Effect of Qianyang Yuyin Granules on Mesenchymal Transdifferentiation of Renal Tubular Epithelial Cells Induced by TGF-β1 / 中国实验方剂学杂志

ObjectiveBy observing the effect of Qianyang Yuyin granules on the phenotype of renal tubule epithelial cells， the intervention of Qianyang Yuyin granule on renal interstitial fibrosis was investigated. MethodThe renal tubular epithelial cells （HK-2） were treated with different concentrations of transforming growth factor （TGF）-β1 （5， 10， 15， 20， 25 μg·L-1） for 24 hours， and cell morphology and growth state were observed with an inverted phase contrast microscope. The 20 μg·L-1 was selected as the most appropriate concentration of TGF-β1 according to Western blot results for subsequent experiments. HK-2 cells were divided into six groups： blank group， TGF-β1 group （concentration of 20 μg·L-1）， low， medium， and high dose Qianyang Yuyin granule groups （concentration of 0.5， 1， 2 g·L-1）， and valsartan group （1 × 10-5 mol·L-1）. The cell activity was measured by cell proliferation and cell counting kit-8 （CCK-8）. The cell migration ability was detected by scratch test. The Transwell method was used to detect the invasiveness of cells. Western blot was used to detect levels of fibronectin （FN）， E-cadherin， α-smooth muscle activator （α-SMA）， Vimentin， collagen type Ⅰ（Col Ⅰ）， collagen type Ⅳ（Col Ⅳ）， and other related proteins. ResultTGF-β1 stimulating epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells was time- and concentration-dependent. Compared with the blank group， higher concentration in the TGF-β1 group indicates longer intervention time and more obvious long spindle change of cells， and the migration and invasion ability of the cells was significantly enhanced. The protein expression level of FN， α-SMA， Vimentin， Col Ⅰ， and Col Ⅳ increased significantly （P<0.05， P<0.01）， while the expression level of E-cadherin protein decreased （P<0.05）. Compared with the TGF-β1 group， Qianyang Yuyin granule groups could maintain normal cell morphology， and the migration and invasion ability of the cells was inhibited. The protein expression level of FN， α-SMA， Vimentin， Col Ⅰ， and Col Ⅳ decreased （P<0.05， P<0.01）， and the expression of E-cadherin protein was significantly restored （P<0.05）. ConclusionQianyang Yuyin granule can reverse TGF-β1-induced interstitial transformation of renal tubular epithelial cells by reducing the phenotypic expression of mesenchymal cells and increasing the phenotypic expression of epithelial cells.

Pathogenesis of Idiopathic Pulmonary Fibrosis and Modulating Effect of Chinese Medicine： A Review / 中国实验方剂学杂志

Idiopathic pulmonary fibrosis （IPF）， as a progressive lung disease， has a poor prognosis and no reliable and effective therapies. IPF is mainly treated by organ transplantation and administration of chemical drugs， which are ineffective and induce side effects， failing to meet the clinical needs. Therefore， developing safer and more effective drugs has become an urgent task， which necessitates clear understanding of the pathogenesis of IPF. The available studies about the pathogenesis of IPF mainly focus on macrophage polarization， epithelial-mesenchymal transition （EMT）， oxidative stress， and autophagy， while few studies systematically explain the principles and links of the pathogeneses. According to the traditional Chinese medicine theory， Qi deficiency and blood stasis and Qi-Yang deficiency are the key pathogeneses of IPF. Therefore， the Chinese medicines or compound prescriptions with the effects of replenishing Qi and activating blood， warming Yang and tonifying Qi， and eliminating stasis and resolving phlegm are often used to treat IPF. Modern pharmacological studies have shown that such medicines play a positive role in inhibiting macrophage polarization， restoring redox balance， inhibiting EMT， and regulating cell autophagy. However， few studies report how Chinese medicines regulate the pathways in the treatment of IPF. By reviewing the latest articles in this field， we elaborate on the pathogenesis of IPF and provide a comprehensive overview of the mechanism of the active ingredients or compound prescriptions of Chinese medicines in regulating IPF. Combining the pathogenesis of IPF with the modulating effects of Chinese medicines， we focus on exploring systemic treatment options for IPF， with a view to providing new ideas for the in-depth study of IPF and the research and development of related drugs.

Immunohistochemical appraisal of epithelial mesenchymal transition type III in gall bladder cancer

Background: Epithelial-mesenchymal transition (EMT) is the heart of invasion. EMT associated with cancer progression and metastasis is known as type III EMT. Beta-catenin, E-cadherin, and MMP9 markers of EMT are routinely employed for diagnostic purposes. Aims: We employed these markers to study EMT by immunohistochemistry (IHC) in gall bladder cancer (GBC) with respect to depth of tumor invasion, clinical outcome, and disease-free survival. Settings and Design: This was a prospective case-control study. Material and Methods: Seventy gall bladders were included (50 GBC and 20 CC). After detailed histology, immunoexpression was studied in terms of percentage and strength of expression. Statistics Analysis Used: Expression was compared between CC and GBC by Student t test and analysis of variance. Kaplan–Meier was used for survival analysis, and the extent of agreement (“Kappa”) was calculated. Results and Conclusions: The age of incidence of GBC was 49.40 (+11.6) years with female predominance (F:M = 4:1). In 88% (44/50) of GBC, the fundus was involved. Moderately differentiated adenocarcinoma was most frequent [54%; 27/50]. Significant downregulation of E-cadherin (P = 0.022) and beta-catenin (P < 0.001) and upregulation in MMP9 (P < 0.001) were seen in GBC with respect to CC with significant association among them. MMP9 expression was significantly associated with higher tumor stage but with chemotherapeutic response. Our results display that epithelial-mesenchymal transition type III plays a role in GBC invasion. MMP9 overexpression and loss of membranous beta-catenin may be considered a marker for poor clinical outcomes and advanced disease.

