Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells

Abstract

Aim: 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.

Conclusion: 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.

Figure 1

Semiquantitative RT-PCR for ZNRD1 and β₂-microglobulin. M: Marker (DL2000); Lane 1, 3: SGC7901 cells; 2, 4: SGC7901/VCR cells.

Figure 2

Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by EcoR V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.

Figure 3

Electrophoresis identification of pcDNA3.1-anti ZNRD1. Lane 1: pcDNA3.1-anti ZNRD1/EcoR V + BamH I; Lane 2: pcDNA3.1-anti ZNRD1; Lane 3: Marker (200 bp).

Figure 4

Detection of ZNRD1 expression in cells by immunocytochemical staining. × 200. A: SGC7901/VCR; B: anti ZNRD1-SGC7901/VCR.

Figure 5

Growth curve of cells.