在单核细胞中干扰SHP-2后对宫颈癌细胞的影响

目的:研究宫颈感染HPV后是否能通过干扰SHP-2从而负调控IFN-β信号通路,最终导致宫颈癌的发生。方法:通过SiHa细胞与THP-1细胞共培养,利用SHP-2 siRNA干扰片段瞬时转染THP-1细胞,干扰SHP-2基因的表达。将SHP-2封闭肽以0、0.5、2.5μg/ml浓度和2、24 h培养THP-1细胞,收集培养后的细胞进行Western Blotting实验,进而检测SHP-2的表达,确定封闭的最佳时间和浓度。收集正常和不同处理因素的THP-1细胞,Western Blotting法检测THP-1细胞中SHP-2蛋白表达。将THP-1细胞及不同处理因素处理好的THP-1细胞分别与SiHa细胞共培养后,在选定的时间和浓度点测其OD值。收集正常THP-1细胞的培养液上清、THP-1细胞给予SHP-2封闭肽处理的培养液上清和THP-1细胞给予SHP-2 siRNA干扰的培养液上清进行ELISA实验。结果:给予SHP-2的SiRNA后SHP-2蛋白表达明显降低,在干扰后48 h、200 pmol时干扰效果最好。给予SHP-2的封闭抗体后SHP-2蛋白表达明显降低,在封闭后24 h、2.5μg/ml时抑制效果最好。THP-1细胞与SiHa细胞相互作用,从两个细胞作用时间来看,THP-1细胞对SiHa细胞杀伤作用随着时间的增长而增强,从24 h始就起明显作用。给予siRNA干扰的THP-1细胞对SiHa细胞的杀伤作用最强(OD值最低),给予封闭肽的次之,无处理因素的最弱(OD值最高)。SiRNA干扰组的浓度值最高,其次是封闭肽组的浓度值,无处理因素组(仅培养THP-1的细胞组)的浓度值最低,SiRNA干扰组与无处理因素组之间差异有统计学意义(P<0.05),封闭肽组与无处理因素组之间差异有统计学意义(P<0.05),SiRNA干扰组与封闭肽组之间差异无统计学意义(P>0.05)。结论:抑制THP-1细胞中SHP-2的表达后,THP-1细胞分泌的IFN-β明显增多,导致它对SiHa细胞的杀伤作用明显增强。SHP-2在宫颈癌的发生中起着重要作用,它发挥作用的机制是通过抑制IFN-β的信号转导,减少I型干扰素IFN-β的生成,使宫颈癌发生成为可能。

Effects of interference of SHP-2 in monocytes on cervical cancer cells

Objective:To investigate whether SHP-2 negatively regulated IFN-β signaling pathway after HPV infection,eventually leading to cervical cancer. Methods:THP-1 cells was co-cultured with SiHa cells.After THP-1 cells were transiently transfected with small interfering RNAs(siRNA) against human SHP-2,gene silencing was monitored at the protein levels by immunoblotting using anti-SHP-2 antibodies.The SHP-2 blocking peptide at different concentrations(0,0.5,2.5 μg / ml) and time(2 h and 24 h),acted on the THP-1 cells.Western blotting was carried out to detect the expression of SHP-2 to determine the closed best time and concentration.THP-1 cells with normal and different treatment factors were collected,SHP-2 protein expression in THP-1 cells was detected by Western blotting.THP-1 cells and the THP-1 cells treated by different factors were cultured with SiHa cells individually,the results were measured in the selected points of time and the concentration.Cell culture supernatants were collected from normal,SHP-2 blocking peptide treated and SHP-2 siRNA treated THP-1 cells.Cell culture supernatants were examined. Results:SHP-2 protein expressions were decreased after SHP-2 siRNA,the effect was best when the interference time was 48 h and concentration was 200 pmol.SHP-2 protein expressions were decreased after blocking peptide,the effect was best when the blocking time was 24 h and concentration was 2.5 μg/ml.OD result in replacement of the THP-1 cell culture medium was higher than that of original culture medium,the difference was statistically significant.From time to look at the two cells,THP-1 cells killing effect on SiHa cells were more and more enhanced from the 24 hours.The interaction for THP-1 cells of siRNA interfering on SiHa cells was strongest,THP-1 cells of blocking peptide was stronger,and nothing done was weakest.There was statistically significant difference in the expression of IFN-β between siRNA interference group and non-treatment factor group(q=3.981,P<0.05) as well as between blocking peptide group and non-treatment factor group(q=3.333,P<0.05).The difference between siRNA interference group and blocking peptide group was not statistically significant(q=0.648,P>0.05). Conclusion:After inhibition expression of SHP-2 in THP-1 cells,IFN-β secretion by THP-1 cells significantly is increased,which leads to its increasing killing effect on SiHa cells.SHP-2 negatively regulated IFN-β signaling pathway after HPV infection,inhibition of IFN-β production,eventually lead to cervical cancer.