Chinese Herbal Medicine Feiyanning Prescription Regulates EMT to Reverse Cisplatin Resistance in Non-small Cell Lung Cancer / 中国实验方剂学杂志

ObjectiveTo observe the effect of Feiyanning prescription （FYN） on cisplatin （DDP） resistance in non-small cell lung cancer （NSCLC） and explore the underlying mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the proliferation of A549 and A549/DDP （DDP-resistant） cells treated by DDP （0， 2.0， 4.0， 6.0， 8.0， 10.0 mg⋅L-1） and the proliferation of A549/DDP cells treated by FYN （0， 100， 200， 300， 400， 500， 600 mg⋅L-1）. Based on immunofluorescence staining and Western blot （WB）， the expression of epithelial mesenchymal transition （EMT）-related proteins in A549 and A549/DDP groups was observed. A549/DDP cells were classified into control group， FYN group （200 mg⋅L-1）， DDP group （6.0 mg⋅L-1）， and combination group ［FYN （200 mg⋅L-1） + DDP （6.0 mg⋅L-1）］ and respectively treated with corresponding drugs. Then， invasion ability of each group was examined by transwell assay， and the expression of EMT-related proteins in each group by WB. Moreover， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and immunofluorescence staining were separately applied to detect the mRNA and protein expression of drug resistance-related factors in each group， respectively. ResultCompared with A549 group， A549/DDP group showed high resistance to DDP （P<0.01）， low expression of E-cadherin， and high protein expression of Vimentin， N-cadherin， and Snail （P<0.05， P<0.01）. As compared with the control group， FYN inhibited the proliferation of A549/DDP cells in a concentration-dependent manner （P<0.01）， and the FYN group， DDP group， and combination group demonstrated low invasion ability （P<0.01）. In addition， the invasion ability in the combination group was particularly lower than that in the DDP group （P<0.01）. The expression of E-cadherin protein was higher and the protein expression of N-cadherin， Vimentin， and Snail was lower in the in FYN group than in the control group （P<0.01）. The protein expression of E-cadherin， N-cadherin， and Vimentin was lower and the expression of Snail was higher in the DDP group than in the control group （P<0.05，P<0.01）. The protein expression of E-cadherin， N-cadherin， Vimentin， and Snail in the combination group decreased as compared with that in the control group （P<0.01）. Compared with the DDP alone， the combination raised the expression of E-cadherin and lowered the protein expression of N-cadherin， Vimentin， and Snail （P<0.01）. The protein and mRNA expression of lung resistance-related protein （LRP） and multidrug resistance 1 （MDR1） was lower and the protein and mRNA expression of topoisomerase Ⅱα （TOPO Ⅱα） was higher in the FYN group than in the control group （P<0.01）. The protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα was higher in the DDP group than in the control group （P<0.01）. The expression of LRP protein and mRNA showed no significant variation， but the protein and mRNA expression of MDR1 and TOPO Ⅱα increased in the combination group compared with those in the control group （P<0.01）. Compared with the DDP group， FYN group and combination group showed low protein and mRNA expression of LRP and MDR1 and high protein and mRNA expression of TOPO Ⅱα （P<0.01）. Compared with FYN， the combination elevated the protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα （P<0.01）. ConclusionFYN prescription can reverse the DDP resistance of NSCLC by modulating EMT.

Astragali Radix-Curcumae Rhizoma combination inhibits proliferation, migration, and invasion of colon cancer HT-29 cells by regulating EMT / 中国中药杂志

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.

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).

The Role of Deubiquitinases in Tumor Epithelial-mesenchymal Transition / 中国生物化学与分子生物学报

Tumor invasion and metastasis are the main reasons for the tumor deterioration and the cause of poor prognosis of patients. In recent years a vast amount of research has been reported that epithelial-mesenchymal transition (EMT) in cancer cells plays a critical role in tumor metastasis. Moreover, EMT is also closely related to tumor stemness, tumor drug resistance and other tumor malignant behaviors. Therefore, effective suppression of EMT has been investigated as potential therapeutic strategy for the treatment of tumor. One of the main functions of the deubiquitinating enzymes (DUBs) is to maintain the dynamic balance of intracellular protein levels via removing conjugated ubiquitin from target proteins, which in turn avoids degradation of target proteins through the ubiquitin protease pathway. DUBs are a class of proteins responsible for regulating protein ubiquitination modification, which abnormal expression or alterations of enzyme activity often results in the initiation of many diseases, including a variety of cancers. Numerous studies have found that some DUBs are unbalanced in the process of tumor invasion and metastasis, and play important roles in the process of tumor metastasis. EMT refers to the dynamic cellular process of transforming epithelial cells to mesenchymal cells, which involves changes in the expression levels of various molecular markers, such as EMT-related transcription factors (Snial1, Slug, ZEB1 etc.), and cell surface proteins (E-cadherin, N-cadherin etc.). Generally, these associated proteins are unstable and easily degraded, meanwhile the occurrence of the EMT process involves the regulation of protein stability. DUBs, as a class of enzymes that maintain protein stability, play important roles in regulating the stability of EMT-related proteins. The occurrence of EMT is also inseparable from the abnormal activation of many signaling pathways in cells such as TGF-β and Wnt pathways. DUBs mediate the activation of these pathways to indirectly regulate the occurrence and development of EMT. DUBs can directly or indirectly affect the progress of EMT by regulating EMT-related molecules or signaling pathways. Therefore, targeting DUBs to inhibit tumor invasion and metastasis will provide new methods and programs for tumor treatment. This review mainly discusses the important roles of DUBs in the EMT process to elucidate that DUBs play critical roles in regulating EMT-related molecules and signal pathways, and may serve as potential therapeutic targets for cancers.

