宫颈癌细胞中HPVE6调控穹窿体RNA2-1-5p的表达

Regulation of HPV E6 on VTRNA2-1-5p expression in cervical cancer cells

目的 HPV E6/E7是宫颈癌发病过程中起主要作用的2种蛋白,文中旨在研究宫颈癌细胞中高表达的穹窿体RNA2-1-5p与HPV E6/E7的相关性。方法实验分为空白对照组、对照组(空载体/阴性对照siRNA)以及实验组(过表达质粒/敲减siRNA),在Si Ha和He La细胞中分别敲减HPV E6/E7,在Ha Ca T细胞中过表达HPV E6,用Western blot检测HPV E6/E7的表达水平,用Real-time PCR检测VTRNA2-1-5p及其前体VTRNA2-1的表达水平。结果在Si Ha和He La细胞,敲减HPV E6后实验组中VTRNA2-1表达[(0.65±0.02)、(0.81±0.11)]较对照组[(1.18±0.10)、(1.07±0.13)]降低(P<0.05),VTRNA2-1-5p表达[(1.54±0.22)、(1.98±0.06)]较对照组[(0.83±0.03)、(1.02±0.06)]升高(P<0.01);敲减HPV E7后VTRNA2-1和VTRNA2-1-5p表达的差异均无统计学意义(P>0.05)。在Ha Ca T细胞,过表达HPV E6后实验组中VTRNA2-1表达较对照组升高[(1.35±0.46)vs(0.98±0.35),P<0.05],VTRNA2-1-5p表达较对照组降低[(1.33±0.21)vs(1.80±0.27),P<0.01]。结论在宫颈癌细胞中,HPV E6可负性调控VTRNA2-1-5p的表达,而HPV E7与VTRNA2-1-5p无作用关系。

Objective HPV E6 and E7 play the main role in the incidence of cervical cancer. This paper aimed to study the correlation between high-expression vault RNA2-1-5p and HPV E6/E7 in cervical cancer cells. Methods HPV E6/E7-siRNA were transfected into SiHa and HeLa cells by lipofectamine 2000, the protein expressions of HPV E6/E7 were detected by Western blot, and the VTRNA2-1 and VTRNA2-1-5p levels were detected by Real-time PCR. The plasmid of HPV E6 were transfected into HaCaT ceils by lipofectamine 2000, the protein expression of HPV E6 were detected by western blot, the VTRNA2-1 and VTRNA2-1-5p levels were detected by Real-time PCR. Results In SiHa cells, the protein levels of HPV16 E6 in E6-siRNA group （0.35 ± 0. 04 ） were significantly decreased compared to the NC group （0.64 ± 0.01 ）. Down-regulation of HPV16 E6 expression decreased the levels of VTRNA2- 1 and increased the levels of VTRNA2-1-Sp （VTRNA2-1 vs NC group, P 〈0.05, n = 3; VTRNA2-1-Sp vs NC group, P 〈0.05, n = 3）. The protein levels of HPV16 E7 in E7-siRNA group （0.72 ± 0.02 ） were significantly decreased compared to the NC group （1.05 ±0.01 ）. Down-regulation of HPV16 E7 expression has no effect on the levels of VTRNA2-1 and VTRNA2-1-5p. In HeLa cells, the protein levels of HPV18 E6 in E6-siRNA group （0.27 ± 0.06） were significantly decreased compared to the NC group （0.57 ± 0.05）. Down-regulation of HPV18 E6 expression decreased the levels of VTRNA2-1 and increased the levels of VTRNA2-1-Sp （VTRNA2-1 vs NC group, P 〈0.05, n =3; VTRNA2-1-Sp vs NC group, P〈0.001, n =3）. The protein levels of HPV18 E7 in E7-siRNA group （0.58 ± 0.02 ） were significantly decreased compared to the NC group （0.97 ± 0.01 ）. Down-regulation of HPV18 E7 expression has no effect on the levels of VTRNA2-1 and VTRNA2-1-5p. In HaCaT cells, the protein levels of HPV16 E6 in pNCMV16e6 group （5.65 ± 0.90） were significantly increased compared to the NC group（ 1.07 ± 0.02）. Up-regulation of HPV16 E6 expression in- creased the levels of VTRNA2-1 and decreased the levels of VTRNA2-1-Sp （ VTRNA2-1 vs NC group, P 〈 0.05, n = 3 ; VTRNA2-1-Sp vs NC group, P 〈 0.01, n = 3）. Conclusion The expression levels of VTRNA2-1-Sp are negatively correlated with HPV E6 and in non-correlation with HPV E7 in cervical cancer cells.