Studies Influence Of High Level Expression Of IL6 On Apoptosis Of Colorectal Cancer Cells Line And The Sensitivity To The 5-Fluorouracil

Objective: Interleukin-6(IL-6) plays an important role in human colorectal cancer occurrence and development. In this study, we overexpressed IL-6 gene on human colorectal cancer Lovo cells by electroporation and detected the effect of IL-6 on proliferation and apoptosis in Lovo cells. Then, the sensitivity of 5-Fluorouracil to IL-6overexpressed Lovo cells was measured by using MTT method.Methods: In this study, Lovo cells were divided into three groups, the normal cells group, no-load plasmid transfected cells group and IL-6 gene transfected cells group. 1) Establishment of IL-6 gene overexpressed Lovo cell line. After got the full-length of IL-6 fragment by PCR, the IL-6 expression vector p ECFP-N1-IL-6 was successfully constructed using molecular cloning methods and sequencing. In order to get a high expression Lovo cell line, electroporation method was used for the transfection of p ECFP-N1-IL-6 and p ECFP-N1-Null. 2) For the detection of IL-6 expression levels in three Lovo cell groups, IL-6 proteinwas measured by sandwich enzyme linked immunosorbent assay(ELISA).3) Proliferation and apoptosis of the three groups were tested by flow cytometry at different points(12h, 36 h and 72h) after electroporation. 4) Proliferation inhibition of the three groups in four different 5-FU concentrations(20ug/ml ? 80ug/ml ? 160ug/ml and320ug/ml) at different time points(12h?36h and 72h) were detected by MTT.Results: 1) IL-6 full-length expression vector p ECFP-N1-IL-6 was constructed successfully. 2) IL-6 protein expression levels were measure by sandwich ELISA. The result showed that the IL-6 expression of normal Lovo cell group, no-load plasmid transfected cells group and IL-6transfected cells group were(34.6103±0.5122pg/ml),(34.7590±1.1456pg/ml) and(78.7118±1.2041 pg/ml) respectively. The expression level of p ECFP-N1-IL-6 transfected cells group was much higher than other groups, there was a statistically significant difference(P<0.05). But IL-6protein expression levels had no statistically significant between normal Lovo cells group with no-load plasmid transfected Lovo cells group(P>0.05). 3) The results of 7-AAD/PE flow cytometry showed that the survival rate of IL-6 high expression group(p ECFP-N1-IL-6 transfected Lovo cells group) was significantly higher than other two groups(P<0.05). But the difference have no statistically significant between the normal and no-load plasmid transfected Lovo cells groups(P>0.05). 4)The results of MTT showed that 5-FU could inhibit the proliferation of all the three groups. The inhibition rate in vitro was on concentration-time dependent(P<0.05).But the proliferation inhibition of high level expression of IL-6 gene Love cells was higher than the normal Lovo cells group and no-load plasmid transfected Lovo cells group, the difference was statistically significant(p<0.05). The proliferation inhibition has no difference between the normal Lovo cells group and no-load plasmid transfected Lovo cells group.Conclusions: 1) we successfully constructed recombinant expression plasmid IL-6-p ECFP-N1 and obtained IL-6 overexpressed Lovo cell line by electroporation. 2) Colorectal cancer Lovo cells could express relatively high level of IL-6 protein by transfect IL-6 gene. 3) The apoptosis rate of IL-6 overexpressed Lovo cell line could been inhibited in vitro. The results showed that IL-6 could inhibit apoptosis of colorectal cancer Lovo cells.4) IL-6 overexpressed Lovo cell line was sensitive to 5-FU, the proliferation inhibition was in a concentration- and timedependent manner.