Study On Regulation Of Cancer Relative Gene ST13, MK And PLK1 Transcription

Cancer is a fearful disease induced by many kinds of factors. If a normal cell is out of control by body it may translate into a cancer. It is known that cancer can be happened and developed by a collection of exterior and internal factors. The exterior factors such as biological, physical and chemical inductor can play action in transforming a normal cell into a cancer cell with internal factors such as immune and gene factors. There is a difference on individual body. A major mechnism of those factors playing action is that cancer relative genes express unconventionally. Cancer-related genes can be divided into two kinds: oncogenes means positive-related cancer and anti-oncogenes means negative-related cancer. Acted oncogenes and deacted anti-oncogenes are always important factors of cancer happening and development. The gene usually plays action on protein. From gene to protein there are two taches of transcription and translation arcording to The Center Theorem. In the complex transcription tache there are four steps inclouding initiation, elongation, termination and mRNA quantity regulation. The initiation and mRNA quantity are most important steps in the gene expression. From the two steps to study cancer relative gene maybe know agene expression, and found out its biological signification. On these a new cancer theraputic method would be invented.ST13 (Suppression of Tumorigenicity 13), MK(Midkine) and PLK1 (polo-like kinase 1) are new genes. The present study showed that there is a close relationship between cancer and the genes. The amount of their expression can influence cancer happening and development. We are going to explore ST13 promoter region and its funcational characteristics on transcriptional initiation. With knock-down MK and PLK1 expression by siRNA (small interference RNA) to realize its regulational funcation on rnRNA quantity and biological signification of anti-tumor.Methods and resultsThe part one: Study of promoter region funcation of cancer related ST13 gene on 5'-flank.Arccording to whole base sequence of ST13 in Genebank to find 5'-flank untranslated region base sequence a funcational base sequence fragment 756bp(-682~+74) was analysized with bioinformatics softwares. There are two CpG islands in a fragment 669bp(-595~+74) inclouding a part of base sequence of the first exon in which +1 is its first base from 5'-flank. They are 206bp(-179~+28) and 271bp(-540~-270). The fragment contains 65 transcription factor-binding sites but no TATA-box. The Spl factor has the most sites.On the result and the former study a series of PCR primers were designed from 5'-flank and 3'-flank of 756bp fragment. The PCRs were running from pGL2-Basic-1894 plasmid. The product fragments were collected by electrophoresis and purify kit. Then they were cloned into pGEMT-Easy vector on TA-base partnership. A positive clone was checked by restriction enzymes and sequencing. Then the fragments of pGEMT-Easy plasmids were subcloned into a series of pGL2 reportervectors, and checked by restriction enzymes to make sure if they were recombined into pGL2 series reporter vectors.That the recombined plasmids and control plasmids including pGL2-control, pGL2-Basic and pSV- B -Galactosidase vectors were purificated and quantitatived. Then they were transfected into SW620 cell with superfectamine reagent. The activity of luciferase and B -Galactosidase was tested after 48 hours respectively. The counts-per-second of luciferase was normalized by OD value of B -Galactosidase activity. With compared normalized counts-per-second of groups it found that 669bp(-595~+74) is a promoter region including an enhancer.ST13 mRNA and protein of cell lines Bcap-37, SMMC-7721 and SW620 were checked with RT-PCR (Real-Time PCR) and Western Blot respectively. There was a difference armong the three cell lines on mRNA but no protein. The recombined plasmid pG12-Basic-669 couold express the most normalized counts-per-second in SW620 than that of Bcap-37 and SMMC-7721.Part two: Study of siRNA knock...