Study The Mechanism On CD44 Promoter Regulating The Positive Expression Of CD44 In Colon Cancer

Objective:CD44 abnormal expression is an important factor in the formation and development of colorectal cancer.Expression of CD44 which as a cell adhesion molecule,and can mediate and participate in the formation of tumor and metastasis.In view of the expression of CD44 in the process of colorectal cancer occurrence and development is important, so the study of the expression and regulation mechanism of CD44 in colorectal cancer Has important significance to Reveal the mechanism of The colorectal cancer’s occurrence and development.But there are few reports of the study about the regulation by CD44 abnormal expression at present.This study through the analysis and detection the regulatory mechanism of CD44 expression in colorectal cancer by CD44 promoter DNA binding protein, CD44 expression regulation function, to explore the related effect of CD44 promoter binding protein, and find the key protein even Protein group who Play a role of regulation in it,thus we can investigate and verify activation and regulation effect of CD44 expression,and the biological behavior of colorectal cancer by CD44 promoter binding protein In the transcription,to lay the foundation for revealing the Transfer development mechanism and recurrence;also provide experimental basis for gene therapy of colorectal cancer, as well as potential therapeutic targets.Methords:CD44 promoter methylation:Respectively extract Extract genome DNA from the negative and positive lymphocytes and LOVO cells (human colon cancer cells)→Bisulfite treatment→BSP primer design synthesis→PCR amplification and purification→PCR Production sequencing→BSP sequencing results visualization analysis.The human CD44 promoter (2021bp fragment upstream of transcription initiation site) was PCR amplified from chromosomal DNA of HMLE cells using primers 5’-AGCTCCTGAATCCATGCTGT-3’(forward)and5’-CTTCGCAGACAGCTCACTTG-3’(reverse),reamplified with primers introducing NheI(5’-ACTATGCTAGCCTGAATCCATGCTG-3’)orXhoI(5’-ATCAACTCGAGGGT GTCCGGAGCGAA-3’) restriction sites. The resulting fragment was cloned into a pGL3 luciferase reporte vector (Promega) and sequence was verified.Augment CD44p by PCR.Get the plasmid with the EasyPureTM Plasmid MiniPrep Kit,and detection with agarose gel electrophoresis.According to the double restriction enzyme recognition sites be introducted,using restriction enzymes NheI and Xhol to fastdigest.And recycle the production of the digestion,and labeling the DNA with 3’EndTag DNA Labeling System be labelled by biotin (long arm)maleimide.Then dectect the efficiency.culture the cryopreserved colo-320,and then add the CD44 antibody for fluorescence-activated cell sorting;and extract the nuclear protein.Carry on the EMSA at last,and Mass spectrum.Results1.BSP sequencing results visualization analysis:Four samples in the upstream of CD44 gene exon 1 (including exon 1) around 2000 bp,no methylation happen, no methylation site.2.CD44p pGL3 Located at 4148 bp.3.The production of the digestion Located at 2000 bp.4.The probe Located at 2000 bp.5.Labelling was succeed.6.EMS A shows:CD44DNA binding protein with lower mobility than any other group.Mass spectrometry analysis shows:The Score> 21,Successful identification, reliability reached significant level.And also shows that:there are five proteins more importentALBU HUMAN、I3L1U9 HUMAN、POTEI HUMANK22E HUMAN、K21C HUMAN。Conclusion:1.Promoter methylation is not the way to regulate CD44 abnormal expression in colorectal cancer.2.CD44p combined with the related proteins is the way to regulate CD44 abnormal expression in colorectal cancer.3.Serum albumin- histone could activated CD44 gene transcription and lead to CD44 abnormal expression in colon cancer;POTE ankyrin domain family member binding CD44p is the key to promote the CD44 abnormal expression in colorectal cancer;Actin and keratin interact with CD44 on colorectal cancer proliferation, infiltration and metastasis;4.CD44 expression and regulation in colorectal cancer to be in-depth study, have important significance on clinical.