LINC00961通过调控PI3K/Akt信号通路对甲状腺癌细胞增殖、迁移和自噬的影响

Effect of LINC00961 on thyroid cancer cell proliferation, migration and autophagy through regulation of PI3K/Akt signaling pathway

【目的】探讨LINC00961是否通过调控PI3K/Akt通路影响甲状腺癌细胞自噬。【方法】体外培养甲状腺癌细胞系(TPC-1,SW579,BCPAP细胞)和正常甲状腺滤泡上皮细胞系(Nthy-ori-3-1甲状腺癌细胞系),实时荧光定量PCR(q RT-PCR)检测LINC00961在细胞中的相对表达;用LINC00961特异性过表达质粒pc DNA3.1-LINC00961和小干扰RNA(si RNA)瞬时转染TPC-1细胞;将TPC-1细胞分为4组:pc DNA3.1-961组(转染pc DNA3.1-961)、si-961组(转染si RNA-961)、pc DNA3.1组(转染pc DNA3.1)和si-NC组(转染si-NC)。用CCK-8检测细胞增殖;免疫荧光共聚焦显微镜观察LC3荧光染色的表达;Western blot检测自噬标志物LC3、p62和PI3K/Akt途径相关蛋白PI3K、p-PI3K、Akt和p-Akt的表达。【结果】LINC00961在3株甲状腺癌细胞中的相对表达显著降低(P<0.05),选择TPC-1细胞为实验细胞株;与pc DNA3.1组相比,pc DNA3.1-961组细胞中LINC00961相对表达、LC3荧光染色斑点数、LC3-II/LC3-I比值显著升高(P<0.05),细胞活力、p62蛋白表达、p-PI3K/PI3K、p-Akt/Akt的相对蛋白表达显著降低(P<0.05);与si-NC组相比,si-961组细胞中LINC00961相对表达、LC3荧光染色斑点数、LC3-II/LC3-I比值显著降低(P<0.05),细胞活力、p62蛋白表达、p-PI3K/PI3K、p-Akt/Akt的相对蛋白表达显著增加(P<0.05)。【结论】LINC00961可能通过抑制PI3K/Akt通路抑制甲状腺癌细胞活性并诱导其自噬。

【Objective】To explore whether LINC00961 affects autophagy in thyroid cancer cells by regulating PI3 K/Akt pathway.【Methods】Thyroid cancer cell line(TPC-1,SW579,BCPAP cells)and normal thyroid follicular epithelial cell line(Nthy-ori-3-1 thyroid cancer cell line)were cultured in vitro.The relative expression of LINC00961 was detected by real-time quantitative PCR(q RTPCR).TPC-1 cells were transiently transfected with LINC00961 overexpression plasmid pc DNA3.1-LINC00961 and small interfering RNA(si RNA).TPC-1 cells were divided into four groups:pc DNA3.1-961 group(transfected with pc DNA3.1-961),si-961 group(transfected with si RNA-961),pc DNA3.1(transfected with pc DNA3.1)and si-NC group(transfected with si-NC).CCK-8 was used to detect cell proliferation.Immunofluorescence confocal microscope was used to observe the expression of LC3 fluorescent staining.Western blot was used to detect the expression of autophagy markers LC3,p62 and PI3 K/Akt pathway related proteins PI3 K,p-PI3 K,Akt and p-Akt.【Results】The relative expression of LINC00961 in three thyroid cancer cells was significantly decreased(P<0.05).TPC-1 cells were selected as experimental cell lines.Compared with pc DNA3.1 group,the relative expression of LINC00961,the number of LC3 fluorescent spots and LC3-II/LC3-I ratio in pc DNA3.1-961 group were significantly increased(P<0.05),andcell viability,p62 protein expression,relative protein expression of p-PI3 K/PI3 K and p-Akt/Akt were significantly decreased(P<0.05).Compared with the si-NC group,the relative expression of LINC00961,the number of LC3 fluorescent spots and LC3-II/LC3-I ratio in the si-961 group were significantly decreased(P<0.05),while cell viability,p62 protein expression,relative protein expression of p-PI3 K/PI3 K and pAkt/Akt were significantly increased(P<0.05).【Conclusion】LINC00961 may inhibit the activity of thyroid cancer cells and induce autophagy by inhibiting PI3 K/Akt pathway.