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).

Effect of interlukin-22 antibody on diabetic nephropathy via inhibition on Snail1 expression / 中国病理生理杂志

AIM:To investigate the effect of interlukin-22(IL-22)on diabetic nephropathy(DN)and its possible mechanism.METHODS: C57BL/6 mice were randomized to normal control(NC)group,DN group, DN+recombinant IL-22(rIL-22)group and DN+IL-22 antibody(anti-IL-22)group.After successful establishment of diabetes model for 8 weeks,the mice in DN+rIL-22 group and DN+anti-IL-22 group were intraperitoneally injected with rIL-22(200 μg/kg)and anti-IL-22(200 μg/kg),respectively,and the mice in NC group and DN group were intraperito-neally injected with 0.1%bovine serum albumin,twice a week for 4 weeks.After the intervention,blood glucose,kidney function,24 h urine microalbumin(m-Alb)and 24 h urine creatinine(Ucr)were measured.The pathological changes of renal tissues were observed under light microscope.The mRNA expression of Snail1 was detected by qPCR.The protein levels of fibronetin(FN)and E-cadherin were determined by Western blot.RESULTS:After the intervention,the ratio of 24 h m-Alb/Ucr increased significantly in other model groups compared with NC group(P<0.05).The levels of 24 h m-Alb and 24 h Ucr increased significantly in DN +rIL-22 group compared with DN group(P<0.05).However,in DN+anti-IL-22 group,the levels of 24 h m-Alb,24 h Ucr and 24 h m-Alb/Ucr ratio were significantly lower than those in DN group and DN+rIL-22 group(P<0.05).The tubular epithelial cell vacuolar degeneration,protein cast formation and glo-merular mesangial expansion in the renal tissues from diabetic mice were observed under light microscope.The lesions were more severe in DN+rIL-22 group,but attenuated in DN+anti-IL-22 group.The mRNA expression of Snail1 increased sig-nificantly in diabetic mice(P<0.05),but decreased significantly after a 4-week intervention by anti-IL-22(P<0.05). The expression of FN,an extracellular matrix protein,increased significantly in DN +rIL-22 group(P<0.05).The ex-pression of E-cadherin,an epithelial-mesenchymal transition marker,decreased significantly in DN +rIL-22 group as well (P<0.05).CONCLUSION: IL-22 neutralizing antibody may attenuate microalbuminuria and delay the progression of DN via inhibition of Snail1 expression in the renal tubular epithelial cells.

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells / 中国病理生理杂志

AIM:To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose,and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS:The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h,and divided into control group,high glucose group,non-targeting (NT) siRNA group,Snail1 RNAi group and IGF-1 RNAi group.The cells were harvested at 48 h and 72 h.Real-time PCR was used to detect the mRNA expression of Snail1,IGF-1,E-cadherin and fibronectin (FN),and the protein levels were determined by immunofluorescence staining.RESULTS:Compared with control group,the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P ＜ 0.01),while that of FN was elevated (P ＜0.01).Meanwhile,the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P ＜0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group (P ＜ 0.01),while that of FN,IGF-l and Snail1 was significantly down-regulated (P ＜ 0.01).The same changes were observed in IGF-1 RNAi group (P ＜0.01).The protein expression of each factor in NT group had no significant change as compared with high glucose group (P ＞ 0.05).Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r =0.852,P ＜ 0.01).CONCLUSION:Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells / 中国病理生理杂志

AIM:To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose,and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS:The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h,and divided into control group,high glucose group,non-targeting (NT) siRNA group,Snail1 RNAi group and IGF-1 RNAi group.The cells were harvested at 48 h and 72 h.Real-time PCR was used to detect the mRNA expression of Snail1,IGF-1,E-cadherin and fibronectin (FN),and the protein levels were determined by immunofluorescence staining.RESULTS:Compared with control group,the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P ＜ 0.01),while that of FN was elevated (P ＜0.01).Meanwhile,the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P ＜0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group (P ＜ 0.01),while that of FN,IGF-l and Snail1 was significantly down-regulated (P ＜ 0.01).The same changes were observed in IGF-1 RNAi group (P ＜0.01).The protein expression of each factor in NT group had no significant change as compared with high glucose group (P ＞ 0.05).Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r =0.852,P ＜ 0.01).CONCLUSION:Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.