Molecular mechanism and treatment of pulmonary fibrosis / 中国临床药理学与治疗学

Pulmonary fibrosis (PF) is a chronic progressive interstitial lung disease. The pathogenesis of PF is not yet clear. The two anti fibrosis drugs approved for IPF treatment, nidanib and pirfenidone, have been proved to reduce the decline of pulmonary function of pF, but both have side effects. So far, there is no obvious and effective treatment to prevent the progress of pF. Therefore, this review focuses on the different cells, molecular mechanisms involved in PF and the current treatment progress of PF, so as to provide theoretical support for a better understanding of these cells, molecular mechanisms and drug development and application in PF.

Effect and mechanism of terminal fucosylation inhibitor on ciclosporin-induced renal epithelial-mesenchymal transition / 器官移植

Objective To evaluate the effect and mechanism of terminal fucosylation inhibitor 2-deoxy-D-galactose (2-D-gal) on ciclosporin (CsA)-induced renal epithelial-mesenchymal transition (EMT). Methods Fifteen male C57BL/6 mice aged 8-10 weeks were randomly and evenly divided into the control group (Ctrl group), CsA group and CsA+2-D-gal group (n=5). The expression levels of fucosyltransferase 1 (FUT1), EMT-associated proteins including E-cadherin, Vimentin, α-smooth muscle actin (α-SMA) in the kidney tissues of the Ctrl and CsA groups were detected by Western blot. The expression levels of terminal fucose in the kidney tissues of Ctrl and CsA groups were determined by immunofluorescence. The renal fibrosis of mice in each group was evaluated by Masson staining. The blood urea nitrogen and serum creatinine levels of mice in each group were detected. The in vitro EMT model of renal tubular epithelial cell HK2 was induced by CsA. HK2 cells were stimulated with 0, 2.5, 5.0 and 10.0 μmol/L CsA for 24 h, respectively. In addition, HK2 cells were divided into the Ctrl, 2-D-gal, CsA and CsA+2-D-gal groups. The morphology of HK2 cells after stimulation with different concentrations of CsA and in each group was observed. The expression levels of FUT1, E-cadherin, Vimentin and α-SMA in HK2 cells after stimulation with different concentrations of CsA and in each group were detected by Western blot. The expression level of terminal fucose in HK2 cells of the Ctrl and CsA groups was measured by immunofluorescence. Results Compared with the Ctrl group, the relative expression of E-cadherin protein was down-regulated, those of FUT1, Vimentin and α-SMA proteins were up-regulated (all P < 0.05), and that of terminal fucose in the mouse kidney tissues was up-regulated in the CsA group. Compared with the Ctrl group, the blood urea nitrogen and serum creatinine levels in the CsA and CsA+2-D-gal groups were up-regulated (all P < 0.05). Compared with the CsA group, the blood urea nitrogen and serum creatinine levels in the CsA+2-D-gal group were down-regulated (both P < 0.05). Compared with the Ctrl group, the collagen fiber deposition was increased and the relative expression of α-SMA protein was up-regulated in the mouse kidney tissues of CsA and CsA+2-D-gal groups (all P < 0.05). Compared with the CsA group, the collagen fiber deposition was decreased and the relative expression of α-SMA protein in the mouse kidney tissues was down-regulated in the CsA+2-D-gal group (both P < 0.05). With the increase of CsA concentration, the morphology of HK2 cells gradually became longer and thinner from original normal cobblestone shape, the relative expression levels of FUT1, Vimentin and α-SMA protein in HK2 cells were up-regulated, and that of E-cadherin protein was down-regulated in a concentration-dependent manner. Compared with the Ctrl group, the expression level of terminal fucose of HK2 cells was up-regulated in the CsA group. After CsA treatment combined with 2-D-gal intervention, the morphology of HK2 cells in the CsA+2-D-gal group was restored to resemble that of normal HK2 cells. Compared with the CsA group, the relative expression of E-cadherin protein in HK2 cells was up-regulated, whereas those of Vimentin and α-SMA proteins were down-regulated in the CsA+2-D-gal group (all P < 0.05). Conclusions CsA may induce EMT both in vivo and in vitro, and the terminal fucosylation is increased. 2-D-gal may inhibit CsA-induced EMT by suppressing the terminal fucosylation.

