Effects Of The Gene Of LOXL3 And HKR3 On Telomerase Activity In Tumor Cell Lines

Telomere, a complex structure present at the ends of eukaryotic chromosomes, consists of tandam arrays of highly conserved hexameric repeats (TTAGGG) in vetebrates. Its function is to protect DNA from degradation of deleterious recombination. The replication-dependent erosion of telomeric DNA can be counteracted by telomerase- mediated addition of single-stranded TTAGGG repeats, and the end replication problem is significantly resolved. Human telomerase is a ribonucleoprotein consisting of at least three subunits, human telomerase RNA (hTR) acting as a template for telomeric DNA synthesis, telomerase associated protein 1(TP1) and telomerase reverse transcriptase (hTERT). Among them, hTERT plays the most important role in the function of telomerase activity. Under the supportion of NSFC(National Scientific Foundation of China), Zhou Ping et al firstly constructed human telomerase reverse transcriptase bait fusion protein vector, and then selected the unknown protein interacting with hTERT from the cDNA library of testicle by two-yeast hybrid array. The sequence of the newly-discoveried protein was contrasted with that in the GenBank. In the end, six kinds of protein including LOXL3,HKR3,T-STAR,par-4,SMARCB1 and NISCH was found. To further investigate the effect of the gene LOXL3 and HKR3 on telomerase of tumor cell lines, sense and antisense eukaryotic expressing vectors of LOXL3 and HKR3 by DNA recombinant technique of molecule biology was constructed and then transfected into three different cell lines (SGC7901,HepG2, SHG44) with DOTAP liposomal transfection method by selection in G418. The obtained transfectants termed 7901-loxl3-sense (7901-loxl3-S), SGC7901-loxl3-antisense (7901-loxl3-AS), 7901-hkr3 –sense (7901-hkr3-S), SGC-hkr3- antisense (7901-hkr3-AS), stably produced sense and antisense LOXL3 or HKR3 respectively. The nomination in HepG2 and SHG44 cell line is similar to them in SGC7901 cell line. Then telomerase activity examined with Telo TAGGG Telomerase PCR ELISA KIT(Roche, America), the expressing level of LOXL3 mRNA , HKR3 mRNA and hTERT mRNA evaluated by methods of RT-PCR in all tumor cells transfected by the sense and antisense gene were analyzed through statistic software SPSS10.0.Compared with untransfected cell lines(N), cancer cells transfected by sense eukaryotic expressing vectors(S) had a mild enhanced LOXL3 or HKR3 mRNA expressing level, however, tumor cells transfected by antisense expressing vectors(AS) manifested a decreased expressing level. In contrast with the control of N, telomerase activity in SGC7901, HepG2 and SHG44 group transfected by pCMV-SPORT6-HKR3-S (hkr3-S) decreased 20.9%,27.4% and 29.4% respectively, and in the group transfected by pCL-neo-HKR3-AS (hkr3-AS) telomerase activity increased 11.9%,9.6% and 15.8%. The expression of hTERT mRNA was significantly higher in the group of hkr3-AS than in the untransfected group and the group of hkr3-S is contrary. The expression of HKR3 mRNA was significantly negative correlation with telomerase activity(r= -0.783,P=0.013). On the other hand, telomerase activity in SGC7901, HepG2 and SHG44 group of loxl3-S had a minus 5.4%,minus 9.3% and plus 7.5%, compared with the group of N. Telomerase activity in SGC7901, HepG2 and SHG44 group of loxl3-AS had a plus7.3%,plus 8.5% and minus 7.7%, compared with the group of N. No significant differnce of hTERT mRNA and telomerase activity were found among the different group transfected by pcDNA3.1(+)- loxl3-S and pcDNA3.1(-)- loxl3-AS.Our results suggest that LOXL3 gene should not show effect on telomerase activity. So the relationship between LOXL3 gene and telomerase activity is not sure. HKR3 gene is significantly negative correlation with telomerase activity and the expression of hTERT parallels to the telomerase activity. HKR3 may be a negative regulator of telomerase. However, more investigation is required to understand the machanism that HKR3 gene regulates telomerase.