The effects of transfection of CXCL10 gene into breast cancer cell line MCF-7 on expression of tumor related genes

Abstract

Objective To construct stable transfected MCF-7 cell line by recombinant plasmid CXCL10-pcDNA3.1 (+) and explore the effects of CXCL10 on expression of tumor related genes. Methods CXCL10 gene, cloned from treated PBMCs by IFN-γ, was used to construct recombinant plasmid CXCL10-pcDNA3.1 (+). Recombinant plasmid CXCL10-pcDNA3.1 (+) and control plasmid PcDNA3.1 (+) were separately transfected into MCF-7 cells by lipifection-2000. The stable transfected cell lines were screened by G418. The transcription, expression and secretion of transfected CXCL10 gene was assayed by RT-PCR, Western blot and ELISA in turn. The cell growth activity of stable transfected cell lines was detected by soft agar cloning formation in experimental group [transfected by CXCL10-pcDNA 3.1 (+) ], control group [transfected by pcDNA3. 1 (+)] and blank group (nothing transfected). Furthermore, the reverse transcriptions of three tumor related genes, included MMP9, VEGF and STAT3, were assayed by RT-PCR. Results The stable MCF-7 cell lines transfected by combined plasmid CXCL10-pcDNA3.1 (+) and plasmid pcDNA3.1 (+) were constructed successfully. There was no significant difference in cell growth activity rate between the experimental group and the control group (χ2=3.8, P>0.05, the rates of the 3 groups were 78.2%, 79.9% and 87.8% in turn). There was significant difference in CXCL10 mRNA expression level between the experimental group and the control group (F=50.5, P 0.05, the gray values of the 3 groups were seperately 0.067±0.003, 0.103±0.007 and 0.085±0.026). Comparing with the control group, the experimental group showed significant lower expression level of VEGF (F=2 232, P<0.01, the gray values of the 3 groups were 0.183±0.003, 0.787±0.019 and 0.789±0.011) and STAT3 ( F=115, P<0.01, the gray values of the 3 groups were 0.573±0.016, 0.707±0.008 and 0.711±0.013). Conclusions CXCL10 can express stablely in MCF-7 cell lines, which resulted in down-regulation of expression of VEGF and STAT3 gene. CXCL10 played an important role in anti-tumor effect. Key words: Breast neoplasms; Cell line; tumor; Chemopkine CXCL10; Gene expression