Modified Xiao Xianxiongtang Inhibits Epithelial-Mesenchymal Transition of Human Gastric Cancer MGC803 Cells via Wnt/β-catenin Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the inhibitory effect of modified Xiao Xianxiongtang on epithelial-mesenchymal transition （EMT） of human gastric cancer MGC803 cells and its relationship with secretory glycoprotein Wnt/β-catenin pathway. MethodThe BALB/c nude mice were implanted with human gastric cancer MGC803 cell suspension in the heterotopic subcutaneous position for inducing tumor. After successful modeling， they were randomly divided into the model group， low-， medium-， and high-dose （16.0，32.0，and 64.0 g·kg-1） groups of modified Xiao Xianxiongtang， and capecitabine （400 mg·kg-1） group， with eight mice in each group， and gavaged with the corresponding drugs， once per day， for 28 consecutive days. Those in the capecitabine group received one-week discontinuation after every two weeks of treatment. The general state and body weight of the nude mice were observed， and the transplanted tumor volume was measured. After being killed， they were weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was carried out for observing the pathological changes in transplanted tumor tissues. The gene and protein expression levels of Wnt1 and β-catenin were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， followed by the determination of matrix metalloproteinase-9 （MMP-9）， vascular endothelial growth factor （VEGF）， N-cadherin， E-cadherin， Vimentin， and Snail protein expression by Western blot. The expression levels of cyclooxygenase 2 （COX2） and prostaglandin E2 （PGE2） were detected by enzyme-linked immunosorbent assay （ELISA）. ResultIt was found that the transplanted tumor in each group showed different growth trends with time， with the most obvious growth observed in the model group. Compared with the model group， the low-， medium-， and high-dose modified Xiao Xianxiongtang groups exhibited reduced tumor volume and slowed growth to varying degrees over time. After medication for days 7，14，21，and 28， the tumor volumes in the low- and high-dose modified Xiao Xianxiongtang groups and capecitabine group declined （P<0.05， P<0.01）， and that in the medium-dose Xiao Xianxiongtang group was also remarkably reduced after medication for days 14，21，and 28 （P<0.01）. Compared with the model group， the high-dose modified Xiao Xianxiongtang group and capecitabine group showed a significant reduction in the relative tumor volume after treatment for days 7，14，21，28 （P<0.01）， and the low- and medium-dose modified Xiao Xianxiongtang groups also presented with decreased relative tumor volume after treatment for days 14，21，28 （P<0.05， P<0.01）. Compared with the model group， the modified Xiao Xianxiongtang at low， medium， and high doses and capecitabine all increased the tumor inhibition rate to varying degrees （P<0.01）， down-regulated the mRNA and protein expression levels of Wnt1 and β-catenin in tumor tissue （P<0.05， P<0.01） and protein expression levels of MMP-9， VEGF， N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）， up-regulated E-cadherin protein expression （P<0.05， P<0.01）， and reduced COX2 and PGE2 contents （P<0.05， P<0.01）. ConclusionModified Xiao Xianxiongtang inhibits the EMT of human gastric cancer MGC803 cell-transplanted tumor， which may be related to Wnt/β-catenin pathway.

Mechanism of Artesunate in Reversing Epithelial-mesenchymal Transition and Akt /Snail Signaling Pathway in Colorectal Carcinoma / 中国实验方剂学杂志

Objective:To investigate the effects of artesunate （ART） on epithelial-mesenchymal transformation （EMT） of colorectal cancer HCT-8 cells，and explore the effects of ART on cell migration，invasion，EMT ability， and protein kinase B （Akt）/Snail signaling pathway of colorectal cancer. Method:3-（4-5-Dimethylthiazol-2-yl）2，5-diphenyltetrazolium bromide （MTT） assay was used to detect the effects of ART at different concentrations on the proliferation of HCT-8 cells. Wound healing assay and Transwell assay were used respectively to detect the effects of ART on migration and invasion of colorectal cancer cells. The effects of different concentrations of ART on the distribution of EMT-related proteins vimentin and E-cadherin in HCT-8 cells were detected by double-immunofluorescent staining. The effects of ART on protein expression levels of EMT markers E-cadherin，vimentin and N-cadherin in HCT-8 cells and the expression of Akt1， p-Akt1， and Snail1 in the Akt/Snail signaling pathway were determined by Western blot. Result:The dose-dependent inhibitory effects of ART on the proliferation of HCT-8 cells were determined and the inhibition rate was calculated. A dose-response curve was plotted accordingly. The half-maximal inhibitory concentration （IC₅₀） of ART on HCT-8 cells was （16.67±1.95） μmol·L^-1. The following four groups were set up： a control group （0 μmol·L^-1），and low-， medium-， and high-dose ART groups（2， 10， 50 μmol·L^-1）. Compared with the results in the control group，ART inhibited the migration and invasion of HCT-8 cells（<italic>P</italic><0.05）. Specifically， the expression of E-cadherin in HCT-8 cells was significantly up-regulated，and that of vimentin and N-cadherin was significantly down-regulated （<italic>P</italic><0.05）. The expression levels of p-Akt1 and Snail1 were significantly decreased after ART treatment，thus inhibiting EMT（<italic>P</italic><0.05）. Conclusion:The findings of this study suggested that ART inhibited the EMT-triggered migration and invasion of HCT-8 cells presumedly by inhibiting the activation of the Akt/Snail pathway to reverse EMT.

