Gene Expression Profile For Treatment Of Traditional Chinese Medicine-liuweidihuang Pills On Postmenopausal Osteoporosis And Analysis Of Altered Gene Expression In Postmenopausal Osteoporosis-related Diseases

Primary osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass,microstructural destruction of bone tissue,increased bone fragility and prone to fractures which can be divided into postmenopausal osteoporosis(POMP)and senile osteoporosis.As the most common type of primary osteoporosis,PMOP usually occurs in women,5-10 years after menopause.According to the International Osteoporosis Foundation,there are currently 200 million osteoporosis patients worldwide,accounting for 60% of the elderly population;the fracture rate of PMOP patients is about 4 times that of normal people and becoming a common cause of disability and death in the elderly women.Although bone loss induced by estrogen deficiency in menopause is the major cause of PMOP,the pathogenesis of PMOP has not yet been fully elucidated.It is a result from a complex effect of a multitude of pathways and cytokines working in a cooperative fashion to regulate osteoclastogenesis and osteoblastogenesis.Therefore,exploring the potential risk factors and pathological molecular mechanisms of PMOP may be the entry point for future PMOP and multi-factor related diseases,which will contribute early detection,early intervention,and timely clinical practice for patients.In recent years,research of traditional Chinese medicine confirmed that bone metabolism-related diseases including osteoporosis were closely associated with kidney deficiency.Many kidney-tonifying prescriptions has been used as adjuvant therapy for bone metabolism-related diseases which help to improve symptoms.As a kidney-tonifying prescription,Liuweidihuang(LWDH)pills has been demonstrated to exert a protective effect on PMOP,and no obvious adverse reactions was found after long-term use.However,the therapeutic mechanism of LWDH on PMOP is not yet clear and needs further research.In addition,research of osteoporosis-related diseases can also help to reveal the molecular mechanism of osteoporosis.Studies have shown that diabetes mellitus and coronary artery disease(CAD)are associated with osteoporosis and there may be a shared pathophysiological mechanism among these three diseases.Both diabetes and CAD are closely associated with genetic and environmental factors that are two disease threatens human health with high morbidity and mortality.The incidence of diabetes mellitus in China is 10.8%.Type 2 diabetes mellitus(T2DM)accounts for more than 90% of diabetic patients,that is a systemic metabolic disorder,and its pathogenesis remain unclear.Moreover,T2 DM and CAD share common genetic susceptibility and pathophysiological risks,and T2 DM is a clear independent risk factor for CAD,which may be the cause of their frequent co-morbidity.Exploring their shared key regulatory genes can provide clues to the pathogenesis of T2 DM,CAD and their related diseases,osteoporosis.As a new discipline formed by the combination of life sciences and computer science,bioinformatics has been widely permeated into the various fields of medical research,and becoming an indispensable approach of biomedical development.With the advent of the post-genome era,bioinformatics-based microarray technology(also known as gene chip)and high-throughput sequencing(Hi-seq)makes it possible to perform a detailed and comprehensive analysis of the transcriptome and genome for each specie,which contributes to a more comprehensive understanding of the complex biological processes of diseases and drugs.Moreover,accumulated evidence indicated that gene expression profile of peripheral blood has been used to explore the molecular mechanism and key regulators of various diseases and drugs.In this study,we identified the key regulatory genes of PMOP as well as key regulatory genes in the treatment of Liuwei Dihuang Pills on PMOP byanalyzing the microarray gene expression datasets and identified the specific and shared key regulatory genes of PMOP-related diseases including T2 DM and CAD by RNA sequencing.Then,a series of bioinformatics analysis methods including Gene ontology(GO)enrichment analysis,Kyoto gene and genome encyclopedia database(KEGG)enrichment analysis,protein-protein interaction relationship(PPI)network construction and prediction of drug target and related pathways was performed to reveal the biological functions of key regulatory genes,which made a contribution for developing potential biomarkers and drug targets,and providing clues