Renoprotective Effect of Tang-Shen-Ning and Its Influence on Notch/Snail1 Pathway in Diabetic Nephropathy KKAy Mice / 世界科学技术-中医药现代化

This paper was aimed to study the renal protective effect of Tang-Shen-Ning (TSN) on diabetic nephropathy (DN) KKAy mice by inhibiting the Notch/snail1 signal transduction pathway.A total of 30 KKAy mice,which were fed with mice-dedicated food for 10 weeks and with the blood glucose over 16.7 mmol· L-1,24-hour urinary albumin larger than 0.4 mg,were made into the DN model.The DN mice were randomly divided into the model group,irbesartan group and TSN group according to their blood glucose and weight.Intragastric administration of medication was given.A total of 10 female C57BL/6J mice were selected as the control group.The general condition,body weight and 24-hour urinary protein quantitation were detected.After 16-week intervention,mice were sacrificed.Levels of blood glucose,blood urea nitrogen (BUN) and serum creatinine (Scr) were detected.HE and Mallory staining were applied to renal tissues.In situ hybridization (ISH) and western blotting were used to detect the Notch/snail 1 pathway,α-SMA,E-Cadherin protein and mRNA expression in renal tissues.Statistical analysis was made by SPSS20.0 software.The results showed that compared with the model group,the rats' general conditions were improved;body weight and 24-hour urinary protein quantitation were significantly decreased (P＜0.01);contents of BUN and Scr were reduced (P＜0.01,P＜0.05).The pathological staining showed significantly reduction on renal interstitial fibrosis.The Notch/snail1 pathway,protein and mRNA expression of α-SMA were significant reduced with statistical significance (P＜0.01);protein and mRNA expression of E-Cad protein were significant increased with statistical significance (P＜0.01).It was concluded that TSN can protect the renal function of DN,delay the disease progression of DN,and inhibit epithelial-mesenchymal transdifferentiation (EMT) of renal tubular epithelial cells and renal interstitial fibrosis.Furthermore,the inhibition on EMT may be through the regulation of the Notch/snail1 pathway.

Effects of Snail1 siRNA on tubular epithelial-to-mesenchymal transition induced by high glucose / 中国病理生理杂志

AIM: To explore the effect of Snail1 siRNA on high-glucose induced tubular epithelial-to-mesenchymal transition (TEMT). METHODS: Subconfluent renal tubular epithelial cells were incubated in serum-free DMEM for 24 h to arrest and synchronize the cell growth. Then cells were treated with normal glucose (5.5 mmol/L D-glucose) or high glucose (25 mmol/L D-glucose) for 72 h. Meanwhile 19.5 mmol/L D-manntiol was used as high osmotic control. Snail1 siRNA was transfected into tubular epithelial cells. In parallel, cells were transfected with non-specific siRNA which served as the control data sets. Cells were then treated with 25 mmol/L D-glucose for 72 h. RNA and cell lysates were collected to determine the protein and mRNA levels of Snail1, TGF-β_1, α-SMA, vimentin and E-cadherin. RESULTS: Transfection caused the decreases in Snail1 at mRNA and protein levels by 62% and 68% respectively as compared to those in untransfected cells cultured in high glucose medium. Western blotting exhibited that Snail1 siRNA transfection restored E-cadherin protein expression by 61% compared to that in high-glucose-treatment cells, whereas it inhibited high-glucose-induced induction of α-SMA protein by 58%. Similarly, RT-PCR revealed that Snail1 siRNA transfection dramatically suppressed the high-glucose-induced mRNA expressions of α-SMA and vimentin by 72% and 61%, respectively, while E-cadherin mRNA increased by 53%. CONCLUSION: Our study provides direct evidence that Snail1 is able to control TEMT.