| Objective:To explore the regulation mechanism of microRNA-708(miR-708)on epithelial-mesenchymal transition,proliferation and apoptosis in melanoma cells by targeting LEF1 through the Wnt signaling pathway.Methods:(1)Selection of cell strains;four melanoma cell strains,including B16,A375,W239 and WM451 were selected to extract total RNA after cultivation.The QRT-PCR kit was further used to conduct reverse transcription aiming to prepare cDNA and design PCR primers of miR-708 and U6.Later,the QRT-PCR method was adopted to detect expression of miR-708 in cells of various groups and select melanoma cell strains with relatively high expressions for the follow-up cell experiments by taking U6 as a reference.(2)Melanoma models and expressions of various indexes;40 clean male Konmin mice were chosen for the experiment after determining the optimal melanoma cell strain,which were further divided into the normal control group and model group.The hair at the left hind limb of mice in the model group was cut off by an eye scissors to be inoculated with 0.2ml single-cell suspension of melanoma cells after sterilization.The mice were killed to peel off the tumor after its diameter achieving about 0.6cm;the mice in the normal control group were not inoculated with B16 cells,while other operation methods and procedures remained the same.① The extracted issues were dyed via HE straining method for further pathology observation.Meanwhile,the immunohistochemical method was used to detect the positive expression rate of LEF1 proteins.Then the number of positive cells was counted under a high-power microscope to calculate the average positive expression rate.② The above mentioned QRT-PCR method was applied to design PCR primers of miR-708,U6,(3-catenin,Wnt3a,bcl-2,N-cadherin,Bax,Caspase3,E-cadherin and GAPDH.The expression level of relevant mRNA in two groups was detected for comparative analysis by taking U6 as the internal reference for the relative expression level of miR-708 and GAPDH as that of LEF1,p-catenin,Wnt3a,bcl-2,N-cadherin,Bax,Caspase3 and E-cadherin.③ The Western blot method was used to detect the relevant protein expression of LEF1,P-catenin,Bcl-2,Bax,Caspase3,Wnt3a,E-cadherin and N-cadherin in the normal control group and model group.The electrophoresis and transfer membrane were carried out after extracting proteins.Later,proteins were combined with their respective specific antibodies.Finally,the second antibodies marked with HRP were adopted to determine the final content.③ Verification of the targeting relationship and cell test;the bioinformatics website and dual-luciferase report method were used to determine the targeting relationship between miR-708 and LEF1 based on the cell and molecular level.The cells were divided into several groups:the control group consisted of normal cells;the blank group was not transfected with any sequences;the NC group,as the negative control,was transfected with miR-708 sequence;the miR-708 mimics group was transfected with miR-708 mimics that can enhance miR-708 expression;the miR-708 inhibitors group was transfected with miR-708 inhibitors that can inhibit miR-708 expression;the siRNA-LEF1 group was transfected with siRNA-LEF1 that can interfere with LEF expression.The miR-708 inhibitors + siRNA-LEF1 group was transfected with miR-708 inhibitors and siRNA-LEF1 that can not only inhibit miR-708 expression,but interfere with LEF expression.① Determination of the targeting relationship;the target-gene analysis was conducted on miR-708 through microRNA.org to verify whether LEF1 was the direct target gene of miR-708.② Similarly,the QRT-PCR method was used to determine mRNA expression levels of miR-708,LEF1,β-catenin,Wnt3a,bcl-2,N-cadherin,bax,E-cadherin and Caspase3 etc in seven groups after cell transfection.The analysis and comparison were further carried out.③ The Western blot method was applied to determine protein expression levels of miR-708,LEF1,p-catenin,Wnt3a,bcl-2,N-cadherin,bax,E-cadherin and Caspase3 etc in seven groups for further comparison and analysis.④ MTT test:the cells were cultivated in a 96-well plate with each one of 3x103～6×103 cells after 48h transfection.Then,the MTT was used to detect OD values at 570nm of various cells.Later,the OD values were taken as the Y-axis to draw a cell viability curve chart so as to determine cell proliferative potential after transfection.⑤ Scratch test:the cells were inoculated on a 6-well plate until the cell confluence reached 90%一 100%after 48h transfection.Then,the scratch test was conducted to detect cell migration in various groups.