to elucidate the pathological molecular mechanisms of PMOP,T2 DM and CAD and the action of LWDH Pills.Accordingly,this study was divided into three parts: the first part-Reveal the altered gene expressions of patients with postmenopausal osteoporosis;the second part-Analysis of altered gene expression in postmenopausal-osteoporosis patients treated with Liuweidihuang pills;the third part-A comparison of gene expression profiles in patients with osteoporosis-related diseases type 2 diabetes,coronary artery disease.Part 1 Reveals the altered gene expressions of patients with Postmenopausal osteoporosisObjective: This present study aimed to identify key genes involved in the pathogenesis of postmenopausal osteoporosis(PMOP)and further explore their biological function.Methods:1.Microarray gene expression data set associated with PMOP was searched and downloaded from the gene expression omnibus(GEO).2.Integrated analysis was performed by using the obtained microarray gene microarray data sets,and the differentially expressed genes(DEGs)between PMOP patients and the normal postmenopausal women were identified.3.By using GENECODIS software,Gene ontology(GO)including biological process(BP),molecular function(MF),and cellular component(CC))was used to perform enrichment analysis for obtained DEGs;Kyotogene and genome encyclopedia(KEGG)enrichment analysis was conducted for DEGs as well.4.To validate the results by our integrated analysis,qRT-PCR was performed to test the expression of candidate DEGs in the peripheral blood of patients with PMOP.Results:1.Four microarray gene expression datasets(GSE100609,GSE56815,GSE13850,and GSE7429)associated POMP were downloaded from GEO and enrolled in in this study.2.Compared with the normal controls,1732 DEGs in POMP were obtained with P<0.05.3.DEGs were significantly enriched for Regulation of transcription,DNA-dependent(P=4.31E-22),Cytoplasm(P=4.14E-108),Protein binding(P=4.19E-115)and Mitogen-activated protein kinase(MAPK)signaling pathway(P=2.58E-09).4.qRT-PCR results indicated that the expression of 4 DEGs including ATF2,FBXW7,RBBP4,and RDX were all consistent with our integrated analysis,generally.Summary:1.Compared with the control group,a total of 1732 DEGs were significantly associated with the molecular mechanism of PMOP.2.MAPK signaling pathway and its mediated pathways may a role in the molecular mechanism of PMOP.3.Four genes including ATF2,FBXW7,RDX and RBBP4 may be the potential regulatory factors and biomarkers of PMOP.Part 2 Analysis of altered gene expression in postmenopausal osteoporosis patients treated with Liuweidihuang pillsObjective: To explore the molecular mechanism for the intervention of Chinese medicine Liuwei Dihuang(LWDH)Pills on postmenopausal osteoporosis(PMOP)patients and to predict its target and pathway of action.Methods:1.Microarray gene expression data set associated with the intervention of LWDH pills on PMOP patients were searched and downloaded from the gene expression omnibus(GEO).2.Firstly,the DEGs in the blood of PMOP patients before and after the intervention of LWDH Pills were obtained.Then,the shared genes not only differentially expressed between PMOP patients and normal controls but also differentially expressed in the blood of PMOP patients before and after the intervention of LWDH Pills were obtained and retained for the following research.3.By using GENECODIS software,Gene ontology(GO)including biological process(BP),molecular function(MF),and cellular component(CC))was used to perform enrichment analysis for the obtained common DEGs;Kyoto gene and genome encyclopedia(KEGG)enrichment analysis was conducted for these common DEGs as well.4.For the obtained shared DEGs,protein-protein interaction(PPI)network was constructed by using the BioGRID(http://thebiogrid.org/)and the Cytoscape software.5.The target genes of LWDH Pills were predicted by using BATMANTCM database(BATMAN-TCM).Results:1.A microarray gene expression dataset(GSE57273)associated with the intervention of LWDH pills on PMOP patients was downloaded from GEO and enrolled in this study.2.A total of 58 common DEGs were found to not only differentially expressed between PMOP patients and normal controls but also differentially expressed in the blood of PMOP patients before and after the intervention of LWDH Pills.3.PPI network showed that CSNK2A1(degree=66),ATF2(degree=33)and FBXW7(degree=26)were hub proteins.4.Based on the BATMAN-TCM database,ATF2,FBXW7,and RDX were regulators associated with the intervention of LWDH pills on PMOPpatients,which were significantly upregulated