沉默Cat D对骨肉瘤细胞增殖、凋亡和侵袭的影响及相关信号通路

Cat D silencing regulates osteosarcoma cell proliferation,apoptosis,and invasion through Akt/mTOR signaling pathway

研究组织蛋白酶D(cathepsin D,Cat D)在骨肉瘤细胞中的表达,并通过shRNA技术沉默Cat D,探讨该分子对骨肉瘤细胞MG63和U2OS增殖、凋亡及侵袭的作用和机制。构建sh-Cat D质粒及阴性对照质粒sh-NC并转染至骨肉瘤细胞MG63和U2OS中,RT-PCR和Western blotting检测Cat D在骨肉瘤细胞中的表达;CCK-8法、克隆形成实验和EdU荧光染色实验检测骨肉瘤细胞的增殖活性;Transwell实验检测肿瘤细胞的侵袭能力;FACS检测肿瘤细胞的凋亡情况;Western blotting检测细胞凋亡相关蛋白(Bax和Bcl-2)的表达及Akt/mTOR信号通路相关蛋白(Akt和mTOR)的磷酸化水平。结果显示,相较健康人成骨细胞hFOB,Cat D在骨肉瘤细胞中的表达显著升高,尤其是MG63和U2OS细胞中Cat D的高表达均显著(均P<0.001)。与sh-NC组相比,Cat D沉默显著抑制MG63和U2OS细胞的增殖和侵袭(均P<0.001)。与sh-NC组相比,Cat D沉默通过上调Bax表达和下调Bcl-2表达促进肿瘤细胞的凋亡(均P<0.001)。同时,Western blotting检测结果显示,与sh-NC组相比,Cat D沉默显著降低肿瘤细胞Akt和mTOR蛋白的磷酸化水平(均P<0.001)。此外,Akt/mTOR信号通路抑制剂(Wortmannin,渥曼青霉素)可增强Cat D沉默对肿瘤细胞凋亡和侵袭的影响,而Akt/mTOR通路激动剂(740 Y-P)可逆转上述影响(均P<0.001)。由此,Cat D在骨肉瘤细胞中高表达,沉默Cat D可以通过抑制Akt/mTOR信号通路的磷酸化水平上调骨肉瘤细胞的凋亡活性并抑制细胞生长,提示Cat D可能成为骨肉瘤的潜在治疗靶点。

This study aimed to investigate the expression of cathepsin D(Cat D)in osteosarcoma cells and to explore the effect and mechanism of Cat D on the proliferation,apoptosis and invasion of osteosarcoma cell lines MG63 and U2OS.Cat D was knocked down by short hairpin RNA(shRNA)-mediated gene silencing.To this end,the sh-Cat D and negative control(sh-NC)plasmids were constructed and transfected into osteosarcoma cell lines MG63 and U2OS.RT-PCR and Western blotting were used to detect the expression of Cat D in osteosarcoma cells.CCK-8 assay,clone formation assay,and EdU fluorescence staining assays were used to detect cell proliferation activity.Transwell assay was employed to evaluate cell invasion.FACS was used to assess cell apoptosis.Western blotting was used to measure the expression of cell apoptosis-related proteins(Bax and Bcl-2)and the phosphorylation levels of Akt/mTOR signaling pathway-related proteins(Akt and mTOR).Compared to that of the normal human osteoblastic hFOB cells,Cat D expression levels in osteosarcoma cells MG63 and U2OS were significantly higher(all P<0.001).Compared to sh-NC,Cat D silencing significantly inhibited the proliferation and invasion of MG63 and U2OS cells(all P<0.001),up-regulated the expression of Bax,down-regulated the expression of Bcl-2 thereby facilitated cell apoptosis(all P<0.001).Meanwhile,Cat D silencing significantly inhibited the phosphorylation of Akt and mTOR(all P<0.001).In addition,the impact of Cat D silencing on the apoptosis and invasion was enhanced by Akt/mTOR signaling pathway inhibitor(Wortmannin)and dampened by Akt/mTOR signaling pathway agonist(740 Y-P)(all P<0.001).In conclusion,Cat D was highly expressed in osteosarcoma cells.Cat D silencing could up-regulate the apoptotic activity and inhibit osteosarcoma cell growth by suppressing the phosphorylation level of Akt/mTOR signaling pathway,suggesting that Cat D may be a potential therapeutic target for osteosarcoma.