vitro carcinogenicity was investigated by sphere-forming assay and clone-forming assay in agar. The transcrip-tional activity and protein expression of Twist1 were an-alyzed using luciferase reporter assay and Western blot. Moreover, the molecular mechanism of BrMC was elucidated via miR-519 mimic transfection and Twist1 gene transduction, respectively. Results Compared with SMMC-7721 cells, miR-519d-3p was low-ex-pressed and Twist1 was over expressed in LCSLCs. And the sphere-forming ratio and the clone-forming ra-tio decreased by treatment with BrMC ( 1.0, 3.0, 10.0 μmol·L-1) in a dose-dependent manner. Fur-thermore, luciferase reporter assay demonstrated miR-519d could directly target the 3′ untranslated region of Twist1 mRNA and regulate protein expression. miR-519d mimic enhanced the effects of BrMC (3.0 μmol ·L-1) . However, Twist1 gene transduction effective-ly reversed the effects of BrMC ( 3.0 μmol·L-1) . Conclusion BrMC inhibits in vivo carcinogenicity via regulating miR-519/Twist1 signal axis in LCSLCs de-rived from SMMC-7721 cell line.

miR-489 inhibits the stem cell phenotype and epithelial-mesenchymal transition of colon cancer by regulating the expression of TWIST 1 / 基础医学与临床

Objective To explore the effects and regulation mechanism of miR-489 on the stem cell phenotype and epithelial-mesenchymal transition of colon cancer cells in vitro.Methods The miR-489 expression of colon cancer cell lines ( HT29, SW480 and SW620, HCT116) and normal intestinal epithelial cells HIEC were detec-ted by RT-qPCR, the miR-489 expression in colon cancer cells was raised by gene transfection technology, the colon cancer stem cell markers CD133, CD44, EpCAM, ALDH1 and epithelial marker E-cadherin, mesenchy-mal cells markers vimentin and N-cadherin were detected by Western blot.The expression of TWIST1 mRNA and protein were detected by RT-qPCR and Western blot.Results The miR-489 relative expression in four kinds of colon cancer cells ( HT29, SW480 and SW620, HCT116) were significantly lower than that of normal HIEC in-testinal epithelial cells, the difference was statistically significant ( P<0.05) .Up-regulation of miR-489 expres-sion in HT29 and HCT116 cells leaded to lower expression of colon cancer stem cell marker CD133, CD44, EpCAM ALDH1 and mesenchymal cells markers Vimentin , N-cadherin, higher expression of epithelial markers ;E-cadherin (P<0.05).Also, up-regulation of miR-489 expression in HT29 and HCT116 cells leaded to lower ex-pression of TWIST 1 mRNA and protein ( P<0.05 ) .Conclusions miR-489 is down-regulated in colon cancer cells and up-regulation of miR-489 expression inhibits stem cell phenotype and epithelial-mesenchymal transition through targeting TWIST1 in colon cancer .

Expression and clinical significance of Twist1 in bone marrow and extramedullary lesions tissues in patients with myeloma / 国际检验医学杂志

Objective To explore the expression and clinical significance of Twist1 in bone marrow and ex-tramedullary lesions of patients with myeloma.Methods 30 cases of patients diagnosed with multiple myelo-ma(MM)treated in our hospital were selected for the study,and other 25 cases patients with MM combined with extramedullary lesions(EMD)were selected as the combined group.The positive expression rate of Twist1 in myeloma nuclei was detected by immunohistochemistry in the MM and EMD tissues,and the ex-pressions of Twist1 were compared in patients with different tumor stages,and the relationship between Twist1 and clinical parameters(gender,age)was analyzed.Results Twist1 could be expressed in the nucleus and cytoplasm of marrow tissue and extramedullary tissue among myeloma patients,and the high expression rate in patients complicated with extramedullary lesions was higher than that in the cytoplasm(P<0.05),and the high expression rate in the nucleus and cytoplasm of extramedullary tissues was higher than that in the myeloma group(P<0.05).The Twist1 expression rate in the nucleus of myeloma tissues was increased with the increase of the stages,but there was no significant difference in the expression rate between the two groups(P>0.05).The relationship analysis of Twist1 expression in the nucleus of bone marrow tissues and the clinicopathological features showed that the high or low Twist 1 expression was not significantly correlated with the gender and age(P>0.05),but had a certain relationship with clinical stages,and was positively cor-related with the disease stages(r=0.316,P<0.05).Conclusion Twist1 is highly expressed in bone marrow of patients with myeloma,especially in the nucleus of extramedullary lesions tissue of patients with combined with extramedullary disease,and the high expression of Twist1 is correlated with pathological staging,and it can be used as one of the clinical indicators for poor prognosis of patients with myeloma.