in PMOP patients compared with normal controls while down-regulated after the intervention of LWDH Pills.5.Based on the BATMAN-TCM database,a total of 10 common targets(ESR1,FGFR2,MED1,PGR,PRKCB,PTGS1,PTGS2,TRIM24,VDR,and WNT4)for the six main traditional Chinese medicine components of LWDH Pills were identified.Among which,ESR1,FGFR2,MED1 and WNT4 were differentially expressed in blood of PMOP patients before and after the intervention of LWDH Pills.Summary:1.A total of 58 common DEGs involved with LWDH pills in intervention of PMOP patients.43 genes were up-regulated in PMOP versus normal control and down-regulated in LWDH treatment versus PMOP;15 genes were up-regulated in LWDH treatment versus PMOP and down-regulated in PMOP versus normal control.2.ATF2,FBXW7 and RDX were key regulators in LWDH pills intervention of PMOP patients.3.Among the 10 co-acting DEGs of the main traditional Chinese medicine components of LWDH pills,ESR1,FGFR2,MED1 and WNT4 may be potential targets in the intervention of LWDH pills on PMOP;all these 10 DEGs suggested that LWDH pills play a potential role in the treatment of endocrine-related diseases.Part 3 A comparison of gene expression profiles in patients with osteoporosis-related diseases type 2 diabetes,coronary artery diseaseObjective: To clarify the hypothesis of whether there is an intrinsic interaction between two osteoporosis-related diseases including type 2diabetes mellitus(T2DM)and coronary artery disease(CAD),and explore the shared regulators of both diseases and their coexisting conditions and further identify whether these two diseases share the same regulatory genes with PMOP.Methods:1.RNA-sequencing was performed on peripheral blood mononuclear cells from patients with T2 DM,CAD,patients with both T2 DM and CAD and normal controls.2.Compared with normal controls,specific and shared DEGs in patients with CAD,T2 DM and patients with both CAD and T2 DM were identified.3.By using GENECODIS software,Gene ontology(GO)including biological process(BP),molecular function(MF),and cellular component(CC)was used to perform enrichment analysis for the obtained specific and common DEGs;Kyoto gene and genome encyclopedia(KEGG)enrichment analysis was conducted for these shared DEGs as well.4.For the obtained DEGs,CAD-specific and T2DM-specific protein-protein interaction(PPI)networks were constructed by using the BioGRID(http://thebiogrid.org/)and the Cytoscape software.5.Microarray gene expression data set associated with T2 DM and CAD was searched and downloaded from the gene expression omnibus(GEO)to validate the expression of DEGs in CAD and/or T2 DM.Results:1.The analysis showed that an overlap of 191 DEGs across CAD,T2 DM and their coexisting conditions,which were significantly enriched in Viral infectious cycle,Anti-apoptosis,Endocrine pancreas development,Innate immune response,and Blood coagulation.2.T2DM-specific PPI networks involving 64 genes,TCF4(Degree =169)、SKP1(Degree = 164)and UBE2W(Degree = 75)were hub genes in T2 DM.3.CAD-specific PPI networks involves 64 genes,HIF1A(Degree = 124),SMAD1(Degree = 112)and SKIL(Degree = 94)were hub genes in CAD.4.According to the GSE23561 expression data from GEO,three DEGs(HIF1A 、 SMAD1 and SKIL)were all significantly up-regulated in CAD,while HIF1 A was down-regulated in T2 DM.Summary:1.A total of 191 shared DEGs may involve with the pathological and molecular mechanisms of CAD and/or T2 DM.2.TCF4,SKP1 and UBE2 W were hub genes of T2 DM,which may play an important role in the occurrence and development of T2 DM.3.HIF1 A,SMAD1 and SKIL were hub genes of CAD,which were up-regulated in CAD compared with normal controls and may play a protective role for ischemia reperfusion in heart.Conclusion:1.ATF2,RDX,FBXW7 and RBBP4 may be potential regulators and biomarkers for PMOP.2.A total of 58 shared DEGs were speculated to involve with both the molecular mechanism of POMP and the therapeutic mechanism of LWDH pills on PMOP.Among which,ATF2,RDX and FBXW7 were key shared regulators for both these two mechanisms.3.There were 10 co-acting DEGs of the main traditional Chinese medicine components of LWDH pills were identified,which suggested that LWDH pills play a potential role in the treatment of endocrine-related diseases.Among them,ESR1,FGFR2,MED1 and WNT4 may be potential targets in the intervention of LWDH pills on PMOP.4.TCF4,SKP1 and UBE2 W were potential key regulators of T2 DM.HIF1A,SMAD1 and SKIL were potential key regulators of CAD.5.T2 DM,CAD and T2DM+CAD have shared DEGs which suggested these three disease status have internal correlation.