Mechanism of Fuzheng Qufeng Prescription in Suppressing Epithelial-mesenchymal Transition of Podocytes in Membranous Nephropathy Rats via TGF-β1/Smad Pathway / 中国实验方剂学杂志

Objective:To study the effect of Fuzheng Qufeng prescription （FZQP） on transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）/Smad signaling pathway and epithelial-mesenchymal transition of podocyte in membranous nephropathy （MN） rats and to explore its molecular mechanism for podocyte protection. Method:The rats were randomly divided into normal control group （NC） and modeling group. Rats in modeling group induced by bovine serum albumin （C-BSA） were randomly divided into model group （MN）， losartan potassium group （LP， 0.05g·kg^-1）， and FZQP high dose （FZQPH， 41 g·kg^-1）， medium dose （FZQPM， 20.5 g·kg^-1）， and low dose （FZQPL， 10.25 g·kg^-1） groups. The administration lasted for 4 weeks. In week 0， 2， and 4 of administration， the levels of 24 hours urine protein （24 h-Upro） were tested. At the end of 4th week， the levels of blood urea nitrogen （BUN） and serum creatinine （SCr） were detected， and the rats in each group were sacrificed and the renal pathological morphology changes were observed by light microscope with hematoxylin-eosin （HE）， Masson and periodic acid-silver metheramine （PASM） staining. The deposition of immune complex， the thickening of glomerular basement membrane （GBM） and podocyte foot process were observed by transmission electron microscope （TEM）. The distribution and expression intensity of Desmin in renal tissues were detected by immunohistochemistry （IHC）. The mRNA and protein expression levels of TGF-<italic>β</italic>₁， Smad2/3， phospho（p）-Smad2/3， Smad7 and Desmin in renal tissues were respectively detected by Western blot （WB） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with NC group， the levels of 24 h-Upro， BUN and SCr significantly increased in model group （<italic>P</italic><0.01）， with increased deposition of immune complex， significantly thickened GBM and fusion of foot processes， significantly increased Desmin mRNA and protein expression （<italic>P</italic><0.01） and increased TGF-<italic>β</italic>₁， Smad2， and Smad3 mRNA and protein expression （<italic>P</italic><0.05）， and decreased Smad7 mRNA and protein expression （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with model group， 24 h-Upro and BUN decreased in FZQP groups and LP group （<italic>P</italic><0.05）， levels of serum SCr in FZQPM group decreased （<italic>P</italic><0.05）， deposition of immune complex， thickening of GBM and fusion of foot process were all alleviated in FZQP groups and LP group. Distribution of Desmin along GBM decreased in FZQPH group， FZQPM group and LP group （<italic>P</italic><0.05）. Both mRNA and protein expression levels of TGF-<italic>β</italic>₁ and p-Smad2/Smad2 in FZQPM group decreased， while mRNA and protein expression levels of Smad7 increased （<italic>P</italic><0.05）. Both mRNA and protein expression levels of p-Smad3/Smad3 in FZQPH group decreased （<italic>P</italic><0.05）. Both mRNA and protein expression levels of Desmin in podocyte in FZQPH group， FZQPM group and LP group decreased （<italic>P</italic><0.05）. Conclusion:FZQP might realize podocyte protection effect in MN via suppressing EMT mediated by overactivated TGF-<italic>β</italic>₁/Smad signaling pathway.

Effect of Zishenwan on Pyroptosis and Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells in Diabetic Nephropathy Mice / 中国实验方剂学杂志

Objective:To study the efficacy and mechanism of Zishenwan （ZSW） against pyroptosis and epithelial-mesenchymal transition （EMT） of renal tubular epithelial cells in diabetic nephropathy （DN） mice， so as to provide evidence for the treatment of DN with ZSW. Method:The <italic>db/db</italic> mice with spontaneous diabetes were randomly divided into the model group， dapagliflozin （1.0 mg·kg^-1） group， and high-， medium-， and low-dose （6.0， 3.0， 1.5 g·kg^-1） ZSW groups. The non-diabetic <italic>db/m</italic> mice were classified into the normal group. The ones in the model and normal groups were given an equal volume of deionized water by gavage， while those in the other groups were intervened with the corresponding drugs for 12 weeks. The fasting blood glucose （FBG） level was tested at tail vein once every two weeks. The levels of urine albumin-creatinine ratio （ACR）， <italic>β</italic>-N-acetyl-D-glucosaminidase （NAG）， and cystatin C （CysC） were detected once every four weeks. After 12 weeks of administration， the blood sampled from eyeballs was used for measuring the blood urea nitrogen （BUN） and serum creatinine （SCr）. The pathological changes in renal tissues were observed by light microscopy and transmission electron microscopy. The expression of EMT markers in the renal tubular epithelium was analyzed by immunohistochemistry （IHC）. The in situ terminal end-labeling （TUNEL） staining was conducted to analyze the nuclear damage of renal tubular epithelial cells. The protein and mRNA expression levels of EMT markers， nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome and pyroptosis-related inflammatory cytokines in renal tissues were separately assayed by Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Result:Compared with the normal group， the model group displayed significantly increased FBG， BUN， serum SCr， ACR， NAG， and CysC （<italic>P</italic><0.01）， impaired renal tissues， altered EMT marker expression intensities and levels （<italic>P</italic><0.01）， and elevated TUNEL-positive rate and protein and mRNA expression levels of pyroptosis-related inflammatory cytokines and NLRP3 inflammasome （<italic>P</italic><0.01）. Compared with the model group， ZSW and dapagliflozin significantly decreased the levels of FBG， BUN， serum SCr， ACR， NAG， and CysC （<italic>P</italic><0.01）， relieved the pathological injuries in renal tissues， changed the EMT marker expression intensities （<italic>P</italic><0.01） and protein and mRNA expression levels （<italic>P</italic><0.05， <italic>P</italic><0.01）， and down-regulated the TUNEL-positive rate （<italic>P</italic><0.01） of renal tubular epithelial cells as well as the protein and mRNA expression levels of pyroptosis-related inflammatory cytokines （<italic>P</italic><0.01） and NLRP3 inflammasome （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:ZSW alleviates DN possibly by inhibiting pyroptosis and EMT in renal tubular epithelial cells.

