Fig 1: Low expression of let-7g was significantly associated with poorer overall survival. (a) Heterogeneous expression of let-7g was detected using in situ hybridization method in HCC tissues. Figure (A) showed that let-7g was low in HCC whereas figure (B) showed let-7g high in HCC. The magnification fold was (×100). (b) Kaplan-Meier survival curves were plotted. There is strikingly significant difference between let-7g positive group and let-7g negative group (P < 0.01, using log rank test), after analysis using ISH in 40 cases of HCC tissues.
Fig 2: Reexpression of let-7g inhibited the proliferation of MHCC97-H and HCCLM3 cells. Cell proliferation of MHCC97-H and HCCLM3 cells after reexpression of let-7g for 0 h, 24 h, 48 h, 72 h, and 96 h were examined by MTT assay. Let-7g can obviously retard the proliferation as compared with control group (**P < 0.01, one-way ANOVA analysis). The mean and standard error from triplicate experiments are indicated.
Fig 3: The endogenous expression level of let-7g in HCC tissues. The basal expression of let-7 family members, including let-7a, let-7b, let-7c, let-7d, let-7e, and let-7f was detected using quantitative real-time Reverse Transcription PCR (qRT-PCR) in 40 paired HCC tissues and their normal controls. Total RNA was extracted using Trizol reagent after 48 h. The relative expression of let-7 family members, normalized to U6, was calculated using the formula 2−ΔΔCt (relative expression). **P < 0.01 versus normal control.
Fig 4: Reexpression of let-7g induced cell apoptosis and changes of cell cycle. (a) Qualification assay of cell apoptosis of MHCC97-H and HCCLM3 after reexpression of let-7g. (b) Quantitation assay of cell apoptosis of MHCC97-H and HCCLM3 after re-expression of let-7g (**P < 0.01, Student's t-test). (c) Qualification assay of cell cycle of MHCC97-H and HCCLM3 after reexpression of let-7g. (d) Quantitation assay of cell cycle of MHCC97-H and HCCLM3 after reexpression of let-7g (**P < 0.01, Student's t-test). (e) Biochemical analysis of proteins that are reported to be involved in the regulation of cell cycle. The representative results out of three times experiments were shown here. 80 μg total protein was loaded per lane when performing Western-blotting.
Fig 5: Reexpression of let-7g inhibited the migration of MHCC97-H and HCCLM3 cells. (a) Qualification of wound-healing assay of MHCC97-H after re-expression of let-7g for 0 and 48 hours. (b) Qualification of wound-healing assay of HCCLM-3 after re-expression of let-7g for 0 and 48 hours. (c) Quantitation of wound-healing assay of MHCC97-H. Migration of MHCC97-H cells was significantly retarded in comparison with scramble sequence control group. There is strikingly significant difference between group transfected with scramble sequence and group transfected with let-7g group (**P < 0.01, one-way ANOVA analysis). (d) Quantitation of wound-healing assay of HCCLM-3. Highly similarly, migration of HCCLM-3 cells was also significantly suppressed compared with scramble sequence group. (**P < 0.01, one-way ANOVA analysis). The mean and standard error from triplicate experiments are indicated.
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