Han Ruiyang, Li Yazhou, Cao Wei
Department of Interventional Radiology, The Second Affiliated Hospital of Air Force Medical University, Xi'an, China.
Department of Hepatobiliary and Vascular Surgery, Hospital of the Chinese Weapons Institutes of Health, Xi'an, China.
Transl Cancer Res. 2020 Jun;9(6):3986-3997. doi: 10.21037/tcr-20-2007.
This study aims to investigate the effect of miR-212-5p overexpression targeting suppressor of cytokine signaling 5 (SOCS5) on the malignant proliferation of liver cancer cells HepG2 and tumor formation in nude mice with transplanted tumors.
Luciferase reporter assay was used to detect the targeted relationship between miR-212-5p and SOCS5, and SOCS5 was overexpressed by the SOCS5 pcDNA vector. MiR-212-5p mimic and pc DNA-SOCS5 were transfected into liver cancer HepG2 cells alone or in combination, and the cells were randomly divided into four groups, the control group, mimic group, SOCS5 group and mimic + SOCS5 group for subsequent experiments. The orthotopic xenograft mouse models were established by using HepG2 cells in BALB/c athymic nude mice.
The results showed that there was a direct targeting relationship between miR-212-5p and SOCS5. Compared with the control group, the clone formation rate, the levels of Ki67, and proliferating cell nuclear antigen (PCNA) protein in the mimic group were significantly lower (P<0.05), but the apoptosis rate was significantly higher (P<0.05). The ratio of Bax/Bcl-2, cleaved Caspase-3/Caspase-3, and cleaved Caspase-9/Caspase-9 was significantly higher (P<0.05), while the ratios of p-phosphatidylinositol 3 kinase (PI3K)/PI3K, p- Protein kinase B (AKT)/AKT, and p-mammalian target of rapamycin (mTOR)/mTOR were significantly reduced (P<0.05). In the SOCS5 group, the result was reversed. Interesting, In the mimic+SOCS5 group the clone formation rate, the protein levels of Ki67, and PCNA were significantly decreased (P<0.05) while the apoptosis rate was significantly increased (P<0.05). The ratio of Bax/Bcl-2, cleaved Caspase-3/Caspase-3, and cleaved Caspase-9/Caspase-9 was significantly increased (P<0.05). The ratios of p-PI3K/PI3K, p-Akt/AKT, and p-mTOR/mTOR were significantly reduced (P<0.05). In vivo, The level of miR-212-5p was significantly increased, with SOCS5 decreased (P<0.05). Furthermore, the number of Ki67 positive cells was significantly reduced (P<0.05), and the apoptosis rate increased significantly (P<0.05). Additionally, the ratio of p-PI3K/PI3K, P-AKT/AKT, P-mTOR/mTOR decreased significantly (P<0.05).
miR-212-5p overexpression down-regulated SOCS5 could inhibit the malignant proliferation of HCC cells HepG2 and tumor formation in nude mice with transplanted tumors.
本研究旨在探讨靶向细胞因子信号转导抑制因子5（SOCS5）的miR-212-5p过表达对肝癌细胞HepG2恶性增殖及裸鼠移植瘤形成的影响。
采用荧光素酶报告基因检测法检测miR-212-5p与SOCS5的靶向关系，并用SOCS5 pcDNA载体过表达SOCS5。将miR-212-5p模拟物和pcDNA-SOCS5单独或联合转染至肝癌HepG2细胞中，细胞随机分为四组，即对照组、模拟物组、SOCS5组和模拟物+SOCS5组，进行后续实验。采用HepG2细胞在BALB/c无胸腺裸鼠中建立原位异种移植小鼠模型。
结果显示miR-212-5p与SOCS5之间存在直接靶向关系。与对照组相比，模拟物组的克隆形成率、Ki67水平和增殖细胞核抗原（PCNA）蛋白水平显著降低（P<0.05），但凋亡率显著升高（P<0.05）。Bax/Bcl-2、裂解的半胱天冬酶-3/半胱天冬酶-3和裂解的半胱天冬酶-9/半胱天冬酶-9的比值显著升高（P<0.05），而磷酸化磷脂酰肌醇3激酶（PI3K）/PI3K、磷酸化蛋白激酶B（AKT）/AKT和磷酸化哺乳动物雷帕霉素靶蛋白（mTOR）/mTOR的比值显著降低（P<0.05）。在SOCS5组中，结果相反。有趣的是，在模拟物+SOCS5组中，克隆形成率、Ki67蛋白水平和PCNA显著降低（P<0.05），而凋亡率显著升高（P<0.05）。Bax/Bcl-2、裂解的半胱天冬酶-3/半胱天冬酶-3和裂解的半胱天冬酶-9/半胱天冬酶-9的比值显著升高（P<0.05）。磷酸化PI3K/PI3K、磷酸化Akt/AKT和磷酸化mTOR/mTOR的比值显著降低（P<0.05）。在体内，miR-212-5p水平显著升高，SOCS5降低（P<0.05）。此外，Ki67阳性细胞数量显著减少（P<0.05），凋亡率显著升高（P<0.05）。另外，磷酸化PI3K/PI3K、磷酸化AKT/AKT、磷酸化mTOR/mTOR的比值显著降低（P<0.05）。
miR-212-5p过表达下调SOCS5可抑制肝癌细胞HepG2的恶性增殖及裸鼠移植瘤的形成。
