Silencing of hsa_circ_0101145 reverses the epithelial-mesenchymal transition in hepatocellular carcinoma via regulation of the miR-548c-3p/LAMC2 axis
Silencing of hsa_circ_0101145 reverses the epithelial-mesenchymal transition in hepatocellular carcinoma via regulation of the miR-548c-3p/LAMC2 axis
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DOI：
10.18632/aging.103324
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发表时间：
2020-06-30
期刊：
影响因子：
5.2
通讯作者：
Wei, Feng
中科院分区：
文献类型：
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作者：
Jin, Jinglan;Liu, Huan;Wei, Feng
肝细胞癌（HCC）是全球癌症相关死亡的主要原因。虽然HCC的治疗和诊断取得了进展，但发病率和死亡率仍在继续上升。一项研究发现，环状RNA作为竞争性内源性RNA发挥作用，并构建了一个基于基因的诺模图来估计HCC患者的总生存率。先前的研究使用高通量测序表明hsa_circ_0101145在HCC中异常表达，但其潜在机制尚不清楚。我们进行RT-qPCR以确定HCC组织中hsa_circ_0101145和miR-548 c-3 p的表达。采用荧光原位杂交（FISH）技术检测hsa_circ_0101145在肝癌组织中的表达及亚细胞定位。hsa_circ_0101145在HCC细胞中的表达被选择性地调节。我们分别通过RT-qPCR和蛋白质印迹分析来确定LAMC 2和EMT mRNA和蛋白质水平。我们采用流式细胞术、CCK 8、Transwell和伤口愈合试验分别监测细胞周期、细胞增殖、侵袭和迁移。我们采用双荧光素酶报告基因和RNA下拉测定来验证hsa_circ_0101145、miR-548 c-3 p和LAMC 2之间的关系。我们使用皮下异种移植模型以及裸鼠静脉尾注射来检查hsa_circ_0101145对HCC细胞转移和增殖的体内作用。数据表明hsa_circ_0101145在HCC组织和细胞系中均显著上调。hsa_circ_0101145高表达与侵袭性HCC表型相关。hsa_circ_0101145的下调通过靶向miR-548 c-3 p/LAMC 2轴抑制HCC增殖以及转移，这使用荧光素酶报告基因和RNA下拉测定来检查。沉默hsa_circ_0101145抑制HCC中的上皮-间质转化。下调miR-548 c-3 p或过表达LAMC 2可恢复hsa_circ_0101145沉默后HCC细胞的迁移和增殖能力。LAMC 2过表达逆转了miR-548 c-3 p诱导的细胞迁移和体外生长抑制。总之，研究结果表明，hsa_circ_0101145沉默通过作为miR-548 c-3 p海绵增强LAMC 2表达来抑制HCC进展。因此，hsa_circ_0101145可能是HCC治疗靶点。
Hepatocellular carcinoma (HCC) is a primary cause of cancer-related deaths globally. While there have been advancements in HCC treatment and diagnosis, incidence and mortality rates continue to rise. One study found that circular RNAs functioned as competing endogenous RNAs, and constructed a gene-based nomogram to estimate overall survival of HCC patients. Previous studies using high-throughput sequencing suggested that hsa_circ_0101145 is abnormally expressed in HCC, but the underlying mechanism is unknown. We performed RT-qPCR to determine hsa_circ_0101145 and miR-548c-3p expression in HCC tissues. We used fluorescence in situ hybridization (FISH) to detect hsa_circ_0101145 expression and hsa_circ_0101145 subcellular localization in HCC tissues. hsa_circ_0101145 expression in HCC cells was selectively regulated. We determined LAMC2 and EMT mRNA and protein levels by RT-qPCR and western blotting analysis, respectively. We employed flow cytometry, and CCK8, Transwell, and wound healing assays to monitor the cell cycle, cell proliferation, invasion, and migration, respectively. We employed dual-luciferase reporter and RNA pulldown assays to verify the relationship among hsa_circ_0101145, miR-548c-3p, and LAMC2. We examined the effects of hsa_circ_0101145 on HCC cell metastasis and proliferation in vivo using a subcutaneous xenograft model as well as intravenous tail injection of nude mice. The data demonstrated that hsa_circ_0101145 was significantly upregulated in both HCC tissues and cell lines. High hsa_circ_0101145 expression was correlated with aggressive HCC phenotypes. Downregulation of hsa_circ_0101145 suppressed HCC proliferation as well as metastasis by targeting the miR-548c-3p/LAMC2 axis, which was examined using luciferase reporter and RNA pulldown assays. Silencing of hsa_circ_0101145 suppressed the epithelial-mesenchymal transition in HCC. Downregulation of miR-548c-3p or overexpression of LAMC2 restored migration and proliferation abilities of HCC cells following hsa_circ_0101145 silencing. LAMC2 overexpression reversed miR-548c-3p-induced cell migration and growth inhibition in vitro. In summary, the findings illustrated that hsa_circ_0101145 silencing suppressed HCC progression by functioning as an miR-548c-3p sponge to enhance LAMC2 expression. Therefore, hsa_circ_0101145 could be an HCC treatment target.