| Obj ective:To explore the inner process during HCC development by using atomic force microscopy (AFM) in hepatocellular carcinoma which was used to study the patten of cytoskeleton remodeling and the correlation to cell invasioin. And exploring the effect of NRP1 gene silencing on atomic mechanical properties of HCC cells.Method:1. The effect of Young’s modulus and cytoskeleton remodeling on cell invasion of hepatocellular carcinoma.(1) HCC cell lines with different potential of metastasis (MHCC-97H cell, MHCC-97L cell, SMMC-7721 cell, Huh-7 cell) were recruited. Topographic images (Height graph) of cell surface were performed by AFM. Young’s modulus of cytoplasm area and nucleus area were analyzed using AFM.(2) HCC cell lines with different potential of metastasis (MHCC-97H cell, MHCC-97L cell, SMMC-7721 cell, Huh-7 cell) were recruited. The pattern of cytoskeleton remodeling was detected by immunofluorescent staining.(3) HCC cell lines with different potential of metastasis (MHCC-97H cell, MHCC-97L cell, SMMC-7721 cell, Huh-7 cell) were recruited. Cell migration ability was observed by cell scratch assay.2. The effect of cytomechanics parameters on NRP1 gene silencing HCC cells.(1) NRP1 gene silencing HCC cell lines (MHCC97H-shNRP1 cell, HCCLM3-shNRP1 cell) and their blank control group (MHCC97H-NC cell, HCCLM3-NC cell) were recruited. Topographic images of cell surface were performed by AFM. Cell surface morphology and average roughness of cytoplasmic region and nucleus region were analyzed by the topographic images.(2) NRP1 gene silencing HCC cell lines (MHCC97H-shNRP1 cell, HCCLM3-shNRP1 cell) and their blank control group (MHCC97H-NC cell, HCCLM3-NC cell) were recruited. Young’s modulus were analyzed using AFM.(3) NRP1 gene silencing HCC cell lines (MHCC97H-shNRP1 cell, HCCLM3- shNRP1 cell) and their blank control group (MHCC97H-NC cell, HCCLM3-NC cell) were recruited. Visco-elastic were analyzed using AFM and expressed by Residual RMS.3. Quantitative data was normality tested using IBM SPSS 21 software. Non-normal distribution data was presented as Median (25%,75%). Comparison between two groups of independent samples was done with wilcoxon test method. Comparison between multiple independent samples was done with Kruskal-wallis H test method. Normal distribution data are presented as mean ± SD. Statistical significance was determined with a one-way ANOVA and T-test. Significance was assigned at P<0.05.Result:1. The effect of Young’s modulus and cytoskeleton remodeling on cell invasion of hepatocellular carcinoma.(1) Young’s modulus (Pa) of HCC cell lines was listed below in the order of cell invasion ability(median (25%,75%)):Young’s modulus of cell cytoplasm area of MHCC-97H cell line was 503.12(366.11,700.31), Young’s modulus of cell cytoplasm area of MHCC-97L cell line was 1026.78 (369.20,2019.96), Young’s modulus of cell cytoplasm area of SMMC-7721 cell line was 1055.28 (367.48,2280.77), Young’s modulus of cell cytoplasm area of Huh-7 cell line was 1608.43 (845.48,3317.25). It showed that Young’s modulus decreased with the increasing trend of cell invasive ability (P<0.05); Young’s modulus (Pa) of HCC cell lines was listed below in the order of cell invasion ability(median (25%,75%)):Young’s modulus of cell nucleus area of MHCC-97H cell line was 655.57 (441.29,943.39), Young’s modulus of cell nucleus area of MHCC-97L cell line was 1285.17 (583.29,1961.19), Young’s modulus of cell nucleus area of SMMC-7721 cell line was 1313.43 (590.71,2678.62), Young’s modulus of cell nucleus area of Huh-7 cell line was 2823.98 (1262.78,4440.07). It showed that Young’s modulus decreased with the increasing trend of cell invasive ability (P<0.01).