Transcriptome Sequencing Explores the Mechanism of Baicalin on Bone Cancer Pain

Figures & data

Figure 1 Baicalin recovered the bone resorption in the Bone metastases cancer. CT was used to detect BMD in SD rats of the NC group, the Bone metastases group and the Bone metastases + Baicalin group. The changes of BMD were calculated by SPSS software.

Figure 2 Baicalin inhibited osteoclast activation and promoted the increase of bone trabeculae in the Bone metastases cancer. Trap is a typical marker of osteoclasts, and the results of Trap staining was that the cytoplasm of osteoclasts was wine red and the nucleus was light blue. Trap staining indicated that osteoclasts count was significantly increased in the Bone metastases group and was decreased after Bone metastases + Baicalin. HE staining is one of the commonly used staining methods in paraffin section technology. The nucleus is dyed purple blue and the cytoplasm is dyed pink. HE staining showed that the number of trabeculae decreased in the Bone metastases group and increased after Bone metastases + Baicalin.

Figure 3 Baicalin inhibited the expression of iNOS in the Bone metastases cancer. iNOS was reported to be related with tumor metastasis. Fluorescein isothiocyanate (FITC) was labeled and showed bright yellow green fluorescence under fluorescence microscope. Immunofluorescence detection revealed that the expression of iNOS was significantly increased in the Bone metastases group and decreased after Bone metastases + Baicalin.

Figure 4 Baicalin inhibited the protein level of inflammatory factors (IL-1β, IL-6, TNF-α and PGE2) in the Bone metastases cancer shown by ELISA. Data were shown as mean ± SD. **p < 0.01, ***p < 0.001 was obtained from the NC group vs the Bone metastases group, the NC group vs the Bone metastases + Baicalin group, the Bone metastases group vs the Bone metastases + Baicalin group.

Table 1 Top 10 Enriched GO Pathways of DEGs Between the Bone Metastases Group and the NC Group

Terms	Pathway Description	Count	p-value
Upregulated
GO.BP:0050794	Regulation of cellular process	411	2.09E-42
GO.BP:0051171	Regulation of nitrogen compound metabolic process	270	9.39E-41
GO.BP:0042254	Ribosome biogenesis	54	2.01E-34
GO.BP:0007049	Cell cycle	99	2.12E-29
GO.BP:0034470	ncRNA processing	55	6.03E-29
GO.BP:0050896	Response to stimulus	299	3.40E-28
GO.BP:0016072	rRNA metabolic process	43	3.82E-28
GO.BP:0006364	rRNA processing	42	7.49E-28
GO.BP:0006996	Organelle organization	169	5.24E-26
GO.BP:0031325	Positive regulation of cellular metabolic process	161	4.05E-25
GO.CC:0005654	Nucleoplasm	234	5.65E-52
GO.CC:0043227	Membrane-bounded organelle	554	3.70E-51
GO.CC:0005737	Cytoplasm	487	1.12E-50
GO.CC:0043231	Intracellular membrane-bounded organelle	521	6.67E-49
GO.CC:0005829	Cytosol	235	5.03E-34
GO.CC:0005739	Mitochondrion	115	8.29E-31
GO.CC:0005740	Mitochondrial_envelope	68	2.00E-23
GO.CC:0031981	Nuclear lumen	271	2.96E-22
GO.CC:0031966	Mitochondrial membrane	62	8.90E-21
GO.CC:0005634	Nucleus	378	2.01E-20
GO.MF:0005515	Protein binding	556	1.88E-76
GO.MF:0003824	Catalytic activity	251	5.28E-24
GO.MF:0042802	Identical protein binding	104	1.18E-15
GO.MF:0003677	DNA binding	118	2.89E-13
GO.MF:0019899	Enzyme binding	100	8.83E-10
GO.MF:0046872	Metal ion binding	158	8.90E-10
GO.MF:0043169	Cation binding	159	3.26E-09
GO.MF:0036094	Small molecule binding	107	4.37E-09
GO.MF:0017111	Nucleoside-triphosphatase activity	47	1.50E-08
GO.MF:0016462	Pyrophosphatase activity	49	1.92E-08
Downregulated
GO.BP:0050794	Regulation of cellular process	547	3.74E-65
GO.BP:0050896	Response to stimulus	443	6.24E-64
GO.BP:0007154	Cell communication	312	5.02E-47
GO.BP:0007165	Signal transduction	294	8.60E-46
GO.BP:0023052	Signaling	306	1.36E-45
GO.BP:0010033	Response to organic substance	207	3.71E-41
GO.BP:0071310	Cellular response to organic substance	175	2.42E-38
GO.BP:0002376	Immune system process	189	7.67E-36
GO.BP:0032502	Developmental process	293	2.71E-33
GO.BP:0034097	Response to cytokine	107	3.91E-33
GO:CC.0016020	Membrane	498	5.88E-68
GO.CC:0005737	Cytoplasm	596	4.77E-52
GO.CC:0012505	Endomembrane_system	292	3.58E-50
GO.CC:0031982	Vesicle	270	1.08E-49
GO.CC:0031224	Intrinsic component of membrane	318	2.11E-45
GO.CC:0071944	Cell periphery	327	2.48E-44
GO.CC:0005886	Plasma membrane	317	1.46E-41
GO.CC:0016021	Integral component of membrane	302	9.88E-41
GO.CC:0005576	Extracellular region	274	1.68E-39
GO.CC:0005615	Extracellular space	232	1.39E-38
GO.MF:0005515	Protein binding	680	1.57E-76
GO.MF:0003824	Catalytic activity	297	5.97E-22
GO.MF:0005102	Signaling receptor binding	116	4.58E-17
GO.MF:0042802	Identical protein binding	120	1.84E-14
GO.MF:0019899	Enzyme binding	132	1.81E-13
GO.MF:0016787	Hydrolase activity	144	5.36E-13
GO.MF:0030234	Enzyme regulator activity	78	6.73E-13
GO.MF:0046872	Metal ion binding	194	4.64E-10
GO.MF:0043169	Cation binding	198	4.73E-10
GO.MF:0022857	Transmembrane transporter activity	72	7.36E-10

