The Study Of Clinical Efficacy And Transcriptional Mechanism Of Quyu Jiedu Decoction In The Treatment Of Colorectal Cancer

ObjectiveIntegrative cancer therapy is emerging as a driving event in colorectal cancer(CRC).Quyujiedu Decoction(QYJD),a Traditional Chinese Medicine(TCM)formula,has been widely served as a complementary and alternative treatment in CRC patients,especially for those at advanced stage.In order to provide theoretical and experimental evidence of assessing the effects of Quyu Jiedu Decoction in the treatment of colorectal cancer,the study was divided in three parts:1.To investigate the association between TCM therapy and survival outcomes in colorectal cancer patients with liver-limited Metastases.2.To analyze the transcripti onal profile of QYJD and luteolin which may provide a framework for determining the potential molecular targets and related mechanisms by which QYJD protects against colorectal cancer.3.Exploring the anticancer potential of LUT and QYJD in colorectal adenocarcinoma HCT116 cells and the epigenetic regulation of the Nrf2/ARE pathway.Methods1.The First Part:The diagnosis and stage classification of CRC was made based on clinical manifestation and clinical guidelines endorsed by the Chinese Colorectal Cancer Association in 2017,and the classification of TCM syndrome differentiation was referred to Integrative Treatment of Chinese and Western Medicine.191 patients(121 females and 70 males)received standard therapy between January 2008 and December 2016,and were treated complying with the National Comprehensive Cancer Network(NCCN)guideline.All the baseline data were carefully in records,including sex,age,TNM stage,primary surgery site,etc.The primary end point was overall survival and the data were obtained by one of the following methods:the hospital medical record system or telephone follow-up.Assessors were blinded to the patients' information in order to reduce assessment bias.All the data were updated to December 30,2018.We collected the information on covariates adjusted for confounding or used for stratification during the baseline period,which including age,sex,the anatomical location of the tumor,tumor differentiation,Number of liver metastases and whether they had received primary surgery,targeted therapy,radiotherapy,local therapy for liver metastases,as well as cycles of chemotherapy completed.Besides,stratified analysis was performed for TCM exposure to detect the effect of TCM exposure on sex,age,liver metastasis,etc.2.The Second Part:The quality and concentration of extracted RNA were determined by an Agilent 2100 Bio-analyzer and a NanoDrop spectrophotometer,respectively.RNA libraries were built from the control and QYJD-treated HCT116 cells.Initiating with 500 ng total RNA,polyA-tailed mRNA was isolated byoligo(dT)and fragmented by adaptive focused acoustic energy(Covaris Inc.,MA,USA).A random hexamer primed,cDNA library of nucleotide sequences(75 bp median fragment size)was created from which millions of short DNA reads were generated in a paired-end orientation.Sequencing was performed on an Illumina NextSeq 500 instrument(Illumina,San Diego,CA,USA)by RUCDU Infinite Biologics.Each lane produced 30 million raw paired reads.Data output in fastq file format containing information about sequences and quality(Pared quality score).Average Pared scores of ? 20 per position were used for alignment.The reads were aligned to the human genome(hg19)with Hisat2v2.1.0 and PCR duplicates were removed.Reads overlapping with genomic features were counted by featureCounts(version 1.5.1)and were analyzed for differential expression with DEGSeq(version 1.14.1)in R(version 3.4.0).A list of DEGs from RNA-Seq platforms was generated using p-value<0.05 and absolute Fold Change>2.Isoforms with Fold Change>2 and p-value were subjected to GO terms(http://www.geneontology.org)and KEGG pathways(based on KEGG,http://www.genome.jp/kegg/).GO analysis was used to determine the biological roles in terms of the molecular functions,biological processes,and cellular components of the DEGs.KEGG analysis mapped the input genes to its knowledge bases and identified the relevant pathway related to the altered mRNAs.3.The Third Part:MTS assay was performed to analyze the viability of HCT116 cells after treatment with LUT or QYJD for 24h,48h and 72h.Anchorage-independent growth capacity is an indicator of tumorigenic and metastatic potential.A soft agar assay was employed to investigate the effect of LUT and QYJD on inhibiting the colony formation of HCT116 cells.Afterwards,luciferase assay,qPCR and Western Blotting were used to detect the effects of QYJD and luteolin on Nrf2 and its target genes on both mRNA and protein levels.Then bisulfite sequencing was applied to investigate whether LUT and QYJD could affect the methylation status of the Nrf2 promoter region.Besides,western blotting was used to examine the effect of LUT and QYJD on the protein expression and enzyme