| Objective:Arsenic is a chemical element that is ubiquitous in the environment.It is a steroidal human carcinogen recognized by the International Agency for Research on Cancer（IARC）and can cause skin cancer,lung cancer,bladder cancer and liver cancer.People are exposed to high arsenic environments primarily through occupational and drinking arsenic-containing groundwater.Because arsenic is excreted in the urine after methylation and metabolism in the body,and the characteristics of the kidney’s bioconcentration of urine,the bladder is exposed to a variety of arsenic compounds with higher concentrations,which can be seen relative to other organs.The risk of bladder cancer may be greater.However,our current mechanism of arsenic-induced bladder cancer remains unclear.Epithelial-mesenchymal transition（EMT）refers to the biological process of epithelial cells transformed into a mesenchymal cell phenotype through a series of cellular processes,and the ability to obtain migration,early bladder cancer production,cells The basis of invasion and transfer.Tumor-associated EMT,its main manifestations are:epithelial cells lose cell polarity,cell adhesion is weakened,such as keratin（Keratin）,E-cadherin（E-cadherin）and other cell adhesion molecules decreased expression,interstitial Cellular phenotype protein expression is enhanced,such as vimentin,N-cadherin and the like.EMT provides us with new ideas for further research on the mechanism of arsenic-induced bladder cancer.Transforming growth factor-β1（TGF-β1）is a type of cytokine that affects cell proliferation,differentiation,apoptosis,adhesion and migration.The TGF-β1/Smad signaling pathway is an important pathway that affects EMT.When TGF-βbinds to TGF-β1 type I receptor（TβRI）and TGF-β1 type II receptor（TβRII）on the cell membrane,it activates Smad2 and Smad3,and binds to Smad4 to form a trimer into the nucleus,and transcription factors.The combination of SNAIL1,SNAIL2 and ZEB induced the decrease of E-cadherin expression,increased expression of N-cadherin and Vimentin,and promoted EMT.MicroRNAs（miRNAs）are capable of targeting a variety of EMT-related proteins.At least 500 or more miRNA expressions can be detected in any human epithelial cell.When EMT occurs,the abundance of most miRNAs changes.The miR-200 family is a typical miRNA that impedes the development of EMT,including five members（miR-200a,miR-200b,miR-200c,miR-141,and miR-429）.Among them,miR-200c is considered to be the most important regulator in EMT,and it is a key factor in inhibiting invasion and metastasis of EMT and many malignant tumors.Therefore,this study was designed to determine long-term low-dose arsenic exposure-induced EMT-related factors in human normal bladder epithelial cells（SV-HUC-1）（E-cadherin,vimentin）and TGF-β1/Smad signaling pathway-related factors,miR-200c in the miR-200 family,which typically inhibits EMT,was used as a research object to explore the role of miR-in the process of EMT induced by chronic arsenic exposure in human bladder epithelial cells.The mechanism of regulation of200c and TGF-β1/Smad signaling pathways.Methods:1.Cell culture;All specific processes of cell culture are strictly aseptically operated in a biological safety cabinet.SV-HUC-1（ATCC,number:CRL-9520,American Type Culture Collection）is a human normal urothelial cell line containing serum,F12K complete medium culture of penicillin and streptomycin was incubated in a 5%CO2 incubator.The exposed group was chronically exposed to a complete concentration of NaAsO2（0.5μmol/L）.When the bottom of the dish was covered with 80%of the cells,it was digested with an appropriate amount of 0.25%EDTA solution.According to the actual amount of cells,it was stored in a ratio of 1:3,and cultured for 40 weeks（about 80 passages）.2.Cell morphology observation and MTT experimentThe cell morphology of the control group and the exposed group was photographed every 10th generation,and the cells were counted by trypsinization,and the cell count was adjusted to 5-10×104/ml.After the cell suspension was prepared,gently mix,add 100 ul of cell suspension per well to a 96-well culture plate,and fill the edge wells with sterile PBS.The MTT reagent was prepared,and 20μmol/L of MTT solution was added to the culture solution,and the mixture was incubated for 4hours.The absorbance（OD）value of each well was measured at 490 nm by a microplate reader.Cell viability is expressed by the reduction rate of MTT.3.Transwell cell migration experimentTake the logarithmic growth of each generation of cells,adjust the cell suspension density to 4×105/ml,add 600μl of complete medium to the lower chamber in a 24-well plate,place in the transwell upper chamber,and add 200μl of cell suspension.After 24 h,the upper chamber was removed and the PBS was rinsed 3times along the upper chamber wall.Fix with 30%paraformaldehyde for 30 min.The PBS was also washed along the upper chamber wall,and the cotton swab was rotated to gently wipe the unmigrated cells.0.1%crystal violet stained for 15 min and washed twice with PBS.The upper chamber was infiltrated in PBS and photographed with a microscope.The crystal violet was completely eluted by decolorization with 33%acetic acid.200μl of the eluate was added to a 96-well plate,shaken at room temperature,and the OD value was measured at 570 nm using a microplate reader to indirectly reflect the number of cells.4.Cell scratch testUse a straight ruler to draw a straight line every 0.5-1cm on the back of the6-well plate,with at least 5 lines per hole.The cells were seeded into a 6-well plate and inoculated with 5x105 cells per well using an automated cell counter,and the cells were well covered after overnight.The next day,the tip of the gun is scratched perpendicular to the horizontal line on the back.The cells were gently washed 3 times with PBS and serum-free medium was added.Put it into the incubator and take photos at 0,6,12,24h.When taking