Effect of Banxia Xiexintang-containing Serum on Epithelial-mesenchymal Transition of Peritoneal Mesothelial Cells Induced by Gastric Cancer-derived Exosomes / 中国实验方剂学杂志

Objective:To investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cell line （HMrSV5） induced by gastric cancer-derived exosomes （Exo）. Method:Banxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes （NCI-N87-Exo） were extracted， followed by their identification by transmission electron microscopy and Western blotting and labeling with 1，1-dioctadecyl-3，3，3，3- tetramethylindocarbocyanine perchlorate （Dil）. The cells were divided into the blank group， model group， and low-， medium-， and high-dose （13.5，27，54 g·kg^-1） Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone， the ones in the model group with 100 mg·L^-1 NCI-N87-Exo， and those in the low-， medium-， and high-dose Banxia Xiexintang groups with 100 mg·L^-1 NCI-N87-Exo plus low-， medium-， and high-dose 10% Banxia Xiexintang-containing serum， respectively. Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h， 48 h and 72 h. Seventy-two hours later， the morphological changes in HMrSV5 cells were observed. The protein expression levels of E-cadherin， cytokeratin 19 （CK19）， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， elastin， and transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）， Smad2/3， and p-Smad2/3 were assayed by Western blot. Result:It was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm. Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed， implying that the NCI-N87-Exo was confirmed. After 24 h， 48 h， 72 h of co-culture， it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells， which was positively correlated with time. Compared with the blank group， Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells， with better effect noticed in the middle- and high-dose Banxia Xiexintang groups（<italic>P</italic><0.05，<italic>P</italic><0.01）. After intervention with Banxia Xiexintang-containing serum， the HMrSV5 cells were arranged densely， and the intercellular space was significantly reduced， with the most obvious changes present in the high-dose Banxia Xiexintang group. Western blot revealed that the protein expression levels of E-cadherin and CK19 in HMrSV5 cells after being intervened with the medium- and high-dose Banxia Xiexintang-containing serum were increased significantly as compared with those in the blank group， whereas the levels of <italic>α</italic>-SMA and Elastin were decreased significantly （<italic>P</italic><0.01）. Banxia Xiexintang-containing serum at the low， medium， and high doses remarkably down-regulated TGF-<italic>β</italic>₁ and p-Smad2/3 protein expression（<italic>P</italic><0.05，<italic>P</italic><0.01）. However， there was no significant change in Smad2/3. Conclusion:NCI-N87-Exo can be taken up by HMrSV5 cells to induce EMT. Banxia Xiexintang can inhibit the uptake of NCI-N87-Exo by HMrSV5 cells and the resulting EMT induced by NCI-N87-Exo， which is related to the regulation of TGF-<italic>β</italic>₁/Smads signaling pathway.

Anti-breast Cancer Activities in Vitro of Oxymatrine Combined with Bevacizumab： An Exploration Focusing on EMT Regulation Through Wnt/β-catenin / 中国实验方剂学杂志

Objective:To observe the effect of oxymatrine （OM） combined with bevacizumab （ BV ） on the proliferation， invasion， and migration of breast cancer MCF-7 cells and explore the mechanism of OM in regulating BV-induced epithelial-mesenchymal transition （EMT） based on the Wnt/<italic>β</italic>-catenin signaling pathway. Method:The effect of different concentrations of OM（0， 0.5， 1.0， 2.0， 4.0， 8.0， 16.0 mmol·L^-1）and BV（0， 0.25×10^-4， 0.50×10^-4， 1.00×10^-4， 2.00×10^-4， 4.00×10^-4， and 8.00×10^-4 mmol·L^-1）on the proliferation of MCF-7 cells were detected by cell counting kit-8（CCK-8）assay. The effect of OM（4.0 mmol·L^-1） combined with BV（2.00×10^-4 mmol·L^-1）on the invasion and migration of MCF-7 cells were observed in transwell and scratch repair tests. Western blot was conducted to investigate the effect of OM（4.0 mmol·L^-1）combined with BV （2.00×10^-4 mmol·L^-1） on proliferation-related proteins in MCF-7 cells， followed by the detection of the expression levels of Wnt/<italic>β</italic>-catenin signaling pathway- and EMT-related proteins. Result:Compared with the blank group， OM （2.0，4.0，8.0，16.0 mmol·L^-1） inhibited the proliferation of MCF-7 cells in a concentration-dependent manner （<italic>P</italic><0.01）， while BV did not show the inhibitory effect against the proliferation of MCF-7 cells. The inhibitory effect of the combination of the two drugs on the proliferation of MCF-7 cells was not significantly different from that of OM. Compared with the blank group， OM significantly reduced the migration distance of MCF-7 cells and the number of invaded cells（<italic>P</italic><0.01）， while BV increased the migration distance of MCF-7 cells and the number of invaded cells （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with BV， its combination with OM significantly inhibited the invasion and migration of MCF-7 cells induced by BV （<italic>P</italic><0.01）. Compared with the blank group， both OM and the combined medication obviously inhibited the phosphorylation of proliferation-related protein kinase B（Akt） and extracellular-signal-regulated protein kinase 1/2 （ERK1/2）in MCF-7 cells （<italic>P</italic><0.01） and down-regulated the protein expression levels of <italic>β</italic>-catenin， proto-oncogene （c-Myc）， CD44， and G₁/S-specific cyclin D₁ in Wnt/<italic>β</italic>-catenin signaling pathway （<italic>P</italic><0.05，<italic>P</italic><0.01）. Besides， OM and the combination of two drugs both significantly reduced the protein expression levels of calcium-dependent cell adhesion protein <italic>N</italic>-cadherin and Vimentin in EMT， whereas increased the expression of calcium-dependent cell adhesion protein E-cadherin（<italic>P</italic><0.01）. However， the expression of the above-mentioned proteins in the BV group was reversed （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:After the combination with BV， OM plays an anti-breast cancer role by effectively inhibiting the activation of Wnt/<italic>β</italic>-catenin pathway induced by BV and reversing EMT.