activity of epigenetic modifying enzymes to investigate the epigenetic mechanism by which LUT and QYJD demethylated the Nrf2 promoter and increased Nrf2 transcription.Results1.The First Part:Between January 2008 and January 2018,221 patients were enrolled into the cohort and 208 data were interpreted.The predefined primary end point in our analysis was OS.The results revealed that the median OS for high-exposure group was 26.13 months compared to the 14.67 months in low-exposure group,indicating that longer-lasting TCM intake was correlated with better overall survival(P<0.01).Uni-variate analysis suggested that the hazard ratio of TCM therapy,tumor differentiation,KPS score,primary/metastasis surgery,local therapy,targeted/chemo-therapy were<1,serving as the protective factors;While the hazard ratios of CEA? 5were>1,serving as the hazard factors.Multivariate analysis indicated that higher exposure of TCM could significantly reduce the risk of death after adjusting for demographic and clinical variables,higher exposure of TCM significantly reduced the risk of death.Because KPS scores were unbalanced between the two groups,we performed exploratory subgroup analysis stratified by KPS status.In subgroup exploratory analysis,patients with higher KPS score were shown to have significantly fewer cancer-related death than patients with lower KPS scores(p<0.05).2.The Second Part:It is revealed by Seq-data that there were 490 differentially expressed genes in QYJD vs Control and 4939 differentially expressed genes in QYJD vs Control;192 genes were up-regulated in either QYJD or LUT,and 77 genes were down-regulated in both groups.According to the fold change,the top 10 up-regulated and down-regulated genes by QYJD and LUT are presented.KRT6A,KRT1,KRT5 and RPPH1 were contained in either group,and their biological functions were related to keratinocyte proliferation,migration of protein synthesis,epithelial cell growth,glucocorticoid receptor regulation and endoribonuclease,respectively.KEGG pathway and GO analysis suggested that the QYJD is mainly associated with the regulation of intracellular oxygen pressure,and is involved injak-stat/TGF-/HIF-1,B cell receptors and cytokine delivery pathway.On the other side,LUT is shown to have significant association with cell growth and metabolic angiogenesis and stability of extracellular matrix,while involved in signaling pathways such as infected lipids and amino acid metabolism.3.The Third Part:The results of the MTS assay showed that LUT and QYJD could significantly reduce cell viability in a dose-dependent manner(p<0.05,p<0.01).Besides it is revealed by soft agar assay that LUT at 7.5,15,and 30 ?M decreased the colony formation ability to 82.09%,81.29%and 44.68%,respectively;While QYJD at 100,200,and 400 ?g/ml significantly decreased the colony formation ability to 82.09%,81.29%and 44.68%,respectively.QYJD induced a higher luciferase activity than the control in a dose-dependent manner at concentrations ranging from 1.25 to 5 mg/ml,and LUT at 40 ?M could significantly induce luciferase activity.Meanwhile,LUT at higher dose could remarkably increase the mRNA and protein expression of Nrf2and NQ01.However,no significant difference of HO-1 protein expression was observed between control and LUT-treated group.QYJD at higher dose significantly induced mRNA expression of Nrf2,HO-1 and NQ01,but failed on protein expression.We performed bisulfite sequencing to investigate whether LUT could affect the methylation status of the Nrf2 promoter region.The results showed that Treatment with a combination of 5-Aza and TSA significantly decreased the CpG methylation level to an average of 45%.Treatment with LUT at 15 u M and 30 u M also showed a pronounced reduction in the methylation of these CpG sites from 60%to 55.0%and 48%.Additionally,4mg/ml and 8mg/ml QYJD showed a pronounced reduction in the methylation of these CpG sites to 54.07%and 44.37%?Conclusion1.Patients who accepted TCM therapy over 6 months experienced a preferable median overall survival of 26.14 months comparing to 14.67 in low-exposure group.Additionally,high-exposure group appeared with a notable decreased death risk(HR=0.47;95%CI=0.34-0.65,P<.01),which is consistent with the adjusted result(adjusted HR=0.488,95%CI=0.248-0.961,P<.05).The subgroup exploratory analysis suggested that higher the KPS score is,which means better general physical condition,the lower decline of death risk was found in higher TCM exposure group.2.To determine the anti-tumor mechanism of QYJD and luteolin against CRC,we performed pathway enrichment and biological process analysis after RNA-seq to screen the global genomic profile to identify genomic loci with differed gene expression HCT116 when exposed to indicating treatment.Subsequently,either QYJD or LUT was identified to activate Nrf2 pathway through epigenetic modification,providing new insights into utilizing QYJD,as well as LUT for cancer prevention and treatment.