Abbreviations: BP, biological process; CC, cellular component; MF, molecular function; DEGs, differentially expressed genes.

Table 2 Top 10 Enriched KEGG Pathways of DEGs Between the Bone Metastases Group and the NC Group

ID	Pathway Description	Count	p-value
Upregulated
hsa03008	Ribosome biogenesis in eukaryotes	15	1.30E-05
hsa04110	Cell cycle	14	2.49E-04
hsa03030	DNA replication	7	3.50E-04
hsa05210	Colorectal cancer	11	3.90E-04
hsa00240	Pyrimidine metabolism	8	1.31E-03
hsa03430	Mismatch repair	5	1.48E-03
hsa01230	Biosynthesis of amino acids	9	2.06E-03
hsa04915	Estrogen signaling pathway	13	2.11E-03
hsa00250	Alanine aspartate and glutamate metabolism	6	2.49E-03
hsa00230	Purine metabolism	12	3.64E-03
Downregulated
hsa04657	IL-17 signaling pathway	21	2.69E-09
hsa04668	TNF signaling pathway	21	7.18E-08
hsa05134	Legionellosis	14	3.61E-07
hsa05164	Influenza A	25	6.41E-07
hsa05133	Pertussis	13	6.34E-05
hsa05165	Human papillomavirus infection	32	1.37E-04
hsa05323	Rheumatoid arthritis	14	1.41E-04
hsa05145	Toxoplasmosis	15	3.11E-04
hsa04060	Cytokine-cytokine receptor interaction	28	4.84E-04
hsa04210	Apoptosis	16	8.62E-04

Abbreviation: DEGs, differentially expressed genes.

Table 3 Top 10 Enriched GO Pathways of DEGs Between the Baicalin Treatment Group and the NC Group

