Together with isocitrate dehydrogenase (IDH) mutation, co-deletion of 1p19q (1p19q codel) is a prerequisite for diagnosis of oligodendroglioma, making it imperative that histopathology laboratories introduce testing for 1p19q codel. To date there is still no consensus reference range and cut-offs that confirm deletion of 1p or 19q. We embarked on determining our reference range in 11 formalinfixed, paraffin-embedded non-neoplastic brain tissue using fluorescence in situ hybridisation (FISH) with the Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Abbott Molecular Inc., USA). At same time we attempted to validate our methodology in 13 histologically-confirmed IDH-mutant oligodendrogliomas. For 1p, percentage cells with deletion (range=8-23%; mean±SD = 15.73±5.50%) and target: control (1p36:1q25) ratio (range = 0.89-0.96; mean±SD = 0.92±0.03) in non-neoplastic brain, differed significantly (p<0.000) from oligodendroglioma (percentage cells with deletion: range = 49-100%; mean±SD = 82.46±15.21%; target:control ratio range:0.50-0.76; mean±SD = 0.59±0.08). For 19q, percentage cells with deletion (range = 7-20%; mean±SD = 12.00±3.49%) and target:control (19q13/19p13) ratio (range:0.90-0.97; mean±SD = 0.94±0.02) in non-neoplastic brain also differed significantly from oligodendroglioma (percentage cells with deletion: range = 45-100%; mean±SD = 82.62±18.13%; target:control ratio range:0.50-0.78; mean±SD = 0.59±0.09). Using recommended calculation method, for diagnosis of 1p deletion, percentage of cells showing deletion should be >32-33% and/or target:control ratio <0.83. For 19q, percentage of cells showing deletion should be >22% and target:control ratio <0.88. Using these cut-offs all 13 oligodendroglioma demonstrated 1p19q codel.

OBJECTIVETo construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.

METHODSEukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.

RESULTSWe found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.

CONCLUSIONThe apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.

Apoptosis ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mutation ; RNA, Catalytic ; genetics ; pharmacology ; Telomerase ; genetics ; pharmacology ; Transfection