Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells.
OBJECTIVE
To investigate the expression level of ZNRD1 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, and to observe the drug sensitizing and proliferation effect of ZNRD1 antisense nucleic acid transduction on SGC7901/VCR cells.


METHODS
Amplification of sequences encoding ZNRD1 from SGC7901/VCR cDNA by PCR. The levels of ZNRD1 mRNA expression were demonstrated using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Eukaryotic expression vector pcDNA3.1-anti ZNRD1 was constructed and transfected into SGC7901/VCR cells by lipofectamine. Immunochemical method was used to detect the expression of protein in SGC7901/VCR cells and transfectants. The cell cycle alteration and the intracellular adriamycin (ADM) accumulation were observed by FACS. Growth curve and drug sensitization of cells for vincristine (VCR) were analyzed with MTT assay.


RESULTS
We cloned the open reading frame of full-length ZNRD1. The expression of ZNRD1 showed higher in SGC7901/VCR than in SGC7901 cells. The antisense ZNRD1 drug-resistant clones were selected after gene transfection. Immunochemical results showed that the expression level of ZNRD1 protein was lower in anti ZNRD1-SGC7901/VCR cells than that in non-transfectants. Comparing to SGC7901/VCR and pcDNA3.1-SGC7901/VCR, anti ZNRD1-SGC7901/VCR showed gradually accumulated in G(1) phase, with a concomitant decrease of cell population in S phase. FACS also suggested intracellular ADM accumulation increased 2fold in SGC7901/VCR cells after transfected with antisense ZNRD1. MTT assay showed that transfectants cells proliferation was lagged and more sensitive to VCR than non-transfectants.


CONCLUSIONS
ZNRD1 gene displayed highly expression in VCR resistant gastric cancer cells. Expression of ZNRD1 protein was effectively blocked in anti ZNRD1-SGC7901/VCR cells by gene transfection. ZNRD1 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to VCR, increased ADM accumulation and inhibited the cells proliferation. ZNRD1 antisense RNA transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree.
Zhang
Yu-Mei
YM
Department of Gastroenterology, Xijing Hospital, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
Zhao
Yan-Qiu
YQ
Pan
Yang-Lin
YL
Shi
Yong-Quan
YQ
Jin
Xiao-Hang
XH
Yi
Hui
H
Fan
Dai-Ming
DM
D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D002455:Cell Division / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics*; D004317:Doxorubicin / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016372:RNA, Antisense / Q000235:genetics* / Q000494:pharmacology*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C406241:POLR1H protein, human; D016372:RNA, Antisense; D004317:Doxorubicin