调控SOX2对HPV-16阳性宫颈癌细胞增殖转移的影响

Effects of SOX2 Gene on the Proliferation and Transfer of HPV-16 Positive Cervical Cancer Cell Lines

目的探讨SOX2基因对HPV-16阳性宫颈癌细胞株增殖转移的影响。方法培养Caski-EGFP细胞后,采用空白腺病毒、SOX2基因沉默的腺病毒载体、SOX2基因过表达的腺病毒载体转染Caski-EGFP细胞并将细胞依次分为C、S、E组。Western blot法及实时荧光定量PCR(RT-PCR)检测SOX2及mRNA水平,检测细胞是否构建成功;CCK-8细胞增殖实验检测各组癌细胞的光密度值,绘制细胞活力曲线,分析SOX2基因对宫颈癌细胞株增殖活力的影响;Transwell侵袭试验分析各组癌细胞侵袭抑制率的改变,探讨SOX2基因对宫颈癌细胞株侵袭转移能力的影响。结果 RT-PCR、Western blot法检测,结果显示mRNA、SOX2蛋白表达量分别为E组>C组>S组,差异有统计学意义(P<0.05),提示构建的细胞构建成功可进行下一步实验。CCK-8细胞增殖实验结果显示,不同时间下,C组、S组、E组吸光度值、细胞活力均呈上升趋势,3组都在72h达到高峰,差异有统计学意义(P<0.01);比较各时间点下3组癌细胞的吸光度值及细胞活力差异,癌细胞培养后第0、24、48及第72h,E组相较于对照组C组而言,癌细胞的吸光度值及细胞活力均显著升高,差异有统计学意义(P<0.01,F_(C-E组间)=3.764,P_(C-E组间)=0.001),S组相较于对照组C组而言,癌细胞的吸光度值及细胞活力均有所降低,差异有统计学意义(P<0.01)。E组相较于对照组S组而言,癌细胞的吸光度值及细胞活力均显著降低,差异有统计学意义(P<0.01,F_(组间)=4.286,P_(组间)=0.000);不同时间点与分组之间存在交互作用(F_(交互)=3.784,P_(交互)=0.001);Transwell小室实验结果显示,以C组实验对照组的细胞侵袭能力设定为100%,则E组的相对侵袭抑制率为146%;S组细胞的相对侵袭抑制率为75%。对比可知,E组的细胞侵袭能力高于实验对照组C组,差异有统计学意义(P<0.01);S组低于实验对照组C组,差异有统计学意义(P<0.05);E组细胞侵袭能力明显高于S组,差异有统计学意义(P<0.01)。结论本研究进一步探讨了SOX2基因对高危型HPV感染(HPV-16)的宫颈癌细胞株增殖能力及侵袭转移能力方面的促进作用,抑制SOX2的表达有望成为一种早期诊断治疗宫颈癌的新途径。

Objective To explore the effect of SOX2 gene on the proliferation and transfer of HPV - 16 positive cervical carcinoma cell lines. Methods After the cultivation of Caski-EGFP cells, the Caski-EGFP cells were transfected with a blank adenovirus, a silent adenovirus vector of SOX2 gene, and the adenovirus vector of SOX2 gene expression, then the cells were divided into C, S and E groups. Protein SOX2 and mRNA levels was measured by Western blot and Real-time PCR （RT-PCR） to detect whether cells were constructed successfully. CCK-8 cell proliferation experiment was used to detect the optical density value of cancer cells in each group.Then we drew the cell vitality curve, and analyzed the effect of SOX2 genes on the proliferation activity of cervical cancer cell lines.Transwell invasion test was conducted to analyze the changes in the inhibitory rate of cancer cells in each group and explore the effect of SOX2 genes on the invasion and metastasis of cervical cancer cell lines. Results RT-PCR and Western blot test results showed：group E group〉C group〉S group, and the difference was statistically significant, so the construction of the cell constructed was successful for the next experiment.CCK 8 cell proliferation experiment results showed that under different time, in group C, S, E group, optical density value, the cell vitality were on the rise, reached a peak in 72h in all three groups, and the difference was statistically significant （P〈0.01） .Compared among three groups, at each time point optical density value of cancer cells and the cell vitality, cancer cells developed after 0, 24, 48 and 72h.Compared with the control group, optical density value of cancer cells and cell viability in group C were significantly higher, and the difference was statistically significant （ P 〈0.01）. Compared with control group,optical density value of cancer cells and cell vitality in C group are reduced, and the difference was statistically significant （P 〈0.01） （ F C-S =5.389, P C-S =0.000）. Compared with the control group, the optical density and cell vitality of cancer cells in E group, decreased significantly and the difference was statistically significant （P 〈0.01） （4.286 F E-S , P E-S =0.000）.Interaction was found between different time points and groups （F interaction =3.784, P interaction =0.001）.The experimental results of the Transwell chamber showed that the control rate of the cells in C group was 100%, and the relative invasion inhibition rate of group E was 146%.The relative invasive inhibition rate of group S cells was 75%.In comparison, the cell invasion ability of group E was higher than that in the control group C group, and the difference was statistically significant （P〈0.01） .S group was lower than the control group C group, and the difference was statistically significant （P〈0.05） .The invasion ability of E group was significantly higher than that of S group, and the difference was statistically significant （P〈0.01） . Conclusion This study further explored the effect of SOX2 gene on the proliferation and invasion of cervical cancer cell lines. Inhibiting the expression of SOX2 is expected to be a new approach to early diagnosis of cervical cancer.