The objective of the present work was to investigate the effect of Periostin (POSTN) silencing on autophagy in osteoblasts, and provide an experimental basis for studying the mechanism of dental eruption. The cells were divided into the following four groups according to their viral number: the NC group, pFU-GW-016PSC53349-1; group KD1, LVpFU-GW-016PSC66471-1; group KD2, LVpFU-GW-016PSC66472-1; and group KD3, LVpFU-GW-016PSC66473-1. The lentiviral vector was infected at MOI = 100 in the ENi.S medium containing 5 g/mL Polybrene. The target gene expression was observed by a Celigo® Image Cytometer at 72 hours after infection, and the positive rate of fluorescence was noted. A two-step method of quantitative real-time PCR (qRT-PCR) was used to detect the silencing effect of POSTN. Western blotting was then performed to assess the expression of autophagy-related proteins Beclin-1 and LC3 in the group showing the best gene silencing effects. The experimental results showed that there was strong green fluorescence in group KD3. As confirmed via qRT-PCR analysis, the POSTN silencing efficiency in group KD3 reached 92.1%. The Western blotting revealed that the expression of Beclin-1 protein in group KD3 was significantly higher than that in the NC group. However, the LC3 protein expression was not significantly different from that of the control group. The lentiviral vector targeting POSTN in osteoblasts was constructed successfully. In addition, the expression of autophagy protein in mouse osteoblasts increased after POSTN silencing. This finding may provide new approaches for understanding the molecular signal transduction of POSTN during the tooth eruption process.

中文翻译：

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骨膜素沉默对成骨细胞自噬的影响。

本研究的目的是研究骨膜素（POSTN）沉默对成骨细胞自噬的影响，并为研究牙齿萌出的机理提供实验依据。根据其病毒数量将细胞分为以下四组：NC组，pFU-GW-016PSC53349-1； NC组，pFU-GW-016PSC53349-1。组KD1，LVpFU-GW-016PSC66471-1; 组KD2，LVpFU-GW-016PSC66472-1; 以及KD3组LVpFU-GW-016PSC66473-1。慢病毒载体在含有5 g / mL Polybrene的ENi.S培养基中以MOI = 100感染。感染后72小时，通过Celigo®Image Cytometer观察靶基因表达，并观察到荧光阳性率。实时荧光定量PCR（qRT-PCR）的两步法用于检测POSTN的沉默效果。然后进行蛋白质印迹以评估自噬相关蛋白Beclin-1和LC3在显示最佳基因沉默效果的组中的表达。实验结果表明，KD3组具有较强的绿色荧光。通过qRT-PCR分析证实，KD3组的POSTN沉默效率达到92.1％。Western blotting显示，KD3组Beclin-1蛋白的表达明显高于NC组。然而，LC3蛋白的表达与对照组没有显着差异。成功构建了针对成骨细胞中POSTN的慢病毒载体。此外，POSTN沉默后，小鼠成骨细胞中自噬蛋白的表达增加。