Aberrant MicroRNA In Tissues At Different Distances From Colorectal Cancer Lesions And Intervetional Effect Of Chinese Medicine Monomer

ObjectiveColorectal cancer(CRC)is one of the most common malignant tumors.Signs and symptoms may include blood in the stool,a change in bowel movements,weight loss,and feeling tired all the time.Due to unhealthy life style and diet custom,its incidence trends to raise year by year in the world.CRC is difficult to be cured by conventional surgery,chemotherapy or radiation therapy,which means effective early detection and target treatment is critical.Finding CRC biomarkers for early diagnosis and individualized treatment is necessary in clinic.Microarray data was analysis in this study to select differently expressed genes which related to CRC.We investigate the relation between selected microRNAs and DNMTs in CRC by several experiments and bioinformatics analysis.Besides,Zuojin pill was investigated to discover its effect on CRC related microRNA.MethodsGene expression data of CRC tissues,cell lines and mice were collected from GEO database to analyse the difference between these three groups.Histological alteration was assessed by HE staining,expression of E-cadherin,Crumbs3,?-SMA,Vimentin was detected by Immunohistochemistry.Tissues resected by surgery were from primary colorectal carcinoma.These samples were collected from three different locations.The normal tissues were collected from more than 10 cm away from colorectal cancer lesions.The para-cancer tissues were collected from about 2cm away from colorectal cancer lesions.And the cancer tissues were collected from colorectal cancer lesions.Microarray data was used to screen distinguishable microRNAs and microRNA targeting DNMTs whose validation assay was performed by quantitative real-time polymerase chain reaction(qRT-PCR).Besides,Microarray data was analysed by bioinformatics methods including target prediction,clustering analysis,GO and Pathway analysis.Compared the gene expression data of human CRC tissue samples,human CRC cell lines and CRC mice models to find out the best experimental subject.According to the comparison result,an in vitro culture of colorectal tissues was established to analyze the effect of Berberine(BER)and Evodiamine(EVO)on the level of hsa-miR-429 and hsa-miR-497-5p.To investigate the relation between microRNAs and DNMTs by dectecting expression of selected microRNAs,DNMT1 and E-cadherin after the treatment of 5-Aza-2'-deoxycytidine(DAC)and antagomir.Results(1)By comparing the gene expression data of human CRC tissue samples,human CRC cell lines and CRC mice models,we found human CRC tissue samples might be the most appropriate subject for our experiments.(2)According to HE staining,the structure of crypts are different in normal tissues,paracancerous tissues and cancer tissues.(3)During CRC process,E-cadherin and Crumbs3 expressd lower in CRC tissues than normal tissues while Vimentin and ?-SMA expressd higher in CRC tissues.(4)Result of validation by qRT-PCR matches microarray data,which indicates microarray data is reliable.There are 13 microRNAs differentially expressed in para-cancer tissues vs normal tissues(Fold change>1.5,P<0.05).And 20 microRNAs differentially expressed in cancer tissues vs para-cancer tissues(Fold change>1.5,P<0.05).45 differently expressed microRNAs were found when comparing cancer tissues with normal tissues(Fold change>1.5,P<0.05).According to bioinformatics analysis,hsa-miR-429 and hsa-miR-497-5p can taget DNMTs and regulate many important process during carcinogenesis.(5)Colorectal tissue culture in vitro was well established as it can survive up to 25 days.After the treatment of BER and EVO,the level of hsa-miR-429 and hsa-miR-497-5p changed.And the difference between cancer tissues and normal tissues was decreased.Higher expression of hsa-miR-429 and hsa-miR-497-5p,less difference between cancer tissues and normal tissues were found after treatment of DAC.With dispose of hsa-miR-429 antagomir,DNMT1 was up-regulated and no significant change in E-cadherin.Conclusion(1)According to microarray data and HE staining,para-cancer tissues and cancer tissues are different from normal tissues.Para-cancer tissue is not appropriate to use as normal one for cancer study.(2)Loss of cell polarity and acquisition of mesenchymal features will change the cell phenotypes during tumorigenesis.(3)Hsa-miR-429 and hsa-miR-497-5p both can target DNMTS and significantly up-regulated in CRC.Aberrant DNA methylation in cancer may caused by cancer related microRNAs.(4)BER and EVO can decrease the difference between cancer tissues and normal tissues of hsa-miR-429 and hsa-miR-497-5p in CRC.They might play a role in anti-tumor activity by regulating expression of cancer related microRNAs.(5)DNA methylation can change the expression of microRNA,while microRNA can regulate DNMTs at the same time.DAC can lower the expression of DNA methyltransferases and regulate CRC related microRNAs,which indicates DAC may be used as a drug in some cases for the treatment of cancer.