Hou Zhouhua, Quan Jun
Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China.
Mol Med Rep. 2017 May;15(5):3285-3291. doi: 10.3892/mmr.2017.6363. Epub 2017 Mar 23.
Hepatitis B virus (HBV) X protein (HBx) is a key regulatory protein that is involved in HBV infection, replication and carcinogenesis of hepatocellular carcinoma (HCC). The aim of the present study was to investigate the role of HBx in the progression and metastasis of liver cancer cells and to determine the underlying molecular mechanism of HBx in metastatic liver cancer cells. HBx protein expression was detected by western blot analysis, and microRNA (miR)‑21 levels were determined by reverse transcription‑quantitative polymerase chain reaction in the highly metastatic MHCC‑97H low metastatic MHCC‑97L and SMMC‑7721 liver cancer cell lines. The results demonstrated that the levels of HBx and miR‑21 were significantly increased in MHCC‑97H cells compared with MHCC‑97L and SMMC‑7721 cells. In addition, three pairs of small interfering (si)RNA specific to HBx were designed and synthesized to interfere with endogenous HBx in liver cancer cells, and the results demonstrated that knockdown HBx was associated with a corresponding decrease in miR‑21 expression. The MTT assay results demonstrated that cell viability significantly decreased in HBx‑siRNA cells compared with scramble siRNA‑transfected cells. In addition, transfection with an miR‑21 inhibitor inhibited MHCC‑97H cell proliferation. Furthermore, Transwell assay results revealed that downregulation of HBx and treatment with miR‑21 inhibitors contributed to the inhibition of MHCC‑97H cell invasion and metastasis. Western blot analysis demonstrated that miR‑21 inhibitors and HBx‑siRNA treatment led to the upregulation of phosphatase and tensin homolog (PTEN), and decreased levels of phosphoinositide 3‑kinase (PI3K), phosphorylated protein kinase B (Akt) and matrix metalloproteinase (MMP)‑2. The results of the present study indicated that HBx was positively associated with miR‑21 expression, and downregulation of miR‑21 and HBx suppressed MMP‑2 activity via the PTEN/PI3K/Akt signaling pathway. Therefore, HBx and miR‑21 may represent novel therapeutic targets for the treatment of HCC.
乙型肝炎病毒（HBV）X蛋白（HBx）是一种关键的调节蛋白，参与HBV感染、复制以及肝细胞癌（HCC）的致癌过程。本研究的目的是探讨HBx在肝癌细胞进展和转移中的作用，并确定HBx在转移性肝癌细胞中的潜在分子机制。通过蛋白质免疫印迹分析检测HBx蛋白表达，采用逆转录-定量聚合酶链反应测定高转移性MHCC-97H、低转移性MHCC-97L和SMMC-7721肝癌细胞系中的微小RNA（miR）-21水平。结果表明，与MHCC-97L和SMMC-7721细胞相比，MHCC-97H细胞中HBx和miR-21水平显著升高。此外，设计并合成了三对针对HBx的小干扰（si）RNA，以干扰肝癌细胞中的内源性HBx，结果表明，敲低HBx与miR-21表达相应降低有关。MTT法检测结果表明，与转染乱序siRNA的细胞相比，HBx-siRNA细胞的细胞活力显著降低。此外，用miR-21抑制剂转染可抑制MHCC-97H细胞增殖。此外，Transwell实验结果显示，下调HBx和用miR-21抑制剂处理有助于抑制MHCC-97H细胞的侵袭和转移。蛋白质免疫印迹分析表明，miR-21抑制剂和HBx-siRNA处理导致磷酸酶和张力蛋白同源物（PTEN）上调，磷脂酰肌醇3激酶（PI3K）、磷酸化蛋白激酶B（Akt）和基质金属蛋白酶（MMP）-2水平降低。本研究结果表明，HBx与miR-21表达呈正相关，下调miR-21和HBx可通过PTEN/PI3K/Akt信号通路抑制MMP-2活性。因此，HBx和miR-21可能是治疗HCC的新治疗靶点。