Effect of Astragalus Polysaccharide on EMT of A549/DDP Lung Adenocarcinoma Xenograft： An Exploration Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of Astragalus polysaccharide （APS） on transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）-induced epithelial mesenchymal transition （EMT） of A549/DDP lung adenocarcinoma xenograft and its potential molecular mechanism. Method:BALB/c nude mice were randomly divided into the non-loading group （A549/DDP cells not loaded with TGF-<italic>β</italic>₁）， model group， cisplatin group， and combined group （A549/DDP cells overexpressing TGF-<italic>β</italic>₁）. Mice in the combined group were treated with intragastric administration of APS （0.3 g·kg^-1·d^-1） and intraperitoneal injection of cisplatin （0.003 5 g·kg^-1）， while those in the cisplatin group only received intraperitoneal injection of cisplatin （0.003 5 g·kg^-1）. After drug intervention， the nude mice were sacrificed and the xenograft and lung were harvested， followed by the weighing of tumor and the calculation of the inhibition rate. The number of tumors metastasizing to the lung was counted under the microscope. The pathological features of tumors and their metastasis to the lung tumor were observed by hematoxylin-eosin （HE） staining. The protein and mRNA expression levels of EMT molecular markers E-cadherin， Vimentin， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） in the xenograft were detected by immunohistochemistry， Western blot， and Real-time polymerase chain reaction （Real-time PCR）. Result:Compared with the non-loading group， the model group exhibited increased tumor weight and pulmonary metastatic nodules （<italic>P</italic><0.05）， sparse tumor cell junctions， long spindle cells， massive metastatic nodules in the lung， down-regulated E-cadherin protein and mRNA expression， and up-regulated Vimentin and <italic>α</italic>-SMA protein and mRNA expression and p-PI3K and p-Akt protein expression （<italic>P</italic><0.05）. Compared with the model group and cisplatin group， the combined group displayed decreased tumor weight and pulmonary metastatic nodules （<italic>P<</italic>0.05）， tight tumor cell junctions， round or oval cells， no obvious lung metastasis， up-regulated E-cadherin protein and mRNA expression （<italic>P</italic><0.05）， and down-regulated Vimentin and <italic>α</italic>-SMA protein and mRNA expression （<italic>P</italic><0.05） and p-PI3K and p-Akt protein expression （<italic>P</italic><0.05）. There was no significant difference in PI3K or Akt protein expression among groups. Conclusion:APS has a certain inhibitory effect against EMT in lung adenocarcinoma A549/DDP cells， which may be related to the inhibition of activated PI3K/Akt protein expression.

Mechanical Heterogeneity of Tumor Tissues and Epithelial-Mesenchymal Transition of Tumor Cells / 医用生物力学

In the process of tumor growth, with the proliferation and expansion of cancer cells, the reconstruction of extracellular matrix (ECM) of cancer tissues, the restriction of surrounding tissues and the flow of cancer tissue interstitial fluid, the special stress environment is formed in the tumor tissues. Significant differences are found in the mechanical environment and mechanical characteristics of different regions of tumor tissues, that is, mechanical heterogeneity. The reseach shows that the mechanical properties of tumor tissue invasion frontier areas are more significant and complex. In particular, the epithelial-mesenchymal transition (EMT) of tumor cells also prefers to concentrate on this area. The mechanical stress generated by the invasion front can induce EMT of tumor cells through TWIST1, TGF-β, WNT and other force signal transduction pathways, and promote tumor cell invasion. From the perspective of tumor biomechanics, this review focuses on the relationship between mechanical heterogeneity of tumor cells and EMT, so as to provide the theoretical basis for mechanoenvironment-targeted therapy of tumors.