a photo,you need to select the same field of view as the first photo to take a photo.Each hole is shot no less than four,and the scratch fusion rate=(24h cell migration distance/The total distance of the scratches was×100%to calculate the cell migration rate.5.Soft agar colony formation experimentThe cells in the logarithmic growth phase were adjusted to a cell density of 1 x106 cells/L.Two low-melting agarose solutions of 1.2%and 0.7%were prepared in PBS and autoclaved.1.2%agarose solution and 2xF12K complete medium were mixed 1:1 as the bottom agar.0.7%agarose and 2xF12K medium were mixed 1:1 in a test tube,and then the cell suspension was added,thoroughly mixed,and injected into the bottom plate to form a double agar layer.After the upper layer of agar was solidified,it was placed in an incubator for 21 days.Always observe and take pictures every 7 days.The number of cell clusters greater than 50 cells was counted under a low power microscope.6.RNA extraction and Real-time PCR detectionThe corresponding cDNA sequence was downloaded from GenBank,and the corresponding mRNA primers were designed and synthesized according to the literature and the special design software Primer 5.The cells in the long-term exposure were extracted by conventional methods and stored in a refrigerator at-80°C.RNA concentration and purity were measured by 260 nm/280 nm spectrophotometry using DEPC water as a blank control.Reverse transcription was carried out according to the instructions using the PrimerScript RT reagent Kit（Perfect Real Time）kit.Then,real-time fluorescence quantitative detection was performed,and amplification was performed by using Q6 Real-Time PCR System.After the reaction was completed,the amplification curve and the fusion curve were confirmed,and the Ct value was calculated.Usingβ-actin as an internal reference,the difference in transcription levels was calculated by comparing the Ct method（ΔΔCt method）.7.Western blotting to detect protein expressionCollect the infected cells,add the cell lysate to lyse the cells,extract the total protein according to the kit instructions,and measure the protein concentration by BCA,and store at-80°C.SDS polyacrylamide gel electrophoresis,transfer to PVDF membrane;after blocking,add primary antibody to incubate to bind to target protein;elute,add fluorescent secondary antibody to incubate,wash away unbound fluorescent secondary antibody;exposure,scanning Quantitative analysis.Result:1.Effect of long-term arsenic treatment on EMT in SV-HUC-1 cells:With the prolongation of NaAsO2 treatment time,the cells changed from a tightly connected flat polymorphic shape to a loose long spindle type;after treatment with 0.5μmol/L NaAsO2 for 20 weeks,the cell viability was significantly enhanced,and the difference was statistically different from that at 10 weeks of treatment.Significance（P<0.01）;Scratch healing experiments showed that after 20 weeks of arsenic treatment,the area of wound healing was also significantly higher than that at 10 weeks（P<0.01）;after30 weeks of arsenic treatment,the migration invasive ability of cells was significantly higher than 10 Weeks（P<0.01）;soft agar colony formation experiments showed that a large number of obvious anchorage colonies appeared on soft agar after 30 weeks of arsenic treatment,and the number of colony formation in arsenic treatment for 20weeks,30 weeks,and 40 weeks was 10 weeks.The groups were 1.85 times,9.79times（P<0.01）and 15.36 times（P<0.01）,and no colonies were generated from the arrays of the arsenic-treated group.With the prolongation of arsenic treatment,compared with the control group,the expression of E-cadherin decreased gradually,and the expression of Vimentin increased gradually,showing a time-effect relationship.2.Effect of long-term arsenic treatment on activation of TGF-β1/Smad signaling pathway in SV-HUC-1 cellsAfter treatment with 0.5μmol/L NaAsO2 for 40 weeks,the expression levels of TGF-β1,p-Smad 2 and p-Smad 3 protein were increased compared with non-arsenic-treated cells（P<0.05）.And the expression levels of Smad 2 and Smad 3did not change.The expression level of EMT-related factors was detected by Western-blot.The expression of E-cad in TGF-β1 inhibitor group was significantly different from that in DMSO group and R40 week group（P<0.05）,and the expression of Vimentin was opposite.3.Effect of long-term arsenic treatment on miR-200c in SV-HUC-1 cellsThe expression of miR-200c was detected by Realtime-PCR after SV-HUC-1 cells were treated with NaAsO2 at 0.5μmol/L for 40 weeks.Compared with the non-arsenic-treated group,the expression of miR-200c was decreased in the R40week group.The expression level of miR-200c in SV-HUC-1 cells transfected with miR-200c mimic for 40 weeks was significantly higher than that in untransfected group and transfected miR-NC mimic group.The expression of EMT-related factor protein was detected by Western-blot.The expression of E-cad in miR-200c mimic group was significantly different from that in transfected NC mimic group and R40week group,and the expression level was significantly increased（p<0.05）.Vimentin expression trend in contrast.4.The role of TGF-β1 in regulating miR-200c in long-term arsenic-treated SV-HUC-1cellsThe expression of miR-200c in the three groups was detected by qPCR.Compared with the R40 week group,the DMSO group had little difference,and the expression of miR-200c in the TGF-β1 inhibition group was significantly increased.Conclusion:1.Long-acting SV-HUC-1 cells with 0.5μmol/L NaAsO2 can gradually metastasize and induce EMT in bladder epithelial cells.2.Long-term treatment of NaAsO2 can activate TGF-/Smad2/3 signaling pathway in SV-HUC-1cells.TGF-β1 is involved in arsenic-induced EMT transformation in SV-HUC-1 cells.3.Long-term arsenic treatment can reduce the level of miR-200c in SV-HUC-1 cells.4.TGF-β1 mediates arsenic-induced EMT transformation of SV-HUC-1 cells by miR-200c. |