⑥ The Transwell experiment was conducted to test cell invasion in various groups.⑦ Detection of cell cycle and apoptosis;the FCM flow cytometry was used to observe cell cycle and apoptosis;the cell cycle was detected under 488nm fluorescence,while the cell apoptosis was detected under 525nm and 620nm by taking 488nm wave length as the exciting light.Results:(1)Selection of cell strains;four melanoma cell strains,including B16,A375,WM239 and Wm451 were selected to conduct the experiment.The results showed that miR-708 was expressed at different degrees in four melanoma cell strains.Thereinto,miR-708 was highly expressed in B16,which was obviously higher than those in the other three cell strains(P<0.05).Thus,the B16 with high miR-708 expression was selected for the follow-up cell test.(2)Melanoma model① The HE straining and pathological observation were conducted on the normal control group and model group after establishing the melanoma model.The results showed that compared with the normal control group,cells in the model group were of varying sizes,relatively large volume and irregular shapes;the melanin grains were only existed in the cytoplasm of few cells.Many cells in the tissue were dead;little inflammatory cell infiltration was seen in the melanoma stroma and around tissues.Meanwhile,the positive expression rate of LEF1 was(20.31 ± 5.28)%in the normal control group,which was obviously lower than(65.35 ± 6.85)%in the model group.The difference had statistical significance(P<0.05).The results indicated that LEF1 protein expression quantity in the B16 melanoma cell model increased affected by the tumor,which was obviously higher than that in the normal control group.The results also implied that LEFT participated in the malignant transformation of melanoma cells.② According to QRT-PCR results,compared with the normal control group,the mRNA expression levels of miR-708,Bax,Caspase3 and E-cadherin were decreased significantly in the model group,while that of LEF1,(3-catenin,Bcl-2,Wnt3a and N-cadherin were improved greatly.The difference had statistical significance(P<0.05).③ In accordance with Western blot results,compared with the normal control group,the expression levels of Bax,Caspase3 and E-cadherin were decreased obviously,while that of LEF1,β-catenin,Bcl-2,Wnt3a and N-cadherin were increased significantly in the model group.The difference had statistical significance(P<0.05).The results also proved that miR-708 as well as Bax,Caspase3,E-cadherin,LEFT,P-catenin,Bcl-2,Wnt3a and N-cadherin etc were closely related to the formation and development of melanoma cells.(3)Verification of the targeting relationship and cell test① Determination of the targeting relationship:the target-gene online analysis on miR-708 was conducted via microRNA.org.The results indicated that a special binding domain was existed between LEF1 gene sequence and miR-708 sequence,of which LEF1 was the target gene of miR-708.Meanwhile,it can be seen from the luciferase report that compared with the NC group,the luciferase signals in the wide Wt-miR-708/LEF1 cotransfection group of the miR-708 mimics transfection group were decreased(P<0.05),while the luciferase activity in the mutant 3’UTR showed no obvious difference(P>0.05),which indicated that miR-708 can bind LEF1 specifically.② The expression levels of related mRNA in various groups were detected after cell transfection.The results showed that compared with the control group,the mRNA expression of miR-708,Bax,Caspase 3 and E-cadherin in the rest of groups were decreased significantly(all P<0.05),while that of LEF1.P-catenin,Bcl-2,Wnt3a and N-cadherin were increased greatly(all P<0.05).Compared with the blank and NC groups,the miR-708 expression in the miR-708 mimics group was increased significantly(all P<0.05),while that in the siRNA-LEF1 group showed no obvious difference(P>0.05).Compared with the blank and NC groups,the mRNA expression quantity of miR-708,Bax,Caspase3 and E-cadherin in the miR-708 inhibitors group was decreased sharply(all P<0.05),while that of LEF1,P-catenin,Bcl-2,Wnt3a and N-cadherin was increased significantly(all P<0.05).Compared with the blank and NC groups,the miR-708 expression quantity in the miR-708 inhibitors +siRNA-LEFl group was decreased significantly(P<0.05.),while the difference of mRNA expression quantity of Bax,Caspase3,E-cadherin,LEF1,P-c’atenin,Bcl-2,Wnt3a and N-cadherin had no statistical significance(all P>0.05).