假基因POU5F1B对宫颈癌SiHa细胞增殖、迁移和侵袭能力的影响

Effects of pseudogene POU5F1B on proliferation, invasion and migration of cervical cancer cell line SiHa

目的探讨POU结构域5类转录因子1B基因(POU5F1B)对宫颈癌细胞增殖、迁移、侵袭的影响及其可能的机制。方法采用定时荧光定量PCR(QPCR)检测正常宫颈上皮End1/e6e7细胞株和宫颈癌细胞株(SiHa、HeLa和C33A)中POU5F1B的表达水平,选取POU5F1B表达量最高的细胞株分别转染POU5F1B特异性小干扰RNA(si-POU5F1B组)和阴性对照序列(si-NC组)。分别采用CCK-8实验、平板克隆形成实验和EdU实验观察细胞增殖情况,细胞划痕实验和侵袭实验观察两组细胞的迁移和侵袭情况,Western blotting实验检测OCT4蛋白的相对表达量,通过动物实验观察下调假基因POU5F1B对裸鼠体内移植瘤质量的影响。结果与End1/e6e7细胞株比较,假基因POU5F1B在宫颈癌SiHa、C33A、HeLa细胞株中的相对表达量分别为3.34±0.23、2.03±0.15和1.13±0.06,选取POU5F1B表达量最高的SiHa细胞进行后续实验。si-POU5F1B组假基因POU5F1B的相对表达量为0.2±0.012,明显低于si-NC组(P<0.01)。CCK-8实验显示,与si-NC组相比,si-POU5F1B组吸光值明显降低(P<0.05);平板克隆形成实验显示,si-POU5F1B组的克隆形成数为(173±15.27)个,低于si-NC组的(318±8.41)个(P<0.01)。EdU检测显示,si-POU5F1B组阳性细胞比例为(18±1.36)%,低于si-NC组的(34±2.63)%(P<0.01)。划痕实验显示,48 h后si-POU5F1B组的划痕愈合率为(45.60±2.27)%,低于si-NC组的(87.15±3.32)%(P<0.01);侵袭实验显示,si-POU5F1B组穿过侵袭下室的细胞数为(303±10.29)个,低于si-NC组的(933±11.88)个(P<0.01);Western blotting检测显示,下调POU5F1B表达后,其亲本基因OCT4的表达水平下降(P<0.05);在动物实验中,si-POU5F1B组裸鼠移植瘤质量为(203.50±54.83)mg,低于si-NC组的(578.06±84.66)mg(P<0.01)。结论下调假基因POU5F1B可抑制宫颈癌细胞的增殖、迁移和侵袭能力,并抑制体内宫颈癌细胞生长,其机制可能与下调其功能基因OCT4的表达有关。

Objective To explore the expression level of POU domain class 5 transcription factor 1B genes(POU5F1B)in cervical cancer cells,and its effect on the proliferation,migration and invasion of cervical cancer cells lines and the possible mechanism.Methods The relative expression of POU5F1B in SiHa,HeLa and C33A cervical cancer cell lines,and normal cer?vical epithelial cells(End1/e6e7)was detected by QPCR.The specific small interfering RNA for POU5F1B(si?POU5F1B group)or negative control sequences(si?NC group)were transfected into the cell lines with highest expression of POU5F1B.The CCK?8 assay,clone formation experiment and EdU assay were respectively employed to detect cell proliferation of the two groups.Cell scratch assay were employed to detect cell migration of the two groups.Matrigel invasion assay was used to observe cell inva?sion of the two groups.Western blotting analysis was performed to detect the effect of down?regulated pseudogene POU5F1B on the expression of functional gene OCT4 protein.The effect of down?regulated pseudogene POU5F1B on the xenograft weight in nude mice was observed by animal experiments.Results In comparison with End1/e6e7 cells,the relative expression of POU5F1B in SiHa,C33A and HeLa cell lines was 3.34±0.23,2.03±0.15 and 1.13±0.06.SiHa cell line was chosen for the following experiments.The relative expression of POU5F1B in si?POU5F1B group was 0.2±0.012,lower than that in si?NC group(P<0.01).CCK?8 assay showed that the absorption value was lower in si?POU5F1B group compared with si?NC group(P<0.05).EdU experiment showed that compared with si?NC group,the ratio of positive cells in si?POU5F1B group was(18±1.36)%,lower than(34±2.63)%of si?NC group(P<0.01).The colony formation experiment showed that the number of col?onies in si?POU5F1B group was 173±15.27,lower than 318±8??41 in si?NC group(P<0.01).Cell scratch experiment showed that the healing rate of si?POU5F1B group after 48 h was(45.60±2.27)%,lower than(87.15±3.32)%of si?NC group(P<0.01).Matrigel invasion assay showed that the number of cells in the invasion chamber of si?POU5F1B group was 303±10.29,lower than 933±11.88 in si?NC group(P<0.01).Western blotting analysis showed that the expression level of OCT4 decreased after the down?regulation of POU5F1B(P<0??05).In animal experiments,the xenograft weight of si?POU5F1B group was(203.50±54.83)mg,lower than(578.06±84.66)mg of si?NC group(P<0.01).Conclusion The down?regulated pseud?ogene POU5F1B can inhibit the proliferation,migration and invasion of cervical cancer cells and inhibit the growth of cervical cancer cells,which may be related to the down?regulation of its functional gene OCT4.