The Silence Of Med-19 Gene By RNA Interference And Its Effect On The Proliferation Of Colorectal Cancer Cell

Colorectal cancer (CRC) is the second most frequent tumor in developed countries,with its growing speed by 4.2% of morbidity and death.in shanghai and guangdong china, which is faster thant western country.The early symptom is so oblivious that many patients may ignorant its threaty,and get the diagnose with a later stage. In Shanghai along 2002, colorectal cancer incidence has gone beyond the first two ranks, second only to lung cancer. Today, the surgical operation is the most important treatment of CRC, supported by chemotherapy, radiotherapy or immunotherapy, as well as traditional Chinese medicine treatment. However, colorectal cancer after 5 years the relapse rate, mortality still remains at a higher level. Finding a positive measures for early detection and effective treatment has become the urgent goal for the medical doctor. So looking for the new tumor markers for early diagnosis of colorectal cancer and new gene for therapeutic targets is a hot spots for basic and clinical research of CRC. This study we are concerned is one important member of Mediator family --- Med-19(mediator complex subunit 19)The technology of RNA interfere is to import the double-stranded RNA into cell which share the same organ with internal mRNA coding zone, then the degradation of the mRNA happens after a series of reaction which making the special gene silence This teconology simple effective as a tool to reseach the gene. In our experiment we design the target sequnce for Med-19,bulid the lentiviral vectors (LVs),which can generate a long and stable silence effict,and make foundation for the relationship of Med-19 gene and colorectal cancer.Mediator is an important components of the transcription apparatus for RNA polymeraseâ…¡in transcription of eukaryotic cells. In 1990 kelleher find it and separate it as an important part of MED famlilay, For these year the function of MED complex has been unveiled.Its function including activing the mRNA tanscription,enhance the basic tanscription.It has been proved that Med-19 has an close relationship with invasive ability.of lung cancer.Our erperment have 3 succesive parts,For colorectal tissue,we excamine the mRNA and protein of colorectal cancer and nomal colorectal tissue using reverse transcription polymerase chain reaction (RT-PCR) amd immunohistochemisty,The rusults shows that the mRNA and protein of Med-19 in tumor is significant higher than the normal tissue. After that we conduct the experiment of Med-19 gene silence, we produce the Med-19-siRNA using lentivirus based vectors at first,the Med-19-siRNA is designed by its message,finding the most proper target,test its infectious ability with 293T cell.After that,we choose colorectal cancer cell line DLD-1 and RKO. After being infected we test infectability of cell with fluroimmunoassay,The infect rate of Med-19-siRNA is over 90%.By the useng of PCR and western blot,we prove the mRNA and protein of KD(konck down)group are lower than the control group (normal colorectal cancer cell) and negative control group (colorectal cell with empty lentivirus based vectors),all of this can prove the Med-19-siRNA can suppress the expresion of Med-19 gene.The cell function experiment including the following parts:MTT,Brdu,Colony formation PI-FACS.The reslts shows that the ability of proliferation is suppressed by the silencing of Med-19 gene.The xenograft experiment is perfomed on 30 nude mices,which divided into 3 groups., The CON(control) group injected with colorectal cancer cell,and NC(negetive control) group injectied with lentivirus vector without any siRNA,with KD(konck down)group injected with lentivirus vector with Med-19siRNA. We observe the tumor size with lenth and width in day 15 and 20 after injected.Turmor weight was compared after the mice was executed 30 days after injected.The results shows that the growth of xenograft tumor is restrained,the number of cell in reproduction is lower comparativelyGenerally speaking, we analysed the expression of Med-19 in colorectal tumor and produce the lentivirus vector with Med-19 siRNA and find that the growth of coloectal cell is restrained by silencing the expression of Med-19 gene.