The application of Bazhen decoction activated the telomere maintenance pathways via up-regulating the DNA helicases and telomere proteins
As we know, telomere function could be the sustaining mechanism for the stem cell function, anti-inflammation, anti-DNA damage induced stress, etc., however, due to the low abundance of the genes in telomere maintenance pathway, it might not be able to stand out as top pathway in the transcriptome. Thus, we further plotted the heatmap for telomere function and DNA damage response related pathways and tried to dissect the function of Bazhen decoction in telomere maintenance.
Interestingly, we found that the telomere end processing related pathways, which is essential for telomere elongation and maintenance, were up-regulated (Figure 2, A, GOBP TELOMERE CAPPING, REACTOME POLYMERASE SWITCHING ON THE C STRAND OF THE TELOMERE, REACTOME TELOMERE C STRAND LAGGING STRAND SYNTHESIS, REACTOME TELOMERE C STRAND SYNTHESIS INITIATION, etc.). And the telomere DNA repair pathway was also up-regulated (Figure 2, A, GOBP TELOMERE MAINTENANCE IN RESPONSE TO DNA DAMAGE), which inhibited the telomere DNA damage induced cellular senescence (Figure 2, A, REACTOME DNA DAMAGE TELOMERE STRESS INDUCED SENESCENCE). We also found the up-regulation of DNA helicase pathways induced by Bazhen decoction (Figure 2, A, GOBP REGULATION OF HELICASE ACTIVITY, GOMF 3 5 DNA HELICASE ACTIVITY, GOMF DNA HELICASE ACTIVITY, GOMF HELICASE ACTIVITY, GOMF SINGLE STRANDED DNA HELICASE ACTIVITY). Interestingly, the telomerase pathway was not obviously regulated by Bazhen decoction (Figure 2, A, REACTOME TELOMERE EXTENSION BY TELOMERASE). We suspected that the telomere maintenance was through the activation of telomere recombination (Figure 2, A, up-regulation of GOBP TELOMERE MAINTENANCE VIA RECOMBINATION, and down-regulation of REACTOME INHIBITION OF DNA RECOMBINATION AT TELOMERE). The score of BZT-L1 was strangely high for some telomere and helicase pathways, but did not impact the tendency of up-regulation of these pathways by Bazhen decoction.
Consistent with this, the DNA damage repair pathways were also up-regulated (Figure 2, A, GOMF DAMAGED DNA BINDING, GOBP DNA SYNTHESIS INVOLVED IN DNA REPAIR, WP DNA REPAIR PATHWAYS FULL NETWORK, KAUFFMANN DNA REPAIR GENES). While the p53 regulated cell cycle arrest pathways were down-regulated by Bazhen decoction (Figure 2, A, REACTOME TP53 REGULATES TRANSCRIPTION OF GENES INVOLVED IN G1 CELL CYCLE ARREST, REACTOME TP53 REGULATES TRANSCRIPTION OF GENES INVOLVED IN G2 CELL CYCLE ARREST, REACTOME TP53 REGULATES TRANSCRIPTION OF CELL CYCLE GENES).
Together these data suggest that Bazhen decoction could promote DNA damage repair and telomere maintenance, reduce DNA replication stress, thus promote cell cycle progression, and prevent cellular senescence.
To verify these data and explore the role of Bazhen decoction in DNA replication and telomere maintenance, we utilized the progeroid MEF cells from Werner syndrome mouse (G3DKO cells, both DNA helicase Wrn and telomerase Terc genes were knocked out), with WT MEF cells as control. From the RNA-seq data, we realized that the low dose (10μg/mL) of Bazhen decoction showed better effect in sustaining cell growth, thus we treated the cells with 10μg/mL, and 20μg/mL Bazhen decoction for further experiments. The Western blot data showed that Bazhen decoction treatment up-regulated the protein level of DNA helicase Blm, Mcm7, Parp1 in both WT (Figure 2, B) and G3DKO cells (Figure 2, C), which are known to unwind DNA G4 structure, and promote DNA replication fork progression, especially in telomere DNA. The telomere protein Terf1, which is known to involve in telomere packaging and c-strand (lagging strand) synthesis, was also up-regulated. While the Pot1, which is known to involve in the negative regulation of telomere length, was down-regulated (Figure 2, B, C). Together these data suggest that the Bazhen decoction might regulate the telomere DNA replication and telomere DNA damage response, and thus regulate telomere lengthening.
At the same time, we also observed that the Bazhen decoction did not obviously affect the tumor suppressor p53 protein level, while the p53 down-stream effector p21 was up-regulated in both WT and G3DKO cells (Figure 2, C, D). Interestingly, another important tumor suppressor Rb family member Rb and p130, which known to be the cell cycle inhibitors, were up-regulated by Bazhen decoction. However, Rb pathway down-stream E2F1 was also up-regulated, which supposes to be the cell cycle promoter. These data suggest a double-side regulation of cell cycle progression by Bazhen decoction (Figure 2, C, D).
Together the above data suggest that Bazhen decoction could up-regulate the expression of DNA helicase, promote the unwinding of G4 structure, thus promote DNA replication, and regulate cell cycle progression.