③ Accordingly,the relevant protein expression after cell transfection was as follows.Compared with the control group,the protein expression of Bax,Caspase3 and E-cadherin in the rest of groups was decreased significantly(all P<0.05),while that of LEFT,P-catenin,Bel-2,Wnt3a and N-cadherin was increased greatly(all P<0.05).The blaak and NC groups showed no obvious difference(P>0.05).Compared with the blank and NC groups,the protein expression quantity of Bax,Caspase3 and E-cadherin in the miR-708 mimics group and siRNA-LEF1 group was increased significantly(all P<0.05),while that of LEFT,P-catenin,Bcl-2,Wnt3a and N-cadherin was decreased greatly(all P<0.05).Compared with the blank and NC groups,the protein expression quantity of Bax,Caspase3 and E-cadherin in the miR-708 inhibitors group was decreased significantly(all P<0.05),while that of LEF1,β-catenin,Bcl-2,Wnt3a and N-cadherin was increased greatly(all P<0.05).Compared with the blank and NC groups,the difference of protein expression quantity of Bax,Caspase3,E-cadherin,LEF1,β-catenin,Bcl-2,Wnt3a and N-cadherin in the miR-708 inhibhitors + siRNA-LEF1 group had no statistical significance(all P>0.05).④ The MTT test results showed that compared with the control group,the OD values of the rest of groups were increased significantly,and the cell proliferative potential got improved after 48h and 72h transfection(all P<0.05).Compared with the blank and NC groups,the OD values in the miR-708 inhibitors group were increased obviously after 48h and 72h transfection(all P<0.05);however,the OD values of the miR-708 mimics group and siRNA-LEF1 group were decreased after 48h and 72h transfection(all P<0.05).The OD value of miR-708 inhibitors +siRNA-LEF1 had no obvious changes(P>0.05).⑤ The scratch test results indicated that the cell migration ability in the blank and NC groups showed no obvious difference(P>0.05).Compared with the blank and NC groups,the cell migration ability in the miR-708 mimics group and siRNA-LEF1 group was weakened significantly(all P<0.05),while that in miR-708 inhibitors was increased greatly(P<0.05);and that in the miR-708 inhibitors +siRNA-LEF1 group showed no obvious changes(P>0.05).⑥ The Transwell experiment was conducted to detect the cell invasion in various groups.The results were as follows;the cell invasion ability in the blank and NC groups showed no obvious difference(P>0.05).Compared with the blank and NC groups,the cell invasion abilities in the miR-708 mimics group and siRNA-LEF1 group were decreased significantly(P<0.05),while that in the miR-708 inhibitors group was increased greatly(P<0.05);and that in the miR-708 inhibitors +siRNA-LEF1 group showed no obvious difference(P>0.05).⑦ Cell cycle:compared with the control group,the cell cycle in the blank group,NC group,miR-708 mimics group,siRNA-LEF1 group,miR-708 inhibitors group as well as the miR-708 inhibitors + siRNA-LEF1 group was shortened in G0/G1 period and prolonged in S period(all P<0.05).Compared with the blank and NC groups,the cell cycle in the miR-708 mimics group and siRNA-LEF1 group was shortened in G0/G1 period and prolonged in S period;the cell cycle in miR-708 inhibitors group was shortened in G0/G1 period and prolonged in S period(P<0.05);the cell cycle in the miR-709 inhibitors + siRNA-LEF1 group showed no obvious difference(P>0.05).⑧ Cell apoptosis:compared with the control group,the cell apoptosis rate of the blank group,NC group,miR-708 mimics group,miR-708 inhibitors group,siRNA-LEF1 group and the miR-708 inhibitors + siRNA-LEF1 group was decreased successively.The difference had obvious statistical significance(P<0.05).Compared with the blank and NC groups,the cell apoptosis in the miR-708 inhibitors group showed decreasing tendency,while that in the miR-708 mimics and siRNA-LEF1 group showed rising tendency(P<0.05).The difference of cell apoptosis in the miR-708 inhibitors + siRNA-LEF1 group had no statistical significance(P>0.05).Conclusion:The miR-708,LEF1,Bax,Caspase3,E-cadherin,β-catenin,Bcl-2,Wnt3a and N-cadherin etc as well as related proteins participated in melanoma cell proliferation,invasion,migration,cycle,apoptosis and regulation processes with important influences on the occurrence and development of melanoma cells.The mRNA and proteins expression levels of miR-708,Bax,Caspase3 and E-cadherin were negatively related with the melanoma development,while those of LEF1,P-catenin,Bcl-2,Wnt3 a and N-cadherin were positively related with the melanoma development.Meanwhile,a fragment on LEF1 sequence can be specially bound with miR-708.The LEF1 is a specific target spot of miR-708.The miR-708 can promote melanoma cell proliferation&EMT,and inhibit cell apoptosis,whose mechanism was related to the target regulating LEF1 expression through Wnt signaling pathway. |