Effects of HDAC5 on the proliferation and apoptosis of the gastric cancer cell line SGC-7901 / 中国肿瘤临床

Objective: To investigate HDAC5 expression in gastric cancer cell lines and its effects on the proliferation and apoptosis of the gastric cancer line SGC-7901. Methods: The expression patterns of HDAC5 and Twist1 in gastric cancer cell lines and normal gastric mucosal cells were detected by Western blot. The effects of HDAC5 and Twist1 on the proliferation and apoptosis of SGC-7901 cells were analyzed by MTT and flow cytometry, respectively. Results: The expression of HDAC5 and Twist1 in gastric cancer cell lines were significantly higher than that in normal gastric mucosal cells (P<0.05). HDAC5 knockdown significantly down-regulated Twist1 expression,inhibited cell proliferation, and induced apoptosis in SGC-7901 cells, whereas HDAC5 overexpression exhibited an opposite effect (P<0.05). Moreover, Twist1 knockdown significantly inhibited cell proliferation and induced apoptosis in SGC-7901 cells (P<0.05). Conclusion:HDAC5 may promote cell proliferation and inhibit apoptosis in gastric cancer cells by upregulating Twist1 expression, thus promoting the initiation and development of gastric cancer.

The Role of Twist1 in Stem Cell Differentiation through Mechanical Cues: A Review and Hypothesis.

Due to the pivotal role of stem cell differentiation in regeneration and disease cure, the study of it has always been a research highlight during the recent years. Stress microenvironment has a great impact on cell growth, proliferation, differentiation and apoptosis. Twist1, as a core epithelial-mesenchymal transition (EMT) regulatory factor, plays an important role in these processes. Moreover, Twist1 gene can express in alveolar bone – periodontal ligament interface and the expression can be regulated by changes in the occlusal force. In this article, we will present a review of Twist1 gene, especially in the aspect of the biological functions in stem cell differentiation under mechanical signals and explore whether Twist1 involved in tissue remodeling in alveolar bone - periodontal membrane interface under stress.

Relation between expression of BCSCs associated genes from swelling axillary lymph nodes of breast cancer with recurrence and metastasis of tumor / 重庆医学

Objective To explore the relation between the expression of breast cancer stem cells(BCSCs) related gene CD44 , OCT4 and Twist1 in breast cancer tissues and swelling axillary lymph nodes with the recurrence and metastasis of breast cancer . Methods The genes and antigen expressions of BCSCs relative factors CD44 ,CD24 ,OCT4 and Twist1 in th breast cancer tissue and axillary lymph nodes were detected by the immunohistochemical method and RT‐PCR .Results Totally 14 cases of preopera‐tively and intraoperatively diagnosed breast cancer had axillary lymph nodes metastasis of cancer cells .The over expression of BC‐SCs relative factors CD44 ,OCT4 and Twist1 antigen positive in the breast cancer tissue and axillary lymph nodes were detected by the immunohistochemical method ,while CD24 was lowly expressed;the gene expressions of BCSCs relative factors CD44 ,OCT4 and Twist1 in the breast cancer tissue and axillary lymph nodes were enhanced by the RT‐PCR detection (P0 .05) .Conclusion The genes and antigen expression of BCSCs related factors CD44 ,OCT4 and Twist1 in the patients with breast cancer complicating swelling axillary lymph nodes are significantly increased ,which might indicate that the BCSCs related factors are related with easily early metastasis and recurrence of breast cancer cells .

The expression of Twist and E -cadherin and its clinical significance in ovarian endometriosis / 预防医学

Objective To know the expression and clinical significance of Twist1,Twist2 and E -cadherin in ovarian endometrium.Methods Immunohistochemistry was used to detect the expression of Twist1,Twist2 and E -cadherin in tissues of ovarian endometrium (EMs I -II,n =24;EMs III -IV,n =30)andnormal endometriums without endometriosis (Control,n =24).Results The expression levels of Twist1,Twist2 were significantly higher in EMs I -II (4.25 ±0.79, 3.83 ±0.96 respectively)and EMs III -IV (4.67 ±0.88,4.30 ±0.95 respectively)while the values of the control group were 3.50 ±0.83,3.54 ±0.88 respectively(both P <0.05),and the group of EMs III -IV had a higher expression of Twist2 (4.30 ±0.95)than that in EMs I -II (3.83 ±0.96)(P <0.05 ).At the same time,the expression of E -cadherin were significantly lower in EMs I -II (3.79 ±0.98)and EMs III -IV (3.57 ±0.73)than the control (4.54 ± 0.93)(P <0.05).The expression of Twist1 and Twist2 was negatively related to that of E -cadherin respectively (both P<0.05),while the expression of Twist1 was positively related to that of Twist2 (P <0.05).Conclusion Twist1,Twist2 and E -cadherin are closely related to the prevalence of endometriosis,while the ability of invasion and metastasis may be associated with the high expression of Twist1 and Twist2,as well as the low expression of E -cadherin.