Human Calcyclin Binding Protein (CacyBP) Encoding Gene And Its Effects On Multiple Drug Resistance Of Gastric Cancer

Human CacyBP encoding gene was found to be highly expressed in the gastric cancer cell which is resistant to ADR(SGC7901/ADR) than the gastric cancer cell which is sensitive to SGC7901 during our former research work,but we do not know the exact function of this gene. To identify the effect of this gene on the mechanism of gastric cancer cell multipule drug resistance(MDR) is an urgent thing. Aim of the study: The eukaryotic vectors of sense and antisense human CacyBP encoding gene were constructed and transinfected into gastric cancer cell SGC7901 and SGC7901/ADR respectively, to study its biological effects on multiple drug resistance of gastric cancer.Methods: RT-PCR was used to amplify hCacyBP cDNA. Primes were designed according to the reported nuclear acid sequence of the gene. Total RNA isolation kit was used to isolate total RNA from cultured human gastric cancer drug resistance cell (SGC7901/ADR) whose CacyBP gene expression is higher than SGC7901 confirmed by our northern blot resuslt. CacyBP mRNA was reversely transcripted into cDNA. Then PCR product was confirmed by DNA sequencing and was cloneddirectionally into eukaryotic expression vector pcDNAS.l by using endonuclease-7-tecnique. The sense and antisence recombinat vectors were identified by endonuclease digestion . The sense recombinat vector and empty carrier were transfected steadily into SGC7901 cell respectively by using lipofectamineZOOO. The antisense recombinat vector and empty carrier were transfected steadily into SGC7901/ADR cell respectively by the same way as before. Using G418 to screen the resistant clone for 8 weeks, picked out the positive clone randomly and amplified the cells. RT-PCR measured the hCacyBPmRNA expression level of SGC7901 and SGC7901 transfected with pcDNA3.1/hCacyBP+ or pcDNA3.1 respectively ;Usign the same method to measure the hCacyBPmRNA expression level of SGC7901/ADR and SGC7901/ADR transfected with pcDNAS.l/hCacyBP- or pcDNA3.1 respectively.MTT measured the drug sensitivity of SGC7901 and SGC7901/ADR, SGC7901 and SGC7901/ADR after transfection respectively. FCM measured the average ADR accumulation concentration and cell cycle of SGC7901 and SGC7901/ADR. SGC7901 and the SGC7901/ ADR after transfection respectively.Results: CD We obtained a 0.686kb fragment by RT-PCR whose sequence was consistent with hCacyBP cDNA registered in genebank(AF:314752). % Northern blot analysis confirmed that the hCacyBPmRNA highly expresses in SGC7901/ADR cell than SGC7901 cell. ?The recombinant sense vector pcDNA3.1/hCacyBP+ generated two fragments of 0.686kb and 6.08kb by Not I and Xba I,the antisense vector pcDNA3.1/hCacyBP- generated two fragments of 0.7kb and 6.06kb by Not I and Nhe Idigestion. The results accord with what we expected. (D RT-PCR showed that the hCacyBPmRNA expression level of SGC7901 transfected with pcDNA3.1/hCacyBP+ was higher than SGC7901 transfected with pcDNAS.l and SGC7901. RT-PCR showed that the hCacyBPmRNA expression level of SGC7901/ADR cell transfected with pcDNAS.l/hCacyBP- was lower than-8-SGC7901 /ADR cell transfected with pcDNAS.l and SGC7901/ADR cell.?MTT showed the survival rate of SGC7901 transfected with the recombinant sense vector pcDNA3.1/hCacyBP+ was higher than the SGC7901 transfected with pcDNAS.l and SGC7901. MTT showed the survival rate of SGC7901/ADR cell transfected with the recombinant antisense vector was lower than the SGC7901/ADR transfected with pcDNA3.1 and SGC7901/ADR cell. ?FCM showed that the average ADR concentration accumulated in SGC7901 transfected with pcDNA3.1/hCacyBP+^ pcDNA3.1 and SGC7901 without transfection was 5.17^ 5.49, 5.64 respectively: FCM showed that the average ADR concentration accumulated in SGC7901 /ADR cell transfected with pcDNA3.1/hCacyBP-> pcDNA3.1 and SGC7901/ADR without transfection was 6.32> 5.62> 5.54 respectively. Ct Using FCM to measure the cell cycle,the results were that Gl phase of SGC7901 transfected with pcDNA3.1/hCacyBP+ or pcDNA3.1 was reduced slightly but G2 and S phases of the cell transfected were increas...