Effect Of Liposome Mediated BCSG1 Antisense Oligodeoxynucleotide On Biological Behaviors Of Esophageal Cancer TE-13 Cells

Objective: Ji etc. cloned a new kind of human gene by direct differential cDNA sequencing in 1997, and discovered it expressed high in invasive breast cancer, but scarcely expressed in normal breast or benign breast diseases, then named it breast cancer specific gene 1, BCSG1. Recently, it was reported that BCSG1 protein had an abnormal expression in a wide range of tumor tissues, containing liver, esophagus, gastric, lung, prostate, cervical cancer etc., and its expression all displayed stage-specific patterns of very low expression in stage I and high expression in stages II-IV, which further showed a strong association between BCSG1 protein expression in primary tumors and tumor distant metastasis.Esophageal cancer is one of the most common malignant tumors in the world, especially in Hebei province. Our experiment used antisense oligodioxunudeotide technique, according to the principle of Watson-Crick and Hoogsteen's base complementrity, and prepared DNA fragment–BCSG1 ASODN, which was idio-complementary to the target gene BCSG1 mRNA. Liposome mediated ASODN was transfected into poorly differentiated squamous cancer TE-13 cells of esophageal cancer, which formed part duplex molecule or triplex nucleonic acid by combining with single strand mRNA or double strands DNA, then they activated the nucleinase H to split themselves or suppressed the mRNA maturity,endochylema conveying or translation process. So the protein composition of the target gene was blocked, then we observe the changes of proliferation, apoptosis and invasion of TE-13 cells, further to explore the function of BCSG1 on biological behaviours of TE-13 cells and approach the probability of target gene on gene therapy of esophageal squamous cancer.Methods: The cultural TE-13 cells in vitro were divided into 3 groups (ASODN group, only liposome group, control group). Three groups were all treated according to transfection process. ASODN group was given liposomes (final concentration was 10μl/ml) and BCSG1 ASODN (final concentration was 0.4μmol/L) at the same time, only liposome group was only given liposomes (final concentration was 10μl/ml), control group was given RPMI-1640 culture solution without fetal calf serum and antibiotics instead of transfection compositions.1 To observe the inhibition of BCSG1 mRNA and protein expression level of TE-13 cells by transfecting BCSG1 ASODN. 48 hours after being transfected, the expression level of BCSG1 mRNA of each group cells was examined by semiquantiative RT-PCR technique. Meanwhile the expression of BCSG1 protein in each group cells was semiquantitately examined by flow cytometry. 2 To detect the cell proliferation inhibition of BCSG1 ASODN by MTT assay.3 To detect the cell apoptosis of each group by FCM.4 To detect the effect of transfecting BCSG1 ASODN on invasion of TE-13 cells by Transwell in-vitro invasion assay.Results:1 RT-PCR detection results: The relative magnitude of BCSG1 mRNA expression in only liposome and control group was 0.539 and 0.566 respectively, and that in ASODN group (0.437) degraded significantly compared with only liposome and control group (p<0.05). There was no significantly different between only liposome and control group ( p>0.05).2 FCM assay results: The BCSG1 FI-value of the ASODN group, only liposome group and control group was 0.703, 0.960, 1.000 respectively, and the expression of BCSG1 protein in ASODN group was the lowest significantly compared with the other two groups ( p<0.05), while the expression of BCSG1 protein in the other two groups showed no statistically significant ( p>0.05).3 MTT assay results: BCSG1 ASODN obviously inhibited the proliferation of TE-13 cells. 48h after BCSG1 ASODN at 0.4μmol/L being transfected, the optical density was 0.380,which showed a significant difference compared with the only liposome group (0.674) and control group (0.717)( p<0.05). While there was no significantly different between only liposome and control group ( p>0.05). 4 The cell apoptosis rate by flow cytometry respectively was:ASODN group 26.68%, only liposome group 3.31%, control group 2.60%. The cell apoptosis rate of ASODN group showed significantly different compared with the other two groups (p<0.05), but there was no significant difference between only liposome and control group (p>0.05).5 Transwell in-vitro invasion assay results: The number of invasive cells in ASODN group decreased significantly (p<0.05), while the difference between only liposome and control group showed no statistically different (p>0.05).Conclusions:1 BCSG1 expresses relatively high in TE-13 Cells of human esophageal poorly differentiated squamous cancer. The BCSG1 ASODN designed in the research inhibited the expression of BCSG1 in TE-13 cell at mRNA transcription and protein translation.2 MTT essay displayed that BCSG1 ASODN can significant- ly inhibit cell proliferation.3 The results of flow cytometry indicated that BCSG1 ASODN induced apoptosis of TE-13 cells.4 Transwell in-vitro invasion assay showed that BCSG1 ASODN depressed cell invasion of TE-13 cells.5 To further suggest that BCSG1 is closely related with the biological behaviors of TE-13 cells, and had an important contribution in cell proliferation, apoptosis and invasion.