Alkannin restrains oral squamous carcinoma cell growth, migration and invasion by regulating microRNA-9/RECK axis

Alkannin (ALK) has anti-inflammatory and anti-tumour activities. We tried to probe the underlying functions of ALK in oral squamous carcinoma (OSCC) cells growth, migration and invasion. OSCC cells viability was investigated after treatment with ALK. Then, BrdU, flow cytometry, Western blot and Transwell assays were executed to appraise proliferation, apoptosis, migration and invasion in OSCC cells with ALK stimulation. The biological functions of miR-9 were explored after miR-9 mimic/inhibitor transfection. The relevance of RECK and miR-9 was predicated by dual luciferase activity assay. JAK1/STAT3 and PI3K/AKT pathways were estimated by Western blot. Tumour formation in vivo was executed by xenograft tumours assay. We found that ALK restrained cell proliferation, facilitated apoptosis, repressed migration and invasion also interdicted JAK1/STAT3 and PI3K/AKT pathways in CAL-27 and SCC-9 cells. miR-9 expression was upgraded in OSCC tissues but decreased in OSCC cells along with ALK administration; meanwhile, overexpressed miR-9 inverted the influences of ALK in OSCC cells growth, migration and invasion. RECK was predicated as a novel target gene of miR-9, and overexpressed RECK hindered JAK1/STAT3 and PI3K/AKT pathways in OSCC cells. ALK prohibited tumour formation in vivo. In conclusion, ALK restrained OSCC cells growth, migration and invasion via adjusting miR-9/RECK axis.Highlights
ALK restrains cell growth, migration and invasion in OSCC cells;
miR-9 is enhanced in OSCC tissues but is repressed by ALK in OSCC cells;
miR-9 inverts the repressive effect of ALK on CAL-27 and SCC-9 cells;
RECK is a novel target of miR-9;
ALK or RECK hinders JAK1/STAT3 and PI3K/AKT pathways in CAL-27 and SCC-9 cells;
ALK prohibits tumour formation in vivo.

ALK restrains cell growth, migration and invasion in OSCC cells;
miR-9 is enhanced in OSCC tissues but is repressed by ALK in OSCC cells;
miR-9 inverts the repressive effect of ALK on CAL-27 and SCC-9 cells;
RECK is a novel target of miR-9;
ALK or RECK hinders JAK1/STAT3 and PI3K/AKT pathways in CAL-27 and SCC-9 cells;
ALK prohibits tumour formation in vivo.

紫草素（Alkannin, ALK）具有抗炎与抗肿瘤活性。本研究旨在探讨ALK在口腔鳞状细胞癌（oral squamous carcinoma, OSCC）细胞增殖、迁移及侵袭过程中的潜在功能。我们检测了ALK处理后OSCC细胞的活力；随后通过溴脱氧尿苷（BrdU）实验、流式细胞术、蛋白质印迹（Western blot）及Transwell小室实验，评估ALK刺激下OSCC细胞的增殖、凋亡、迁移与侵袭能力。在转染微小RNA-9（miR-9）模拟物/抑制剂后，我们探究了miR-9的生物学功能；通过双荧光素酶报告基因活性实验预测了RECK与miR-9的靶向相关性；采用蛋白质印迹法检测JAK1/STAT3与PI3K/AKT信号通路的活化水平；并通过异种移植瘤实验开展体内成瘤实验。

研究结果显示，在CAL-27与SCC-9细胞中，ALK可抑制细胞增殖、促进细胞凋亡，同时抑制细胞迁移与侵袭，并阻断JAK1/STAT3及PI3K/AKT信号通路。miR-9在OSCC组织中表达上调，但在ALK处理的OSCC细胞中表达下调；此外，过表达miR-9可逆转ALK对OSCC细胞增殖、迁移与侵袭的抑制作用。实验证实RECK是miR-9的新型靶基因，过表达RECK可抑制OSCC细胞中的JAK1/STAT3及PI3K/AKT信号通路。体内实验表明，ALK可抑制体内肿瘤形成。综上，ALK可通过调控miR-9/RECK轴抑制OSCC细胞的增殖、迁移与侵袭。

研究亮点
1. ALK可抑制OSCC细胞的增殖、迁移与侵袭；
2. miR-9在OSCC组织中表达上调，但可被ALK在OSCC细胞中下调；
3. miR-9可逆转ALK对CAL-27及SCC-9细胞的抑制作用；
4. RECK是miR-9的新型靶基因；
5. ALK或RECK可抑制CAL-27与SCC-9细胞中的JAK1/STAT3及PI3K/AKT信号通路；
6. ALK可抑制体内肿瘤形成。

ALK可抑制OSCC细胞的增殖、迁移与侵袭；
miR-9在OSCC组织中表达上调，但可被ALK在OSCC细胞中下调；
miR-9可逆转ALK对CAL-27及SCC-9细胞的抑制作用；
RECK是miR-9的新型靶基因；
ALK或RECK可抑制CAL-27与SCC-9细胞中的JAK1/STAT3及PI3K/AKT信号通路；
ALK可抑制体内肿瘤形成。