Ⅱ型多磷酸肌醇4-磷酸酶在甲状腺乳头状癌中的作用及其机制

The role of inositol polyphosphate 4-phosphatase typeⅡin papillary thyroid carcinoma and its mechanism

目的观察Ⅱ型多磷酸肌醇4-磷酸酶(INPP4B)在人甲状腺乳头状癌组织及其癌旁正常组织的表达,观察INPP4B表达对甲状腺乳头状癌细胞增殖、迁移和侵袭等生物学行为的影响,探讨其调节机制。方法采用定量反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测2019年1月至2019年12月期间30例甲状腺乳头状癌患者癌组织及其癌旁正常组织中INPP4B的表达水平。在甲状腺乳头状癌细胞系IHH4细胞中敲减INPP4B表达,采用系列体外试验检查INPP4B表达水平改变对IHH4细胞功能的影响。计量资料用两独立性t检验,并进行Mann-Whitney U检验非参数分析。结果RT-PCR结果显示,在30例甲状腺乳头状癌组织中,有22例INPP4B mRNA水平明显升高,癌组织中INPP4B的高表达率为73.3%(22/30),显著高于癌旁正常组织(3.3%,1/30)(P<0.01)。Western blot结果显示,在30例甲状腺乳头状癌组织中21例INPP4B蛋白呈上调表达,与RT-PCR检测结果符合率为95.5%。在IHH4细胞中敲减INPP4B表达可上调上皮型钙黏蛋白(E-cadherin)表达3.61倍(χ^2=33.800,P<0.05);下调磷脂酰肌醇3-激酶(PI3K)表达0.24倍(χ^2=14.300,P<0.05)、下调血清/糖皮质激素调节激酶3(SGK3)表达0.18倍(χ^2=19.400,P<0.05)、下调卷曲蛋白(Twist)表达0.31倍(χ^2=8.700,P<0.05)和下调波形蛋白(Vimentin)表达0.25倍(χ^2=15.800,P<0.05);但敲减INPP4B表达对磷酸化蛋白激酶B(p-Akt)表达影响很小,仅下调0.96倍(χ^2=0.000,P>0.05)。噻唑蓝(MTT)增殖试验表明,敲减INPP4B表达可显著抑制IHH4细胞的增殖能力。细胞克隆形成试验结果显示,与对照组[(138±11)个]比较,敲减INPP4B表达组[(76±9)个]的体外集落数量显著减少,差异有统计学意义(t=9.692,P<0.05)。刮痕愈合试验结果表明,敲减INPP4B表达组和对照组的IHH4细胞刮痕愈合率分别为(33.48±0.05)%和(93.29±0.07)%,差异有统计学意义(t=68.582,P<0.01)。Transwell试验结果显示,敲减INPP4B表达组和对照组的IHH4细胞迁移细胞数分别为8(4,16)个和129(94,186)个,差异有统计学意义(U=0,P<0.01)。结论INPP4B在甲状腺乳头状癌组织中呈高表达,提示其可能对甲状腺癌的发生发展有重要作用;体外细胞水平敲减INPP4B表达能够抑制癌细胞增殖、迁移和侵袭能力,提示INPP4B在甲状腺乳头状癌中起促癌作用。

Objective The expression of inositol polyphosphate 4-phosphatase typeⅡ(INPP4B)in human thyroid cancer tissues and their adjacent normal tissues was investigated at the tissue level.At the in vitro cell level,the effects of INPP4B on the biological behavior of papillary thyroid cancer cells such as proliferation,migration and invasion were observed,and the possible regulatory mechanisms were explored.Methods Quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting techniques were used to detect the expression of INPP4B in cancer tissues and adjacent normal tissues of 30 patients with papillary thyroid carcinoma.After knockdown expression of INPP4B in papillary thyroid carcinoma cell line IHH4 cells,the effect of INPP4B expression level on the cell function of IHH4 was examined by a series of in vitro experiments.Results The results of RT-PCR and western blot detection indicated that the twenty-two of thirty thyroid cancer tissue samples had high INPP4B mRNA level and high protein level(73.3%,22/30),which was significantly higher than that in adjacent normal tissues(3.3%,1/30)(P<0.01).The results of Western blot showed that the expression of INPP4B protein was up-regulated in 21 of 30 cases with thyroid carcinoma,which was 95.5%consistent with the RT-PCR results.After knockdown expression of INPP4B in IHH4 cells,E-cadherin protein was upregulated by 3.61 times(χ^2=33.800,P<0.05);phosphatidylinositol 3 kinase(PI3K)protein was downregulated by 0.24 times(χ^2=14.300,P<0.05),SGK3 protein was downregulated by 0.18 times(χ^2=19.400,P<0.05),Twist was downregulated by 0.31 times(χ^2=8.700,P<0.05)and Vimentin protein was downregulated by 0.25 times(χ^2=15.800,P<0.05);The knockdown expression of INPP4B has little effect on the expression of phosphorylated protein kinase B(p-Akt)protein,which was downregulated by 0.96 times(χ^2=0.000,P>0.05).Methyl thiazolyl tetrazolium(MTT)proliferation experiments showed that the knockdown expression of INPP4B could significantly inhibit the proliferation ability of IHH4 cells.The results of cell clone formation experiments showed that compared with the control group[(138±11)],the number of colonies in vitro in the knockdown expression of INPP4B group[(76±9)]decreased significantly(t=9.692,P<0.05).The scratch healing experiments showed that,the healing rates of IHH4 cells in the knockdown expression of INPP4B group and control group were(33.48±0.05)%and(93.29±0.07)%,respectively.Differences were statistically significant(t=68.582,P<0.01).Transwell experiments results showed,the number of IHH4 cell migration cells in the knockdown expression of INPP4B group and the control group was 8(4,16)cells and 129(94,186)cells,differences were statistically significant(U=0,P<0.01).Conclusion INPP4B is highly expressed in papillary thyroid carcinoma tissues,suggesting that it may play an important role in the tumorigenesis and development of thyroid cancer;the knockdown expression of INPP4B at cell level in vitro can inhibit the proliferation,migration and invasion of cancer cells,suggesting that INPP4B plays a role in promoting progression in papillary thyroid carcinoma.