Résumé
Colorectal cancer (CRC) is a disease with high incidence worldwide. As of 2018, it is the second leading cause of cancer deaths in the world. In Saudi Arabia, the incidence of this disease has been increasing in the younger population. Both genetic and lifestyle factors may have contributed to its increased incidence and pathogenesis. Monosodium glutamate (MSG) is a food flavor enhancer that can be found in many commercial foods, and it can sometimes be used as a substitute to table salt. MSG has been investigated for its possible genotoxicity, yielding controversial results. In the present study, the effect of MSG on cell viability and its effect on expression of APC, BECN1, and TP53 genes in SW620 and SW480 colon cancer cell lines were studied. TP53 is a tumor suppressor gene that functions in modifying DNA errors and/or inducing apoptosis of damaged cells, and both APC and BECN1 genes are involved in CRC and are of importance in cellular growth and metastasis. Cancer cell viability was analyzed using MTT assay, and the results showed a significant increase in the number of viable cells after 24h of treatment with MSG with different concentrations (0.5, 1.0, 10, 50, and 100mM). Moreover, gene expression results showed a significant increase in the expression levels of APC and BECN1 under specified conditions in both cell lines; conversely, TP53 showed a significant decrease in expression in SW620 cells. Thus, it can be concluded that MSG possibly confers a pro-proliferative effect on CRC cells.
O câncer colorretal (CCR) é uma doença com alta incidência mundial. Desde 2018, é a segunda principal causa de mortes por câncer no mundo. Na Arábia Saudita, a incidência dessa doença vem aumentando na população mais jovem. Tanto fatores genéticos quanto de estilo de vida podem ter contribuído para o aumento da sua incidência e patogênese. O glutamato monossódico (MSG) é um intensificador de sabor de alimentos que pode ser encontrado em muitos alimentos comerciais e às vezes pode ser usado como um substituto do sal de cozinha. O MSG tem sido investigado por sua possível genotoxicidade, produzindo resultados controversos. Neste estudo, foram estudados o efeito do MSG na viabilidade celular e seu efeito na expressão dos genes APC, BECN1 e TP53 em linhas de células de câncer de cólon SW620 e SW480. TP53 é um gene supressor de tumor que atua modificando erros de DNA e/ou induzindo apoptose de células danificadas, estando os genes APC e BECN1 envolvidos no CRC e sendo importantes no crescimento celular e metástase. A viabilidade das células cancerosas foi analisada por meio do ensaio MTT, e os resultados mostraram um aumento significativo no número de células viáveis após 24 h de tratamento com MSG em diferentes concentrações (0,5; 1,0; 10; 50 e 100mM). Além disso, os resultados da expressão gênica mostraram um aumento significativo nos níveis de expressão de APC e BECN1 sob condições especificadas em ambas as linhagens celulares. Por outro lado, TP53 mostrou uma diminuição significativa na expressão em células SW620. Assim, pode-se concluir que, possivelmente, o MSG confere um efeito pró-proliferativo às células CRC.
Sujets)
Humains , Gènes APC , Glutamate de sodium/toxicité , Tumeurs colorectales/génétiqueRésumé
Abstract Colorectal cancer (CRC) is a disease with high incidence worldwide. As of 2018, it is the second leading cause of cancer deaths in the world. In Saudi Arabia, the incidence of this disease has been increasing in the younger population. Both genetic and lifestyle factors may have contributed to its increased incidence and pathogenesis. Monosodium glutamate (MSG) is a food flavor enhancer that can be found in many commercial foods, and it can sometimes be used as a substitute to table salt. MSG has been investigated for its possible genotoxicity, yielding controversial results. In the present study, the effect of MSG on cell viability and its effect on expression of APC, BECN1, and TP53 genes in SW620 and SW480 colon cancer cell lines were studied. TP53 is a tumor suppressor gene that functions in modifying DNA errors and/or inducing apoptosis of damaged cells, and both APC and BECN1 genes are involved in CRC and are of importance in cellular growth and metastasis. Cancer cell viability was analyzed using MTT assay, and the results showed a significant increase in the number of viable cells after 24 h of treatment with MSG with different concentrations (0.5, 1.0, 10, 50, and 100mM). Moreover, gene expression results showed a significant increase in the expression levels of APC and BECN1 under specified conditions in both cell lines; conversely, TP53 showed a significant decrease in expression in SW620 cells. Thus, it can be concluded that MSG possibly confers a pro-proliferative effect on CRC cells.