Epithelial Mesenchymal Transformation and Stem Ceil Self-renewal and Differentiation / 中国生物化学与分子生物学报

The lineage transition between epithelium and mesenchyme is a process known as epithelial- mesenchymal transition (EMT) , by which polarized epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes.EMT is commonly found in embryonic development, wound healing, organ fibrosis and tumor metastasis.EMT and mesenchymal-epithelial transition (MET) are involved in the early and late development of embryo, such as implantation, gastrulation, heart development and so on.EMT and MET are involved in the regulation of stem cell phenotype changes and cell migration, which are important mechanisms of cell differentiation and three-dimensional tissue construction.The expression of cell adhesion molecules is transformed from E-cadherin to N-cadherin, which is an important sign of EMT.By interacting with (3-catenin, p 120-catenin and a-catenin, E-cadherin can affect the activation of Wnt, small GTPase superfamily and other signaling pathways and regulate cytoskeleton movement.TGF, Notch, Wnt, BMP, FGF, and other signaling pathways, Snail, Twist, Zeb and other transcription factors, together with epigenetic modifying enzymes, participate in the initiation and regulation of EMT in a coordinated way.In vitro models showed that E-cadherin was involved in self-renewal of stem cells.Somatic cell reprogramming could he regarded as MET, and the expression of E- cadherin can he obtained in somatic cells after transfection of reprogramming factors.In vitro studies have found that EMT and its related molecules (such as E-eadherin, Snail, Twist, Zeb, etc.) were involved in the early triploblast differentiation and the formation of specific cell types in the late stage.The study on the mechanism of EMT will help to improve the differentiation efficiency of stem cells in vitro and promote the induction of organoids.

Screening of Differential Genes for Endometriosis and Expression Validation of the Potential Target GAS6 / 中国生物化学与分子生物学报

Growth arrest specific protein 6 (GAS6) plays an important role in the occurrence and development of tumors, and its signal transduction is involved in cell proliferation, adhesion and migration, but its related functions and molecular mechanisms in endometriosis (EMs) are still unclear. In this study, we searched and downloaded the transcriptome datasets of EMs from GEO database and performed GEO online analysis, and then screened out the differentially expressed genes and performed cluster analysis based on GO and KEGG pathway. The mRNA levels of the differentially expressed genes shared by more than three datasets were verified by qRT-PCR in the endometrium of ten women with no endometriosis and no clear disease and the ectopic endometrium of 11 patients with ovarian chocolate cysts. Immunohistochemistry and qRT-PCR were used to verify the expression of GAS6 and epithelial mesenchymal transition (EMT) marker genes, and immunofluorescence was used to co-label GAS6 and E-cadherin in endometriosis clinical samples. In this study, a total of 47 differentially expressed genes were screened out of the four transcriptome datasets, which were mainly enriched in processes such as cell migration and related signal pathways such as MAPK, PI3K-AKT, and tight junction. The mRNA levels of the nine differentially expressed genes shared by more than three datasets in endometriosis patients were consistent with the results of bioinformatics analysis. GAS6 expression levels in ectopic endometrium of EMs patients are higher than the control group (P < 0. 05), and EMs patients have the characteristics of EMT in the ectopic endometrial tissue, that is, the expression of E-cadherin is down-regulated (P < 0. 05) and the expression of vimentin is up-regulated (P < 0. 01). The expression of E-cadherin in the ectopic endometrial glandular epithelial cells of EMs patients is low while the expression of GAS6 is up-regulated, suggesting that GAS6 may mediate the EMT process in endometriosis. In conclusion, this study reveals that GAS6 is highly expressed in endometriosis patients and may mediate the EMT process to participate in the occurrence and development of endometriosis, providing a potential target for clinical treatment of endometriosis.

TMED2 is Regulated by miR-5583-5p to Elicit an Oncogene Effect in Tongue Squamous Cell Carcinoma / 中国生物化学与分子生物学报

Tongue squamous cell carcinoma (TSCC) is the most common type of oral squamous cell carcinoma (OSCC) with high morbidity and mortality. Many studies have shown that microRNA (miRNA) are small non-coding RNA that regulate the post-transcriptional processing of target genes, resulting in the degradation and translation inhibition of target mRNA. However, how the transmembrane p24 trafficking protein 2 (TMED2) is regulated by miR-5583-5p on migration, invasion, proliferation and epithelial-mesenchymal transition (EMT) of TSCC Cal-27 cells is unclear. In this study, a database was used to analyze the expression of TMED2 in HNSCC (P <0. 001) in head and neck cancer (HNC). Western blot showed that the expression of TMED2 protein was up-regulated in 6 cases of TSCC tissues and cell lines such as SCC-9, SCC-25 and CAL-27. After the Cal-27 cells transfected with TMED2 interference plasmid (SiTMED2) the expression of E-cadherin was up-regulated, and N-cadherin and Vimentin was down-regulated. Migration and invasion experiments showed that the number of cells transfused into the basement membrane of the cells was lower than that of the control group (P<0. 05). The results of EdU showed that the proliferation of Cal-27 cells transfected with SiTMED2 was decreased (P<0. 05). The results of dual luciferase experiment showed that TMED2 had a binding target to miR-5583-5p, and the expression of miR-5583-5p in Cal-27 cell was lower than that in Hoec cells. The expression of miR-5583-5p was increased and TMED2 protein was decreased after the Cal-27 cells were transfected with miR-5583-5p plasmid (P < 0. 05). In conclusion, TMED2 is regulated by miR-5583-5p and promoted the migration, invasion, proliferation and EMT of tongue squamous cell carcinoma cell Cal-27.