Fig 2: FEN1 serves as a direct miR-134-3p target in SKOV-3 and OVCAR-3 cells. (A) The putative binding site between FEN1 and miR-134-3p was predicted by starBase. (B) Dual-luciferase reporter assay was performed to reveal the association between miR-134-3p and FEN1. (C) Reverse transcription-quantitative PCR was performed to assess the mRNA expression of FEN1. (D) Western blot analysis was employed to assess the protein expression levels of FEN1 in SKOV-3 and OVCAR-3 cells transfected with miR-134-3p mimic. The data are presented as the mean ± SD (n=3). **P<0.01 vs. NC mimic. miR, microRNA; NC, negative control; FEN1, flap structure-specific endonuclease 1; WT, wild-type; MUT, mutant.

Fig 3: The enhanced anticancer effect of Andrographis and berberine is mediated by the suppression of the DNA replication pathway in CRC. (A) The volcano plot obtained from whole-transcriptome profiling analyses of RKO and HT-29 cells after treatment with berberine (|log2 fold change| > 1, p < 0.05); (B) The bubble diagram of the KEGG pathway analysis based on the significantly dysregulated genes; (C) The heatmap of the common differentially expressed genes in both RKO and HT-29 cells and the heatmap of the transcriptional expression of DNA replication-specific genes; (D) A bar graph showing the relative mRNA expression of FEN1, MCM7, PRIM1, MCM5, POLA1, MCM4, and PCNA in RKO and HT-29 cells after being treated by berberine, Andrographis and their combination. Statistical Significance: * p < 0.05, ** p < 0.01, *** p < 0.001. BBR, berberine; Andro, Andrographis.

Fig 4: Andrographis and berberine mutually enhance the inhibition of DNA replication in mouse xenografts and patient-derived tumor organoids. (A) A bar graph showing the relative mRNA expression of FEN1, MCM7, PRIM1, MCM5, POLA1, MCM4, and PCNA in dissected tumor xenografts after being treated with berberine and/or Andrographis; (B) A bar graph showing the relative mRNA expression of DNA-replication-related genes in two strains of patient-derived tumor organoids; (C,D) The protein immunoblot analysis of DNA-replication-related proteins in RKO cells and dissected tumor xenografts treated with berberine and/or Andrographis. Statistical Significance: * p < 0.05, ** p < 0.01, *** p < 0.001. BBR, berberine; Andro, Andrographis.

Fig 5: Expression of host DNA repair factors in the MmuPV1-induced tumor and MmuPV1-infected, tumor-free tissues.A, Distributions of mapped RNA-seq reads for H2ax, Fen1, Cdk1, Plk1, Polθ, and Mdc1 in tumor (Sample #4) and tumor-free (sample #11) ear tissues (see Fig 2B) to the mouse genome were visualized by IGV. B, Quantitative RNA expression for H2ax, Fen1, Cdk1, Plk1, Polθ, and Mdc1 from ear tissues by RT-qPCR. Total RNA extracted from MmuPV1 tumor (sample #4) and tumor-free (sample #11) ear tissues from animal #2 (See Fig 2B). The RNA expression levels of the indicated genes were determined by RT-qPCR using mouse gene-specific TaqMan probes. C, Phosphor-H2AX (S139) (γH2AX), FEN1, CtIP, and phosphor-CtIP (S327) in tumor samples #1 and #4 and tumor-free samples #10 and #12) (see Fig 2B) were determined by Western blot using the indicated antibodies. Tubulin served as a sample loading control.