Resumo O câncer colorretal (CCR) é uma doença com alta incidência mundial. Desde 2018, é a segunda principal causa de mortes por câncer no mundo. Na Arábia Saudita, a incidência dessa doença vem aumentando na população mais jovem. Tanto fatores genéticos quanto de estilo de vida podem ter contribuído para o aumento da sua incidência e patogênese. O glutamato monossódico (MSG) é um intensificador de sabor de alimentos que pode ser encontrado em muitos alimentos comerciais e às vezes pode ser usado como um substituto do sal de cozinha. O MSG tem sido investigado por sua possível genotoxicidade, produzindo resultados controversos. Neste estudo, foram estudados o efeito do MSG na viabilidade celular e seu efeito na expressão dos genes APC, BECN1 e TP53 em linhas de células de câncer de cólon SW620 e SW480. TP53 é um gene supressor de tumor que atua modificando erros de DNA e/ou induzindo apoptose de células danificadas, estando os genes APC e BECN1 envolvidos no CRC e sendo importantes no crescimento celular e metástase. A viabilidade das células cancerosas foi analisada por meio do ensaio MTT, e os resultados mostraram um aumento significativo no número de células viáveis após 24 h de tratamento com MSG em diferentes concentrações (0,5; 1,0; 10; 50 e 100mM). Além disso, os resultados da expressão gênica mostraram um aumento significativo nos níveis de expressão de APC e BECN1 sob condições especificadas em ambas as linhagens celulares. Por outro lado, TP53 mostrou uma diminuição significativa na expressão em células SW620. Assim, pode-se concluir que, possivelmente, o MSG confere um efeito pró-proliferativo às células CRC.
Résumé
Objective To investigate the role of autophagy in the development of diabetic cataract by detecting the expression of autophagy-related factors (BECN1,LC3B,P62) in diabetic mouse lens epithelial cells.Methods Eighty C57BL/6 male mice were selected.Fifty C57 mice were consecutively dosed with streptozotocin (STZ) 50 mg/ (kg · d) by intraperitoneal injection to induce type 1 diabetes mellitus model,and served as the model group;the remaining 30 mice were injected with appropriate dose of citrate buffer,and served as the control group.The fasting plasma glucose was tested by collecting the caudal vein blood in the model group.The morphological changes of autophagy of lens epithelial cells were observed by transmission electron microscopy.The expression and localization of LC3B and P62 protein were detected by immunohistochemistry.PCR was used to detect the expression of BECN1,LC3B and P62 mRNA in the anterior capsule.The relative expression of autophagy-related proteins in the anterior capsule was detected by Western blot.The use of animals complied with Regulations on the Management of Experimental Ainimals from Shandong Eye Institute.Results Compared with the control group,transmission electron microscopy revealed that the autophagosomes of lens epithelial cells in model group was large and contained more mitochondria.Immunohistochemical method showed that the expression of LC3B and P62 proteins in the anterior capsule tissue of experiment group was stronger than that of the control group.The relative expression level of BECN1,LC3B and P62 mRNA in the experiment group was 1.48±0.10,2.62±0.15 and 1.89±0.20,respectively,which was higher than 1.10±0.02,1.10±0.05 and 1.01±0.01 in the control group,with significant differences between the two groups (t =6.64,14.25,6.14;all at P ＜ 0.05).The relative expression of BECN1,LC3B and P62 protein in the experiment group was 1.50±0.10,1.24±0.09 and 3.19± 1.04,respectively,which was higher than 1.00±0.00,1.00±0.00 and 1.00±0.00 in the control group,with significant differences between the two groups (t =8.75,6.10,3.65;all at P＜0.05).Conclusions The phenomenon of autophagy in lens epithelial cells of diabetic mice is abnormal,and autophagy dysfunction may play an important role in the formation of diabetic cataract.
