长链非编码RNA COL11A1-208对口腔鳞癌细胞增殖及侵袭的影响

Effect of long non-coding RNA COL11A1-208 on proliferation and invasion in oral squamous cell carcinoma cells

目的观察长链非编码RNA(lncRNA)COL11A1-208在口腔鳞状细胞癌(OSCC)细胞中的表达和定位,及其对OSCC细胞增殖和侵袭的影响。方法采用实时荧光定量PCR(qPCR)检测COL11A1-208在CAL27、HN4、HN6等OSCC细胞系中的表达情况;通过RNA核质分离和荧光原位杂交(FISH)实验检测COL11A1-208在OSCC细胞中的定位。采用CAL27、HN4细胞建立COL11A1-208敲减组(SS-COL11A1-208组)和敲减对照组(SS-NC组);采用HN4、HN6细胞建立COL11A1-208过表达组(LV-COL11A1-208组)和过表达对照组(LV-NC组)。采用CCK-8实验、平板克隆形成实验以及Transwell实验检测COL11A1-208表达改变对各组细胞增殖活性、克隆形成能力、迁移和侵袭能力的影响。qPCR和Western blotting检测COL11A1-208表达改变对COL11A1基因及上皮-间充质转化相关蛋白(上皮钙黏素、波形蛋白)表达的影响。采用裸鼠皮下移植瘤模型观察COL11A1-208表达对OSCC细胞体内增殖能力的影响。结果qPCR结果显示,COL11A1-208在7个OSCC细胞系的相对表达量均明显高于正常对照细胞(P<0.05)。核质分离结果显示,CAL27、HN4、HN6细胞内COL11A1-208的胞核占比明显高于胞质占比(P<0.01);FISH实验结果显示,COL11A1-208主要定位于细胞核。与SS-NC组比较,SS-COL11A1-208组COL11A1-208相对表达量明显降低(CAL27:0.225±0.030 vs.1.000±0.000;HN4:0.393±0.028 vs.1.000±0.000;P<0.01);细胞增殖活性减弱(P<0.05),细胞克隆形成能力降低(P<0.0001),细胞穿膜数减少(P<0.05)。与LV-NC组比较,LV-COL11A1-208组COL11A1-208相对表达量明显升高(HN6:6524.216±3395.926 vs.1.000±0.000;HN4:3486.230±743.908 vs.1.000±0.000;P<0.05),细胞增殖活性增强(P<0.05),细胞克隆形成能力增高(P<0.01),细胞穿膜数增多(P<0.0001)。SS-COL11A1-208组COL11A1蛋白相对表达量明显低于SS-NC组(P<0.05),而LV-COL11A1-208组则明显高于LV-NC组(P<0.01)。另外,与相应对照组比较,SS-COL11A1-208组上皮钙黏素表达增高(P<0.05),波形蛋白表达降低(P<0.05);LV-COL11A1-208组上皮钙黏素表达降低(P<0.05),波形蛋白表达增高(P<0.01)。裸鼠皮下移植瘤模型实验显示,与相应对照组比较,ASO-COL11A1-208组皮下移植瘤重量和体积均减小(P<0.05),LV-COL11A1-208组皮下移植瘤重量和体积均增大(P<0.05)。结论COL11A1-208可促进OSCC细胞的增殖和侵袭能力,在OSCC发生与进展中发挥重要作用。

Objective To investigate the expression and subcellular localization of long non-coding RNA(lncRNA)COL11A1-208,as well as the effect on the proliferation and invasion of oral squamous cell carcinoma(OSCC)cells.Methods The differential expression of COL11A1-208 in seven OSCC cell lines compared with primary cultured normal oral epithelial cells was detected by quantitative real-time PCR(qPCR),and the subcellular localization of COL11A1-208 was evaluated by cell nuclear/cytoplasmic fractionation and fluorescence in situ hybridization(FISH).CAL27 and HN4 cells were respectively transfected with COL11A1-208 Smart Silencer(SS-COL11A1-208 group)and negative control(NC)Smart Silencer(SS-NC group),while HN4 and HN6 cells were stably infected by COL11A1-208 lentivirus overexpression vector(LV-COL11A1-208 group)and NC lentivirus overexpression vector(LV-NC group).Cell proliferation in each group was detected by CCK-8 assay and plate colony formation assay,while cell migration and invasion ability in each group were detected by Transwell assays.Then,the expression of COL11A1 were detected by qPCR and Western blotting assays,while the protein expression of E-cadherin and vimentin was detected by Western blotting assay.Finally,the effect of COL11A1-208 on OSCC cells in vivo was studied by xenograft formation assay.Results COL11A1-208 was highly expressed in OSCC cell lines compared with the normal cells(P<0.05).The results of nuclear and cytoplasmic RNA isolation assay showed that the nuclear proportion of COL11A1-208 in CAL27,HN4 and HN6 cells was significantly higher than that in cytoplasm(P<0.01);similarly,FISH results showed that COL11A1-208 was primarily localized within the nucleus of OSCC cells.The relative expression of COL11A1-208 in SS-COL11A1-208 group was significantly lower than that in the SS-NC group(CAL27:0.225±0.030 vs.1.000±0.000;HN4:0.393±0.028 vs.1.000±0.000;P<0.01).Compared with the SS-NC group,proliferation activity,colonizing ability,migration and invasion abilities of SS-COL11A1-208 group decreased significantly(P<0.05).The relative expression of COL11A1-208 in LV-COL11A1-208 group was significantly higher than that in the LV-NC group(HN6:6524.216±3395.926 vs.1.000±0.000;HN4:3486.230±743.908 vs.1.000±0.000;P<0.05).Compared with the LV-NC group,proliferation activity,colonizing ability,migration and invasion abilities of LV-COL11A1-208 group were significantly increased(P<0.05).Compared with the SS-NC group,the relative expression of COL11A1 protein in SS-COL11A1-208 group decreased significantly(P<0.05),while the relative expression of COL11A1 protein in LV-COL11A1-208 group increased significantly than that in the LV-NC group(P<0.01).In addition,compared with the SS-NC group,the relative expression of E-cadherin in SS-COL11A1-208 group increased significantly(P<0.05),and the relative expression of vimentin decreased significantly(P<0.05).In LV-COL11A1-208 group,the relative expression of E-cadherin decreased significantly(P<0.05),and the relative expression of vimentin increased significantly(P<0.01).In vivo experiments showed that the xenograft tumor weight and volume of ASO-COL11A1-208 group were significantly reduced(P<0.05),while the xenograft weight and volume of LV-COL11A1-208 group were significantly enhanced(P<0.05).Conclusion COL11A1-208 could facilitate the cell proliferation and invasion of OSCC,which plays an important role in the development and progression of OSCCs.