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BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported. OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells. METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy. RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.
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Objective To explore the expression of ANLN in HCC and study the clinical value of ANLN expression for HCC patients after liver transplantation.Methods The protein and mRNA expression of ANLN was detected by immunohistochemistry and RNA-seq from TCGA respectively.Chi-square test and t test were used for correlation analysis between ANLN expression and clinicopathological characteristics.The predictive value of ANLN expression for HCC patients after liver transplantation was estimated by log-rank test and cox proportional hazards regression model.Results The positive protein expression rate of ANLN in HCC detected by immunohistochemistry was 37.0％ (34/92),significantly higher than 6.5 ％ (6/92) in para-carcinoma non-tumor tissue (P＜0.001,x2 =25.044).Upregulation of ANLN mRNA expression in HCC was also detected by the analysis of RNA-seq from the TCGA (P ＜ 0.000 1).The positive ANLN protein expression was positively correlated with AFP＞400 ng/L (P＜0.001,x2 =11.952) and tumor size ＞8 cm (P =0.034,x2 =4.506).The independent risk factors for poorer 5-year survival of patients after liver transplantation were confirmed,including positive ANLN protein expression (P =0.031,OR =1.965,95 ％CI =1.064-3.630),tumor size ＞8 em (P =0.003,OR =2.841,95 ％CI =1.437-5.617),worse differentiation degree (P =0.001,OR =3.613,95％ CI =1.646-7.928),peritumor intravascular cancer emboli (P =0.041,OR =1.896,95％CI =1.028-3.498) and tumor necrosis or hemorrhage (P=0.010,OR=2.195,95 ％CI=1.211-3.979).Conclusion The expression of ANLN in HCC is upregulated and the positive protein expression indicates the poor prognosis for long-term survival of patients after liver transplantation.
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Objective To identify the miR-217 targeted gene ANLN by experiment.Methods Bioinformatic algorithms were used to predict the potential targets of miR-217.Then,ANLN binding with miR217 and mutant ANLN (mutANLN) sequence were designed and synthesized,and their amplified fragments were inserted into plasmid psiCHECK-2,and recombinant plasmid psiCHECK-2-ANLN and psiCHECK-2-mutANLN were reconstructed.The two recombinant plasmids were co-transfected into pancreatic cancer cell line PANC1 with miR-217,miR-217 inhibitor,NC,NC inhibitor by liposome,respectively.Dual luciferase reporter system was used to determine the luciferase activity,and Western blot was used to measure the expression of ANLN protein.Results The luciferase activities of psiCHECK-2-ANLN,psiCHECK-2-ANLN +miR-217,psiCHECK-2ANLN + miR-217 inhibitor,psiCHECK-2ANLN + NC,psiCHECK-2-ANLN + NC inhibitor were 2.221 ± 0.188,0.769 ± 0.061,3.764 ± 0.371,2.265 ± 0.201,2.242 ± 0.018,and the difference among these groups was statistically significant (F =77.405,P ＜0.001),but the difference among psiCHECK-2ANLN group,psiCHECK-2-ANLN + NC group and psiCHECK-2-ANLN + NC inhibitor group was not statistically significant.However,luciferase activities of psiCHECK-2-ANLN + miR-217 group were significantly decreased when compared with other 3 groups,and luciferase activity of psiCHECK-2-ANLN +miR-217 inhibitor group were significantly increased when compared with other 4 groups (all P ＜0.001).Luciferase activities of groups transfected with psiCHECK-2-mutANLN was not significantly different (P =0.053).The expression of ANLN protein in PANC1 with psiCHECK-2-ANLN + miR-217 co-transfection was significantly down-regulated when compared with that with psiCHECK-2-ANLN transfection alone.Conclusions ANLN is one of the direct target genes of miR-217 in PANC1 cells.