(2) Cytoskeletal integrity and regularity was reduced with the increasing trend of cell invasive ability as showed in Figure 4. Moreover, MHCC-97H, the most invasive cell of these four HCC cells, even performed a phenomenon of microfilaments fracture. And filaments of nucleus region were much more compact than cytoplasmic region in these four cells.(3) In the cell scratch assay, cell migration rate of MHCC-97H was 46.67% in 24h and 86.47% in 48h. cell migration rate of MHCC-97L was 45.70% in 24h and 82.86% in 48h. cell migration rate of SMMC-7721 was 39.41% in 24h and 79.85% in 48h. cell migration rate of Huh-7 was 34.60% in 24h and 72.09% in 48h.2. The effect of cytomechanics parameters on NRP1 gene silencing HCC cells.(1) Cell height of MHCC97H-NC cells was about 5.5μm with rough uneven surfaces, and the average roughness of the cytoplasmic domain was 283.25±31.26nm, while the average roughness of the nucleus area of 382.78±78.10nm. Cell height of NRP1 knockdown cells MHCC97H-shNRP1 was about 4.5μm with rough uneven surfaces, and the average roughness of the cytoplasmic domain was 30.23±7.17 nm, while the average roughness of the nucleus area of 271.75±74.59 nm. MHCC97H-NC cells have a greater roughness range, while NRP1 knockdown cells MHCC97H-shNRP1 have a lesser roughness range which showed that NRP1 knockdown cells MHCC97H-shNRP1 has a relatively smooth surface; Cell height of HCCLM3-NC cells was about 6.5 μm with rough uneven surfaces, and the average roughness of the cytoplasmic domain was 147.95±15.53nm, while the average roughness of the nucleus area of 315.83±57.55nm. Cell height of NRP1 knockdown cells HCCLM3-shNRPl was about 5.7μm with rough uneven surfaces, and the average roughness of the cytoplasmic domain was 16.03±2.18nm, while the average roughness of the nucleus area of 135.70±29.69nm. HCCLM3-NC cells have a greater roughness range, while NRP1 knockdown cells HCCLM3-shNRP1 have a lesser roughness range which showed that NRP1 knockdown cells HCCLM3-shNRPl has a relatively smooth surface.(2) Young’s modulus of MHCC97H-NC cells were 3888.91±110.17 Pa. Young’s modulus of MHCC97H- shNRP1 cells were 4687.06±123.03 Pa. NRP1 gene silencing treated cell lines significantly increased in Young’s modulus in both cell lines (P<0.01); Young’s modulus of HCCLM3-NC cells were 1982.01±66.13 Pa. Young’s modulus of HCCLM3-shNRP1 cells were 2863.61±55.27 Pa. NRP1 gene silencing treated cell lines significantly increased in Young’s modulus in both cell lines (P<0.01).(3) Visco-elastic of MHCC97H-NC cells were 81.55±2.53 pN. Visco-elastic of MHCC97H-shNRP1 cells were 65.6±2.41 pN. NRP1 gene silencing treated cell lines significantly declined in Visco-elastic in both cell lines (P<0.05); Visco-elastic of HCCLM3-NC cells were 123.18±2.90 pN. Visco-elastic of HCCLM3-shNRP1 cells were 97.89 ± 3.56 pN. NRP1 gene silencing treated cell lines significantly declined in Visco-elastic in both cell lines (P<0.01)Conclusions:Young’s modulus decreased with the increasing trend of cell invasive ability in HCC cell lines. Cytoskeleton integrity and regularity also reduced while cell stiffness and stability were decreasing, vice versa. Silencing of NRPl gene can decrease the roughness of HCC cell membrane. Silencing of NRP1 gene can obviously increase Young’s modulus and decrease Visco-elastic in HCC cells. Young’s modulus of tumor cells may indicate tumor malignant degree, cell invasion ability increaed with the lower Young’s Modulus and higher deformability. Cytoskeleton remodeling and/or destroy of tumor cells effect its metastatic potential. Changes of cytomechanical properties represent invasive potential of tumor cells and can be used as a new indicator of cancer development and prognosis. |
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