Fig 1: SP1 was conformed as a transcriptional factor of RNASEH2A and modulate RNASEH2A expression. A. The targeting sequences between SP1 and RNASEH2A were presented. B. Overexpression of SP1 promoted luciferase activity, while mutated MUT-1 or MUT-2 resulted in diminished luciferase activity, **p < 0.01 vs. WT group. C. After si-con or si-SP1 treatment, Ch-IP assay was carried out, and RNASEH2A was detected by qPCR. D. The interference efficiency of si-SP1 and overexpression efficiency of SP1-OE were presented, knocked down of SP1 reduced RNASEH2A expression, while overexpression of SP1 increased RNASEH2A expression, **p < 0.01 vs. si-con, ##p < 0.01 vs. vector. E-F. The promoting effects of SP1-OE on the OD values of HepG2 and Hep3B cells were reduced after co-treatment of SP1-OE and si-RNASEH2A. *p < 0.05, **p < 0.01 vs. con, #p < 0.05, ##p < 0.01 vs. SP1-OE.
Fig 2: Depletion of RNASEH2A antagonized the effect of SP1-OE in modulating HCC cell proliferation, invasion and migration. A. The protein levels of RNASEH2A and SP1 were measured in HepG2 and Hep3B cells. B. The promoting effects of SP1-OE on the colony number of HCC cells were diminished after co-treatment of SP1-OE and si-RNASEH2A. C. Upregulation of SP1 elevated the invasion number of HCC cells, while the function of SP1-OE was suppressed by si-RNASEH2A treatment. D. Overexpression of SP1 increased the migrated distance of HepG2 and Hep3B cells, while the effect of SP1-OE was diminished by si-RNASEH2A treatment. **p < 0.01 vs. con, ##p < 0.01 vs. SP1-OE. Scale bar = 50 μm.
Fig 3: Depletion of RNASEH2A significantly blocked the cell cycle, invasion and migration in HepG2 and Hep3B cells. A. In HepG2 and Hep3B cells, si-RNASEH2A-1 or si-RNASEH2A-2 stimulation obviously decreased the protein levels of CDK1 and CDK2. B. In HepG2 and Hep3B cells, si-RNASEH2A-1 or si-RNASEH2A-2 stimulation obviously reduced the number of cell invaded. C. In HepG2 and Hep3B cells, si-RNASEH2A-1 or si-RNASEH2A-2 stimulation obviously reduced the cell migration distance. **p < 0.01 vs. si-con. Scale bar = 50 μm.
Fig 4: A role of DDX3X in ribonucleotide excision repair. (A) RER reactions in the presence of DDX3X (lanes 2–3), Pol β, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5, 7: re-digestion by RNaseH2 of the heat inactivated reactions. RER reactions in the presence of DDX3X, Pol δ, PCNA, dNTPs (lanes 8–9), Fen-1 and DNA ligase 1 (lanes 10–11). Lanes 9 and 11: re-digestion by RNaseH2. Lane 1: Substrate *D39R1D15: D55 alone. (B) RER reactions in the presence of DDX3X (lanes 2–3), Pol λ, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate *D39R1D15: D55 alone. (C) Western blot for DDX3X and β-actin. L, molecular weight markers. NSC, non-silencing vector control cells; shDDX3X, stable DDX3X-silenced cells. (D) Quantification of genomic rNMPs by the riboassay. NSC untreated cells signal was set as 1 fluorescence arbitrary unit. Different treatments were: untreated genomic DNA (–), genomic DNA treated with 1 U of E. coli RNaseH2 or with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments. (E) Western blot for vinculin, DDX3X and RNaseH2A. L, molecular weight markers. siCNT, non-silencing smart-pool siRNAs; siDDX3X, DDX3X-silenced cells, siRH2A, RNaseH2A-silenced cells. (F) Quantification of genomic rNMPs by the riboassay. siCNT (control) cells signal was set as 1 fluorescence arbitrary unit. As above, different treatments were: untreated genomic DNA (–) and genomic DNA treated with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments.
Fig 5: RNASEH2A was highly expressed in HCC cell lines and promoted the proliferation of HCC cells. A-B. The mRNA and protein levels of RNASEH2A were significantly increased in HCC cell lines, **p < 0.01 vs. L-02. C-D. In HepG2 and Hep3B cells, transfection with si-RNASEH2A-1 or si-RNASEH2A-2 significantly decreased the mRNA and protein levels of RNASEH2A. E-F. Transfection with si-RNASEH2A-1 or si-RNASEH2A-2 obviously reduced the viability in HepG2 and Hep3B cells. G. In HepG2 and Hep3B cells, treatment with si-RNASEH2A-1 or si-RNASEH2A-2 obviously decreased the number of cell clones. *p < 0.05, **p < 0.01 vs. si-con.
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