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Objective To investigate the regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes and their expressions in rats with experimental autoimmune uveitis (EAU).Methods Application of dual luciferase report system was conducted to detect the regulatory roles of rno-miR-30b-5p in Atg5,Atg12 and Becn1 gene expression.Lewis rats were randomly divided into control group and EAU group,with 6 rats in each group.Next,rats in EAU group were immunized to establish EAU model.After treatments,Genesis-D fundus camera was used to observe the fundus inflammation every day.After immunization for 12 days,the pathological features of rat ciliary body and retina were detected,and meanwhile,the spleen and lymph nodes in both groups were isolated to detect the expressions of rno-miR-30b-5p,Atg5,Atg12 and Becn1 genes by quantitative PCR (Q-PCR);the levels of autophagy related proteins were determined by ELISA.Results Dual luciferase report gene expression assay confirmed that Atg5,Atg12 and Becn1 were target genes of rno-miR-30b-5p.Twelve days after immunization,compared with the control group,rats in the EAU group had severe iris adhesions and severe blood vessel swelling,and pathological examination revealed massive infiltration of inflammatory cells in the ciliary body and retina.Furthermore,rno-miR-30b-5p mRNA was 0.46 ±0.01,0.29 ±0.17in the spleen and lymph nodes in EAU group,respectively,which was down-regulated when compared with the control group (P ＜0.01);whereas the expressions of Atg5,Atg12 and Becn1 were significantly upregulated,and the differences were statistically significant (all P ＜ 0.05).ELISA results showed that Atg5,Atg 12 and Becn 1 protein expression levels in the spleen and lymph nodes in EAU rats were significantly higher than those in the control group (all P ＜ 0.05).Conclusion rno-miR-30b-5p can regulate the expressions of Atg5,Atg12 and Becn1 autophagy-related genes.The down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes in EAU rats can significantly up-regulate the expressions of Atg5,Atg12 and Becn1 genes,thereby regulating the pathogenesis of uveitis.
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@# Objective: To investigate the effect of miR-124 on the invasion and migration of esophageal cancer KYSE170 cells by regulating autophagy. Methods: miR-124 mimic was transfected into esophageal cancer KYSE170 cells. Transwell assay was used to detect the change of invasion and migration ability of cells. Dual luciferase reporter gene assay was used to verify the targeted regulation of BECN1 (Beclin1) by miR-124, and Western blotting was used to analyze the expressions of BECN1, P62 and LC3 protein. siRNA targeting BECN1 was transfeted into KYSE170 cells, and then the cell invasion and migration ability was calculated by Transwell assay. The expressions of BECN1, P62 and LC3 protein were detected by Western blotting. miR-124 mimic and BECN1 over-expression plasmid were co-transfected into KYSE170 cells, and then Transwell assay was used to detect the changes of cell invasion and migration ability, and Western blotting to examine the expression levels of autophagy-related gene. Results: The invasion and migration ability of KYSE170 cells were significantly inhibited after transfection with miR-124 mimic (All P<0.05). The expression of autophagyrelated protein P62 was increased, and the expression of BECN1 and LC3 was significantly decreased (All P<0.01); in addition, the activity of luciferase reporter gene was also significantly reduced (P<0.01). Silencing BECN1 expression inhibited the invasion and migration of esophageal cancer KYSE170 cells (P<0.01). However, after co-transfection with BECN1 over-expression plasmids, the effects of miR-124 mimic on the autophagy, invasion and migration of esophageal carcinoma KYSE170 cells were significantly weakened (P<0.01), it was also accompanied with lower P62 expression, and higher LC3 expression (P<0.01). Conclusion: miR-124 mimic can inhibit the invasion and migration of esophageal carcinoma cells. The mechanism may be related to the autophagy-related gene BECN1 expression.
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ABSTRACT:Objective To observe the influence of saikosaponin-d (SSd)on the proliferation and the function of autophagy of human hepatocellular carcinoma (HCC)cell line SMMC-7721 to explore the possible mechanisms. Methods SMMC-7721 was cultured invitro and then treated with SSd of various concentrations (5.0,7.5,10.0, 12.5,15.0 and 17.5 mg/L)for 24,48 and 72 h.We used MTT to detect cell proliferation,selected the optimal concentration and time,and detected the expressions of BECN1 at mRNA and protein levels by PCR and Western blot.Results The inhibition rate of the proliferation of SMMC-7721 cell line increased with the increase of the concentration of SSd,and the highest inhibition rate (60%)appeared when the concentration reached 12.5 mg/L. The expression of BECN1 in the group with SSd was obviously higher than that in the control group (P<0.05). 3-MA decreased not only the expressions of BECN1 at mRNA and protein levels but also the expression of BECN1 when used in conjunction with SSd.Conclusion The inhibiting function of SSd on SMMC-7721 presents a dependency between drug concentration and function time,basically in line with the drug dose-effect relationship. SSd induces the occurrence of autophagic cell death through up-regulating the expression of BECN1 ,thus inhibiting the proliferation of SMMC-7 7 2 1 .
