PO.ET03.01 · 实验与分子治疗
PGRMC1诱导的脂质代谢重编程导致肝细胞癌对索拉非尼耐药
PGRMC1 induced lipid metabolic reprogramming leads to sorafenib resistance in hepatocellular carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：肝细胞癌（HCC）是最主要的肝癌类型，常在晚期才被确诊，导致高死亡率。索拉非尼是一线药物，可使晚期患者的生存期平均延长10.7个月。然而，大量研究表明，六个月后患者会对索拉非尼治疗产生耐药。因此，理解索拉非尼耐药（SR）的分子机制并确定治疗HCC最有前景的治疗靶点至关重要。在导致SR发生的多种通路中，HCC脂质稳态的紊乱是其中之一。PGRMC1是一种调控脂质代谢和血红素堆积的蛋白，近来在多种恶性肿瘤中被鉴定为促癌靶点。因此，我们研究了PGRMC1在HCC的SR中的作用。
方法：我们通过逐步提高索拉非尼剂量建立了SR-HCC细胞系（Hep3B、HepG2和Huh7）。评估了THLE3（正常肝细胞系）、亲本HCC及SR-HCC细胞系中PGRMC1的表达水平。对过表达或沉默PGRMC1的基因修饰HCC细胞系，采用细胞活力、凋亡、集落形成、迁移和侵袭实验评估其SR。为探究PGRMC1相关SR的机制，我们使用聚焦于脂蛋白信号和胆固醇合成通路的微阵列进行了RT-PCR。采用RT2 PCR、免疫印迹和免疫荧光验证PGRMC1在与SR相关的脂质代谢中的作用。
结果：与HCC细胞和正常肝细胞相比，SR-HCC细胞系中PGRMC1表达更高。此外，过表达PGRMC1增加了SR，而在HCC和SR-HCC中沉默PGRMC1则产生相反效果。值得注意的是，我们的研究揭示，与亲本HCC细胞相比，SR-HCC和ovPGRMC1-GFP-Hep3B细胞表现出更高水平的膜胆固醇和脂质过氧化。此外，我们的发现表明PGRMC1通过上调APOC3、HMGCS2、NR0B2、STARD3和SREBF2，并下调APOD，改变了HCC中的脂质代谢。另外，我们的数据还证明，抑制PGRMC1通过改变HCC中的脂质代谢有效增强了索拉非尼的抗癌作用。
结论：我们得出结论，PGRMC1是通过重编程脂质代谢来提高索拉非尼敏感性的可行靶点。
查看英文原文 English abstract
Background: Hepatocellular Carcinoma (HCC) is the most predominant type of liver cancer and are often diagnosed at advanced stage resulting in high rates of mortality. Sorafenib is a first-line medication which increases survival by an average of 10.7 months in patients with advanced patients. However, numerous studies have shown that after six months, patients develop resistance to sorafenib treatment. Therefore, understanding the molecular mechanisms of sorafenib resistance (SR) and determining the most promising therapeutic target for the treatment of HCC is of vital importance. Among the various pathways that lead to the development of SR are disruptions in the lipid homeostasis of HCC. PGRMC1, a protein that regulates lipid metabolism and heme stacking, has been recently identified as an onco-target in a number of malignancies. Thus, we have investigated PGRMC1's role in SR in HCC.
Methods: We established SR-HCC cell lines (Hep3B, HepG2, and Huh7) through a gradual rise in sorafenib dosage. PGRMC1 expression levels in THLE3 (normal liver cell line), parental HCC, and SR-HCC cell lines was assessed. Genetically modified HCC cell lines with PGRMC1 overexpression or silencing were evaluated for SR using cell viability, apoptosis, colony formation, migration, and invasion assays. To investigate the mechanism of PGRMC1-related SR, we conducted RTPCR using lipoprotein signaling and cholesterol synthesis pathway focused microarray. RT 2 PCR, immunoblot, and immunofluorescence were utilized to validate the role of PGRMC1 in lipid metabolism associated with SR.
Results: PGRMC1 expression was higher in SR-HCC cell lines in comparison to HCC cells and normal liver cells. Furthermore, overexpression of PGRMC1 increased SR, but the silencing of PGRMC1 in HCC and SR-HCC led to the opposite effect. It is interesting to note that our studies revealed that SR-HCC and ovPGRMC1-GFP-Hep3B cells exhibited higher levels of membrane cholesterol and lipid peroxidation in comparison to parental HCC cells. Moreover, our findings indicate that PGRMC1alters lipid metabolism in HCC by upregulating APOC3, HMGCS2, NR0B2, STARD3, and SREBF2, and downregulating APOD. In addition, our data also demonstrates that inhibition of PGRMC1 effectively enhanced the anticancer effect of sorafenib by altering lipid metabolism in HCC.
Conclusions: We conclude that PGRMC1 is a feasible target for increasing sorafenib sensitivity through reprogramming lipid metabolism.
利益披露 Disclosure
P. Narayanan, None..
A. Ramirez, None..
J. Lopez, None..
K. Nguyen, None..
M. Amador Rojo, None..
K. Garcia, None..
K. Soni, None..
G. Hua, None..
R. Lakshmanaswamy, None..
R. Subramani, None.