PinX1 Suppresses The Proliferation Of Gastric Carcinoma Cells And Enhances The Sensitivity Of Gastric Carcinoma Cells To 5-Fluorouracil Through Mad1/c-Myc Pathway

Objective Since telomeres and telomerase play crucial roles in maintaining cell immortalization and cancer progression, they may be targets for anticancer treatment. The telomerase level is high in most human cancer while it is low or no in the somal cells. The telomerase reverse transcriptase (hTERT) is the key of telomerase activity regulation. PinX1 was reported in 2001 in human cervical cancer using the yeast two-hybrid method. The previous studies showed that PinX1 is a potent inhibitor of telomerase activity and its TID domain could suppress telomerase activity by coupling telomerase directly. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. We transfected PinX1 gene into the gastric carcinoma line MKN28 without expression of PinX1 and transfected PinX1 siRNA plasmids into the gastric carcinoma line BGC823 with a high level expression of PinX1. drug-resistant clones were screened with G418 in the study. Then we observed the biological behaviour and Mad1/c-Myc expressions in MKN28 cells before and after gene transfection, and explored the possible novel mechanism.Methods 1.we constructed the eukaryotic expression vector pIRES2-EGFP contained PinX1 gene by molecular cloning technology. We also constructed PinX1 RNAi plasmids including interference sequence and negative control sequence. 2. we examined the expression of PinX1 in three different gastric carcinoma cell lines including MKN28,SGC7901 and BGC823 using RT-PCR and Western Blotting. Then, we transferred the recombinant plasmids into gastric carcinoma cell lines using lipofectamineTM 2000. drug-resistant clones were screened with G418. 3. We observed the change of PinX1 expression level before and after gene transfection by semi-quantitative RT-PCR and Western Blotting. 4. The Cell morphology was observed by HE staining and transmission electron microscopy. 5. MTT assay was used to observe cell growth and to test the sensitivety of gastric carcinoma cell lines to 5-FU. 6. Cell cycle and cell apoptosis were analyzed by FCM. 7. Mad1/c-Myc expressions were detected with semi-quantitative RT-PCR and Western Blotting. 8. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP).Results 1. Sequencing suggested that the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 was correct. 2. PINX1 expression was negative in MKN28 cells,weak positive in SGC7901 cells,but strong positive in BGC823 cells. 3. we transfected PinX1 into MKN28 cell and the void vector was transfected as control. we also transfected siRNA eukaryotic expression vectors into BGC823 and the negative sequence vector was transfected as control. Drug-resistant clones were screened with G418 and established their stable transfected cell line. The PinX1 expression was detected by semi-quantitative RT-PCR and western blotting. It was indicated that the PinX1 was transfected into MKN28 successfully. Two siRNA vectors could suppress the expressive level of PinX1 and the interference 2 was more effective. 4. PinX1-transfected cells became larger in size and the coenocyte was common. Apoptotic body with meniscus heterochromatin could be seen in the tranfected cells under transmission electron microscope. At the same time, the karyoplasm proportion was increased and the nucleolus was prominent before gene transfection. 5. PinX1-transfected MKN28 cells grew more slowly than the cells not transfected or transfected with void vectors by MTT examination (P<0.05). 6. The IC50 value(0.253±0.021mg/L) decreased markedly in PinX1-transfected cells than that in the PinX1-untransfected cells or transfected with void vectors(0.560±0.017\0.590±0.026 mg/L)by MTT examination (P<0.05). 7. The apoptosis rate was higher in PinX1-transfected MKN28 than that in PinX1-untransfected cells or transfected with void vectors only by FCM. The growth of PinX1-transfected cells was retarded, and was blocked into G0/G1 stage. 8. PinX1-transfected MKN28 showed up-regulation of Mad1 and, down-regulation of c-Myc, whereas RNAi led to down -regulation of Mad1 and up-regulation of c-Myc in BGC823 cells. 9. The telomerase activity reduced obviously after PinX1 transfection while it was elevated when PinX1 gene was suppressed by PinX1 RNAi.Conclusions 1. we constructed the recombinant eukaryotic expression vector and siRNA vectors targeting PinX1 gene and which were tranfected in to gastric cancer cells successfully. 2. Two RNAi vectors were indentified to suppress the expression of PinX1 specifically and the interference 2 was more effective. 3. Flow cytometric analysis displayed that PinX1 transfanction inhibited gastric cancer MKN28 cells growth,and induced them apoptosis and an arrests of G0/G1 phase. 4. The IC50 value decreased markedly in PinX1-transfected cells,suggesting that PinX1 transfection enhances the sensitivity of MKN28 cells to 5-FU. 5. The PinX1 gene mayasuppressor of telomerase activity. 6. PinX1 may suppress telomerase activity through Mad1/c-Myc pathway.