Résumé
INTRODUÇÃO: As doenças retinianas associadas à neovascularização, tais como a degeneração macular relacionada à idade e as retinopatias diabética e da prematuridade são as principais e mais importantes causas da cegueira em todo o mundo. Nos últimos anos, injeções intravítreas de fármacos com ação antiangiogênica, como o bevacizumabe (BVZ), têm sido de grande valia tanto em pacientes na fase adulta quanto nos recém-natos. Todavia, estudos experimentais in vitro e in vivo sugerem que essas drogas promovam efeitos adversos sobre alguns processos celulares, interferindo diretamente em mecanismos fisiológicos que mantém a homeostase do tecido retiniano, incluindo os mecanismos de proliferação, diferenciação e morte celular. OBJETIVO: investigar o efeito do BVZ nos processos de transcrição e tradução de marcadores da gliose: GFAP e vimentina, de morte celular, caspase-3 e beclina-1, e dos proteoglicanos relacionados à manutenção e desenvolvimento de tecido retiniano: neurocam, fosfacam e sindecam-3. MÉTODOS: Dois modelos experimentais foram usados nesse estudo: 1) linhagem celular de Müller de Glia humana adulta (MIO-M1), cultivada em meio de cultura D-MEM na presença e ausência de BVZ por 12 e 24 horas nas concentrações de 0,25 mg/mL e 0,50 mg/mL e 2) explantes de retinas de ratos 2 dias pós-nascidos submetidos à 0,50 mg/mL da droga por 48 horas. Durante este período foram mantidos a 5% de dióxido de carbono à temperatura de 37°C. A análise de proteínas foi realizada por imunocitohistoquímica e Western Blotting e a expressão de RNAm, pela reação em cadeia da polimerase em tempo real (PCR Real Time). Foi utilizado o Teste de ANOVA - fator único para a comparação entre os grupos controle e tratados com BVZ de um mesmo período (12h ou 24h) e o teste t de Student para a comparação entre as mesmas concentrações de 12h e 24h, e para a comparação entre os grupos controle e tratado com BVZ dos explantes (p < 0,05). RESULTADOS...
Backgraound: Vasoproliferative retinal disorders such as age-related macular, degeneration, diabetic retinopathy and retinopathy of prematurity are major causes of blindness in the world. In recent years, intravitreal injections of drugs with antiangiogenic action, as bevacizumab, have been very useful for both patients in adulthood and in newborns. However, experimental studies, in vivo and in vitro, suggest that antiangiogenic drugs may promote side effects in cellular proceedings, interfering directly in physiological mechanisms of cellular proliferation, differentiation and death. POURPOSE: Investigate the bevacizumab effects in transcription and translation processes of gliosis, GFAP and vimentin, cellular death markers, caspase-3 and beclin-1, and proteoglycans involved in retinal tissue maintenance and development, neurocan, phosphacan and syndecan-3. METHODS: Two experimental models were used on this research: cellular lineage of adult and human Müller glial cell(MIO-M1) were cultivated on D-MEM medium with 0,25 and 0,50 mg/mL bevacizumab for 12 and 24 hours, and two days old rat retinal explants submitted to 0,50 mg/mL for 48 hours. During this period were stored in laboratory ovens at 5% carbon dioxide pressure and 37 °C average temperature. Molecular techniques were used to evaluate gene expression and protein content. Protein assessments were performed by immunocytochemistry and western blotting analysis, while Real Time PCR was used to measure mRNA content. ANOVA tests one factor were applied to compare the control and BVZ groups of the same period (12h or 24h) and t test from Student to compare the same conditions of 12h and 24h, and to compare the control and BVZ retinal explants groups (p<0.05). RESULTS...