Study On The Design Of Antisense Oligonucleotides Targeting To Ku70 MRNA And The Linfluence To Radiosensitivity In Hela Cell Line

Cervical cancer is one of the most frequent female malignancy,it'sincidence at the second place and only lower than breast cancer,so cervicalcancer is a serious disease that threatens the women's health and life .Inrecent years,the incidence of cervical cancer is not only still high but alsorises steadily,the proportion of young patients is on the rise,and thecervical adenocarcinoma is more and more familiar.Because the youngpatients lack alert to the disease, adenocarcinoma is easy to occur lymphousnodes transfer early and the radiosensitivity of adenocarcinoma is low ,it isdifficult to treat cervical adenocarcinoma and the prognosis is notideal.Radiotheraphy is a very efficient means that is widely used to treatcancer and is usually applied for cervical cancer.The radiosensitivity ofdifferent types of cancer is obviously different and it is one of the factorsthat decide the radiotherapy effect. The radiosensitivity of cervicaladenocarcinoma is lower than squamous cell carcinoma,the tranditionalradiotherapy have severe damage and many syndromes because of the largeradiation doses,so people hope to find a credible indicator that can reflecttumor cells'radiosensitivity, measure the tumor's radiosensitivity in advanceby examing the indicator before radiotheraphy,and improve theradiotheraphy project and decision-making,then change the tumor cells'radiosensitivity by intervening the expression of the indicator,so enhance thecurative effect of each patient.DNA is the most important target of radiation inside cells,DNAdouble-strand break(DSB)is the most basic and crucial damage caused byradiation ,and the quantity of DNA DSB has correlation withcells'mortality,that means the quantity of DNA DSB caused by one Gy inradiosensitive cells is more than in radioresistant cells.Not all cells that haveDSB would die because there is a set of integrated mechanism in cells,onlywhen the cells lack the ability to repair the DSB and the integrality of DNAis destroyed they will die ,so the ability to repair the DNA DSB is one of thedeterminants of cell's radiosensitivity,and the cell is sensitive to radiation aslong as it lacks the ability to rejoin the DSB. The indicators ofradiosensitivity include:1.The clone-livability of cells parting frombody,SF2 has obvious relation with the radiotherapy prognosis of cervicalcancer.2.The ability to repair the DNA DSB: The level of DNA DSB repairis accordant with the cell's high radiosensitivity.So we could predict theradiosensitivity of Hela cells by evaluating the aforementioned indicators. In mammal cells, NHEJ is the primary approach of DNA DSBrepair ,and DNA-PK is the key enzyme in NHEJ, consisting of a catalyticsubunit(DNA-PKCS )and a heterodimetric regulatory complex Ku,Kuheterodimer can combine the free ends and promote the DSB torecombine.So the DNA-PKCS/Ku-deficient cells have dysfunction in DNADSB repairing and are hypersensitive to radiation. Ku70 is one of thesubunit of Ku heterodimer ,many studies show ,Ku70 is correlative with theradiosensitivity or chemosensitivity of several cancers,so it may become aspecial target of radiosensitizing therapy of cervical adenocarcinoma. We detected the expression of Ku70mRNA with RT-PCR,in thisexperiment, Ku70mRNA expression were detected in Hela cell line,anddetected the Hela cell's radiosensitivity by clone-forming method,the resultofshows SF2=0.50. Antisense oligonucleotide is the crude or artificial oligonucleotidesequence,it combine specially with cellular target nucleic acid byWatson-Crick base-pairing principle,and regulate the expression of targetgene at transcribing or translating level.Antisene technique is one of theeffectual measures that can control the expression of abnormal genes. The design of the antisense involves selecting different sites on targetmRNA. According to statistics, a nucleotide with the length of 17nt has oneopportunity to appear in human genome, therefore the familiar length ofantisense oligonucleotide is 17~21nt. The phosphorothioate deriver is theprimary type of antisense because of its character of anti-nuclease and itseasiness to enter cells. The important step to antisense technique is theprediction of the secondary structure and screen for target sites. theoreticalprediction of RNA target sites for active oligonicleotides is related to thedevelopment of algorithms that can locate single-stranded regions in RNAsecondary structure models.The secondary structure was computed withRNAdraw and Sfold.. Because of the complicated structure of mRNA invivo, the key of antisense design is to select the accessible sites of thesurface of mRNA molecules. However the determination of RNA structureis very difficult, so the selecting rule is unclear. One of the familiar means isto predict the secondary structure of mRNA using certain software.Although such predicted structure could not entirely represent the realconfiguration, the physiological structure is nearly correlative with thepredicted one, basing on the same principle of the least free energy ofbase-pair. Therefore, to select effective targets basing on software is asimple and fast method which has a significance of guidance. According to calculation of mRNA structure with the use of two kindsof software, RNAdraw and Sfold,and the calculation and statistical analysisof mRNA structure had been performed in the studies.Olionucleotides weredesigned to target non-structured mRNA region and 6 oligonucleotides wereselected and transfected into Hela cells by lipofectin.The unspecial toxicityof these olionucleotides is measured with MTT assay. Screens out the NO.1antisense ODNs which toxicity is the lowest as the target sequence and cansurpress the expression of gene Ku7.All these results implied that theapproach to designing antisense ODNs by means of calculation of the twokinds softwares was effective and feasible in the studies. After transfected NO.1 antisense ODNs(at the concentraton of100nmol/l) by lipofectin,we distilled the total RNA,and measured theKu70 mRNA level by RT-PCR , the result showed NO.1 coulddown-regulate the Ku70 mRNA level.Then we detected the Hela cells'radiosensitivity after transfected,and the results of both clone-formationand MTT methods showed the NO.1 antisense ODNs obviously increasedHela cell's radiosensitivity in a dose-dependent manner. The results above proved that the cause of Ku70 NO.1 antisense ODNsincreasing the Hela cell's radiosensitivity was relative to it's restraining thetranscription of Ku70 mRNA and farther decreasing the translation of Ku70...