Atg4B基因调控自噬在高危型HPV致宫颈癌中的作用和机制研究

Study on the role and mechanism of Atg4B gene regulating autophagy in cervical cancer induced by high-risk HPV

目的研究自噬相关蛋白4同源物B(Atg4B)基因调控自噬在高危型人乳头瘤病毒(HPV)致宫颈癌中的作用和机制。方法培养宫颈癌C33a细胞,构建pcDNA3.1-Atg4B质粒并转染C33a细胞。采用免疫印迹实验检测Atg4B蛋白的表达水平,采用^(3)H-胸腺嘧啶核苷酸渗入法检测C33a细胞增殖数目,采用Transwell实验检测细胞迁移数目,采用透射电镜观察自噬小体的形成数目。结果HR-HPV E6组C33a细胞增殖数目、Atg4B蛋白表达水平及自噬小体数目分别为(5208.12±859.33)cpm/孔、(0.62±0.13)及(7.67±0.58)个/视野,HR-HPV E6 mut组C33a细胞增殖数目、Atg4B蛋白表达水平及自噬小体数目分别为(2095.41±194.67)cpm/孔、(0.14±0.06)及(0.67±0.58)个/视野,Mock组C33a细胞增殖数目、Atg4B蛋白表达水平及自噬小体数目分别为(3496.89±568.14)cpm/孔、(0.32±0.08)及(3.67±0.58)个/视野,与mock组比较,HR-HPV E6组C33a细胞增殖数目、Atg4B蛋白表达水平及自噬小体数目显著增加(t=2.878,3.320,8.485,均P<0.05),HR-HPV E6 mut组C33a细胞增殖数目、Atg4B蛋白表达水平及自噬小体数目显著减少(t=4.040,3.118,6.364,均P<0.05)。pcDNA3.1 Atg4B组C33a细胞增殖数目和自噬小体数目分别为(4917.23±425.16)cpm/孔和(5.67±0.58)个/视野,pcDNA3.1空载体组C33a细胞增殖数目和自噬小体数目分别为(1989.76±243.15)cpm/孔和(1.67±0.57)个/视野,两组比较差异均有统计学意义(t=10.352,8.485,均P<0.05)。HR-HPV E6+BafA1组C33a细胞增殖、迁移及自噬小体数目分别为(1667.22±203.76)cpm/孔、(189.15±20.21)个及(2.67±0.58)个/视野,HR-HPV E6+DMSO组C33a细胞增殖、迁移及自噬小体数目分别为(4583.81±592.24)cpm/孔、(467.73±58.63)个及(7.33±0.58)个/视野,两组比较差异均有统计学意义(t=8.066,7.752,9.899,均P<0.05)。结论Atg4B基因调控细胞自噬小体的形成,进而促进宫颈癌细胞的增殖、迁移,在HR-HPV致宫颈癌发生、发展中发挥重要作用。

Objective To research the role and mechanism of autophagy related protein 4 homology B(Atg4 B)gene regulating autophagy in cervical cancer induced by high-risk human papillomavirus(HPV).Methods Cervical cancer C33 a cells were cultured,pcDNA3.1-Atg4 B plasmid was constructed and transfected into C33 a cells.Western blotting was used to detect the expression levels of Atg4 B protein,;H-thymine nucleotide infiltration method was used to detect the proliferation number of C33 a cells,Transwell assay was used to detect cell migration number,transmission electron microscope was used to observe the number of autophagosome.Results The proliferation number of C33 a cells,the expression level of Atg4 B protein,and the number of autophagosome in HR-HPV E6 group were(5208.12±859.33)cpm/hole,(0.62±0.13),and(7.67±0.58)per view,respectively;the proliferation number of C33 a cells,the expression level of Atg4 B protein,and the number of autophagosome in HR-HPV E6 mut group were(2095.41±194.67)cpm/hole,(0.14±0.06),and(0.67±0.58)per view,respectively;the proliferation number of C33 a cells,the expression level of Atg4 B protein,and the number of autophagosome in Mock group were(3496.89±568.14)cpm/hole,(0.32±0.08),and(3.67±0.58)per view,respectively.Compared with mock group,the proliferation number of C33 a cells,the expression level of Atg4 B protein,and the number of autophagosome in HR-HPV E6 group increased significantly(t=2.878,3.320,8.485,all P<0.05),the proliferation number of C33 a cells,the expression level of Atg4 B protein,and the number of autophagosome in HR-HPV E6 mut group decreased significantly(t=4.040,3.118,6.364,all P<0.05).The proliferation number of C33 a cells and the number of autophagosome in pcDNA3.1 Atg4 B group were(4917.23±425.16)cpm/hole and(5.67±0.58)per view,respectively;the proliferation number of C33 a cells and the number of autophagosome in pcDNA3.1 empty vector group were(1989.76±243.15)cpm/hole and(1.67±0.57)per view,respectively,there were statistically significant differences between the two groups(t=10.352,8.485,both P<0.05).The proliferation number of C33 a cells,the migration rate of C33 a cells,and the number of autophagosome in HR-HPV E6+BafA1 group were(1667.22±203.76)cpm/hole,(189.15±20.21),and(2.67±0.58)per view,respectively;the proliferation number of C33 a cells,the migration rate of C33 a cells,and the number of autophagosome in HR-HPV E6+DMSO group were(4583.81±592.24)cpm/hole,(467.73±58.63),and(7.33±0.58)per view,respectively,there were statistically significant differences between the two groups(t=8.066,7.752,9.899,all P<0.05).Conclusion Atg4 B gene regulates the formation of autophagy,promotes the proliferation and migration of cervical cancer cells,which plays an important role in occurrence and development of cervical cancer caused by HR-HPV.