Study Of The Effect On Telomerase Activity Of MDCC-MSB1 Cell By RNAi

Marek′s disease is a malignant lymphoproliferative disease of domestic chickens caused by MD virus, in which lymphoproliferactive infiltration in visceral organs, muscles, and peripheral nerves are common features. Every year, MD causes heavy economic losses in the poultry industry. Though new types of vaccine were applied, very gvirulent (VV) and very virulent plus (VV+) strains still being isolated and the large-scale breakout of MD has been reported worldwide. The prevention and control of MD has become one of the crucial ticklers for the poultry industry. As known, the tumor cell of gallinaceous MD possesses a comparably high telomerase activation. Besides, the fact that telomerase activity of MDCC-MSB1 (derived from lymphoblastoid cells inverted by MDV) is 4.5 times higher than that of the normal chicken cell ellustrates that MDV has a property of activating telomerase activity. Therefore, the studies on telomerase activity of relevant genes in MD tumor would be very important to elucidate the association between telomerse and MD.Today, telomerse is one of the noticeable anti-tumor treats target. Research discovered that any type of in vitro cultured tumor cell strain and above 85% of human malignant tumor possess high telomerase activity, yet the majority of normal tissues and benign tumor cells lack or present rare telomerase activity excluding certain normal embryo stem cells cells like germ cells, hematopoietic cells etc which owns high telomerase activity. These indicate a close correlation between telomerase activity and tumor occurrence, development, treatment. Telomerase mainly consists of two compositions: Telomerase RNA (TR) and Telomerase reverse transcriptase (TERT). TR is the template of synthesis repetitive sequence of telomere, also a essential core ingredient of telomerse. TERT plays a significant role in regulating telomerase activity, merely exists in the positive cells of expressing telomerase activity, and TERT's expression is the committed step of telomerase activity activation. This research directly takes chTR and chTERT as target genes, aims to study their function on regulating telomerase activity and the relations between telomerase and tumor.In this research, we applied small interfering RNA (siRNA) technology, took the secondary structure of chTR (the template domain, the pseudoknot domain and the CR4-CR5 domain )as the target sites, designed a short hairpin structure of two strands of oligonucleotide as the following order: Apa I site, 19nt positive-sense sequence, 9nt loop sequence, 19nt antisense sequence, the terminator of RNA polymerase III(TTTTTT) and EcoR I site. A plasmid vector was built by a double-stands DNA inserting into the pSilencer 1.0-U6 vector (clone sits: Apa I and the EcoR I, T4 ligase). Then the plasmid were transfected into E.coli, after extracting the plasmid DNA (indentification by XhoⅠ, Hind III respectively), the sequences were analysised by agarose electrophoresis and DNA sequencing. The sequencing identification confirmed that the target sequence had been inserted into the predicted site precisely and the recombinant plasmid vectors were successfully constructed.The recombinant plasmid vectors were transfected into MDCC-MSB1 by LipofectamineTM2000. We conducted nine groups dividing into pSi-chTR-sh1, pSi-chTR-sh2, pSi-chTR-sh3, pSi-chTERT-sh, pSi-chTERT-sh and pSi-chTR-sh1, pSi-chTERT-sh and pSi-chTR-sh2, pSi-chTERT-sh and pSi-chTR-sh3, liposome group and control grouop. The proliferation activity of MDCC-MSB1 was determined by MTT assay. Comparing with nontreated control and oligofectamineTM, the OD value presented a drop tendency at 24h after transfected; decrease became obvious at 48h; descent also displayed at 72h; yet OD values started to restore at 96h, but the values were lower than that in the control group. The telomerase activity was tested by TRAP assay. Results showed comparing with control group, telomerase activity decreased in transfected recombiant, and was lower in co-transfected group than the activity in single transfected group. The difference between chTR transfected groups and chTERT transfected group was not significant. But the telomerase activity of pSi-chTR-sh1 transfected group were lower compared with that of pSi-chTR-sh2 or pSi-chTR-sh3 transfected group. Cell cycle assay was examined by flow cytometry. Results showed the proportion of G0/G1 phase of MDCC-MSB1 which transfected by recombinant plasmid vectors increased and the proportion of S phase depressed. These indicated that shRNA targeting chTR could suppress the telomerase activity of MDCC-MSB1 and this ability becomes notable when transfected shRNA with chTERT simultaneously.In this research, We taken the secondary structure of chTR (the template domain, the pseudoknot domain and the CR4-CR5 domain )as the target sites and constructed pSi-chTR-shRNAs plasmid vector for the first time at home and abroad. Results showed shRNA with both the chTR, chTERT respectively transfected MDCC-MSB1 and chTR, chTERT co-transfected MDCC-MSB1 had capabiliy in inhibiting telomerase activity. This would be helpful in elucidating the regulation function of telomerase activity of chTR as well as the interaction between chTR and chTERT. Thus provides a possible path way in finding a more effective shRNA for telomerase activity inhibition and a possible treatment for oncotherapy.