Terms	Pathway Description	Count	p-value
Upregulated
GO.BP:0050794	Regulation of cellular process	167	1.21E-18
GO.BP:0050896	Response to stimulus	127	5.94E-15
GO.BP:0023051	Regulation of signaling	70	4.99E-13
GO.BP:0051171	Regulation of nitrogen compound metabolic process	96	1.49E-11
GO.BP:0009966	Regulation of signal transduction	62	1.83E-11
GO.BP:0009968	Negative regulation of signal transduction	37	6.02E-11
GO.BP:0009893	Positive regulation of metabolic process	72	2.40E-10
GO.BP:0032502	Developmental process	89	3.97E-10
GO.BP:0050790	Regulation of catalytic activity	50	5.13E-10
GO.BP:0007275	Multicellular organism development	79	7.73E-10
GO.CC:0016020	Membrane	149	3.23E-18
GO.CC:0005737	Cytoplasm	189	9.24E-18
GO.CC:0043227	Membrane-bounded organelle	210	9.24E-15
GO.CC:0012505	Endomembrane system	87	3.62E-14
GO.CC:0031982	Vesicle	75	1.29E-11
GO.CC:0043231	Intracellular membrane-bounded organelle	188	7.27E-11
GO.CC:0005576	Extracellular region	76	4.87E-09
GO.CC:0031090	Organelle membrane	62	1.90E-08
GO.CC:0071944	Cell periphery	86	4.09E-08
GO.CC:0099503	Secretory vesicle	28	4.14E-08
GO.MF:0005515	Protein_binding	236	3.12E-37
GO.MF:0003824	Catalytic_activity	105	2.71E-11
GO.MF:0042802	Identical_protein_binding	46	1.21E-08
GO.MF:0043169	Cation binding	68	2.81E-05
GO.MF:0004857	Enzyme inhibitor activity	14	2.85E-05
GO.MF:0016740	Transferase activity	43	4.70E-05
GO.MF:0046872	Metal ion binding	65	7.93E-05
GO.MF:0030234	Enzyme regulator activity	24	1.35E-04
GO.MF:0008427	Calcium-dependent protein kinase inhibitor activity	2	2.73E-04
GO.MF:0008454	Alpha-1,3-mannosylglycoprotein 4-beta-N-acetylglucosaminyltransferase activity	2	2.73E-04
Downregulated
GO.BP:0050794	Regulation of cellular process	208	3.04E-28
GO.BP:0051171	Regulation of nitrogen compound metabolic process	121	5.28E-17
GO.BP:0007049	Cell cycle	49	4.65E-16
GO.BP:0000278	Mitotic cell cycle	35	1.28E-15
GO.BP:0051726	Regulation of cell cycle	45	1.70E-14
GO.BP:0031325	Positive regulation of cellular metabolic process	78	1.38E-13
GO.BP:0051252	Regulation of RNA metabolic process	84	3.03E-13
GO.BP:1903506	Regulation of_nucleic_acid-templated_transcription	80	3.24E-13
GO.BP:0006355	Regulation of transcription, DNA-templated	79	3.49E-13
GO.BP:2001141	Regulation of RNA biosynthetic_process	80	3.53E-13
GO.CC:0005737	Cytoplasm	223	5.24E-22
GO.CC:0005829	Cytosol	114	1.39E-18
GO.CC:0043227	Membrane-bounded organelle	242	5.54E-16
GO.CC:0005654	Nucleoplasm	87	2.43E-13
GO.CC:0043231	Intracellular membrane-bounded organelle	216	1.69E-11
GO.CC:0016020	Membrane	147	1.47E-10
GO.CC:0012505	Endomembrane system	86	6.92E-10
GO.CC:0005856	Cytoskeleton	52	6.85E-09
GO.CC:0015630	Microtubule cytoskeleton	36	8.45E-09
GO.CC:0031090	Organelle membrane	68	4.64E-08
GO.MF:0005515	Protein binding	259	4.97E-35
GO.MF:0046872	Metal ion binding	82	1.24E-07
GO.MF:0043169	Cation binding	82	3.80E-07
GO.MF:0003824	Catalytic activity	98	7.76E-06
GO.MF:0140110	Transcription regulator activity	41	8.11E-05
GO.MF:0045294	Alpha-catenin binding	3	1.46E-04
GO.MF:0008092	Cytoskeletal protein binding	24	2.58E-04
GO.MF:0003677	DNA binding	47	2.64E-04
GO.MF:0030332	Cyclin binding	4	3.05E-04
GO.MF:0030234	Enzyme regulator activity	25	3.87E-04

Abbreviations: BP, biological process; CC, cellular component; MF, molecular function; DEGs, differentially expressed genes.

Table 4 Top 10 Enriched KEGG Pathways of DEGs Between the Baicalin Treatment Group and the NC Group

ID	Pathway Description	Count	p-value
Upregulated
hsa04640	Hematopoietic cell lineage	6	0.005
hsa05205	Proteoglycans in cancer	9	0.006
hsa05145	Toxoplasmosis	6	0.009
hsa04610	Complement and coagulation cascades	5	0.011
hsa01100	Metabolic pathways	32	0.045
hsa00270	Cysteine and methionine metabolism	3	0.046
hsa00330	Arginine and proline metabolism	3	0.046
hsa00510	N-Glycan biosynthesis	3	0.046
hsa05323	Rheumatoid arthritis	4	0.062
hsa00790	Folate biosynthesis	2	0.064
Downregulated
hsa04933	AGE-RAGE signaling pathway in diabetic complications	7	0.001
hsa05168	Herpes simplex virus 1 infection	17	0.001
hsa05202	Transcriptional misregulation in cancer	9	0.003
hsa05132	Salmonella infection	10	0.005
hsa04064	NF-kappa B signaling pathway	6	0.005
hsa04144	Endocytosis	10	0.005
hsa05144	Malaria	4	0.007
hsa04668	TNF signaling pathway	6	0.007
hsa05020	Prion disease	10	0.009
hsa04218	Cellular senescence	7	0.010

Abbreviation: DEGs, differentially expressed genes.

Figure 5 RT-qPCR results indicated that the mRNA expression level of ALPP, DUSP1, CYR61, ALPPL2, SPP1 and TLR4 were significantly up-regulated in the Bone metastases group compared with the NC group. By comparing the Bone metastases + Baicalin group and the NC group, the expression of these genes were significantly down-regulated. The RT-qPCR results of these genes were consistent with the results of the transcriptomic sequencing results. Data were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 was obtained from the NC group vs the Bone metastases group or the Bone metastases + Baicalin group. ^###p < 0.001 was obtained from the Bone metastases group vs the Bone metastases + Baicalin group.