肝癌细胞质内，real-time PCR和Western blot检测表明肝癌细胞系BEL-7402和HepG2 HSP60表达水平较高，而SMMC7721、Huh7和SK-HEP1表达水平较低。（3）HSP60表达与患者血清AFP（P<0.001）和肿瘤分化程度（P =0.006）显著相关。（4）HSP60表达水平与肝癌患者术后总生存和无复发生存无显著相关性。
3. HSP60对肝癌生物学特性的影响：过表达HSP60可显著促进肝癌细胞系SK-HEP1凋亡（P <0.05），抑制细胞迁移（P <0.05）和侵袭（P <0.05），但对细胞增殖无显著影响；干扰HSP60表达可显著抑制肝癌细胞系BEL-7402凋亡（P <0.05），促进细胞迁移（P <0.05）和侵袭（P <0.05），但对细胞增殖不显著相关。
【结论】
1. 肝癌细胞中线粒体标志分子COX IV和HSP60表达均下降，提示肝癌发生发展过程中线粒体生成可能存在异常改变。
2. COX IV表达下降在肝癌的发生发展中可能起重要作用，COX IV的表达可作为肝癌患者术后生存的预测指标。
3. HSP60表达下降可以减少肝癌细胞凋亡，促进侵袭和转移。
reactive materials studiesExpression and functional studies of Mitochondria marker molecular COX IV and
HSP60 in HCC
Abstract
Key words：COXIV，HSP60，Hepatocellular carcinoma，expression，prognosis，function
Hepatocellular carcinoma (HCC) accounts for the third most cancer-related deaths worldwide and second most cancer-related deaths in China, thus HCC makes a great challenge of public health in China. Despite great advance in the clinical studies on HCC, current clinical staging system of HCC is insufficient to precisely predict the prognosis of patients. Therefore, screening novel biomarkers is important to well understand the molecular mechanisms of hepatocarcinogenesis, improve prognostic prediction and decision making for treatment, and invent novel effective therapeutic strategies for HCC. Mitochondria are only organelles that own independent genomics in the eukaryocytes, which play crucial roles in the processing of bioenergenesis, apoptosis, reactive oxygen species (ROS) generation and calcium homeostasis regulation. Recently, the relationship between mitochondrial abnormalty and cancer development has been becoming a hot spot of cancer research. It is generally considered that abnormal alterations of mitochondria would affect multiple biological functions of cance
r cells. Cytochrome c oxidase (COX) is the terminal step of electron transport of oxidating repiratory chain in the mitochondria. There are 13 subunits in one COX complex, in which subunit IV (COX IV) play the rate limiting role in the assembly of COX. Therefore, COX IV can serve as a molecular biomarker of mitochondria. Previous studies have demonstrated that the abnormal
expression of several COX subunits are associated with the proliferation, invasion and chemoresistance of cancer cells. However, the expression of COX IV in HCC and its clinical significance have never been reported. Heat shock protein 60 (HSP60) is a nuclear-encoded mitochondrial chaperon, which play important roles in the transport and folding of mitochondrial proteins. Therefore, HSP60 may serve as another biomarker of mitochondria. A number of studies have demonstrated expression alterations of HSP60 in many types of malignancies. Moreover, the expression level of HSP60 is closely associated with the clinical characteristics of HCC and the prognosis of patients. However, the expression of HSP60 and its clinical correlation are unknown, and the impact of HSP60 expression on the biological characteristics of HCC cells has not been investigated. 【Objectives】
This study aimed to examine the expression of two mitochondrial biomarkers, COX IV and HSP60, in HCC tissues and its clinical significance. We also investigated the impact of HSP60 expression alterati
ons on the biological characteristics of HCC cell lines. 【Methods】
1. Examination of COX IV expression in HCC: Real-time PCR was used to examine the expression of COX IV mRNA in 30 HCC tissues and corresponding adjacent pericancer tissues. Immunohistochemistry （IHC）was used to examined the protein expression of COX IV in 323 HCC tissues and paired noncancerous liver tissues. The association of COX IV expression with clinicopathological characteristics and clinical outcome of HCC was analyzed.
2. Examination of HSP60 expression in HCC: Real-time PCR and western blot were used to examine the expression of HSP60 mRNA in 30 HCC tissues and corresponding adjacent pericancer tissues. Immunohistochemistry (IHC) was used to examined the protein expression of HSP60 in 331 HCC tissues and paired noncancerous liver tissues. The association of HSP60 expression with clinicopathological characteristics and clinical outcome of HCC was analyzed. Real-time PCR, western blot and immunofluorescence were used to examine the expression of HSP60 in HCC cell lines.
3. pcDNA3.1-HSP60 vectors and HSP60-specific siRNA were transfected into HCC cells
by cationic liposomes. The proliferation, apoptosis, migration and invasion of HCC cell lines were assessed after transfection with HSP60 overexpression or silencing by RNA interference.
【Results】
1. Expression of COX IV in HCC and its clinical significance: (1) real-time PCR results demonstrated that the expression of COX IV mRNA was significantly lower than that in pericancer tissues (P <0.05). IHC further confirmed that the expression of COX IV protein was significantly reduced in HCC tissues (P<0.001). (2) Low expression of COX IV was significantly associated with positive HBsAg (P=0.045)，high serum α-fetal protein (AFP) (P=0.012) and low differentiation grade (P=0.030) of HCC. (3) Kaplan-Meier curve analysis showed that low COX IV expression was significantly associated with poor relapse-free survival of HCC patients (P=0.024). Cox analysis further confirmed that low COX IV expression was significantly associated with increased relapse risk of HCC（OR =0.774；95% CI，(0.563-0.982)；P =0.037）. (4) Low COX IV expression was significantly associated with increased risk of portal vein tumor thrombus (PVTT) （OR =
2.323；95% CI，(1.044-5.169)；P =0.039）.
2. Expression of HSP60 in HCC and its clinical significance: (1) real-time PCR results demonstrated that the expression of HSP60 mRNA was significantly lower than that in pericancer tissues (P =0.006 ). IHC further confirmed that the expression of HSP60 protein was significantly reduced in HCC tissues (
P=0.0003). Western blot analysis further demonstrated that the expression of HSP60 was remarkably reduced in HCC cells.
(2) Immunofluorescence demonstrated that HSP60 was mainly expressed in the cytoplasm of HCC cells. Real-time PCR and Western blot analyses showed that HSP60 was highly expressed in BEL-7402 and HepG2 cell lines，and lowly expressed in SMMC7721、Huh7 and SK-HEP1 cell lines. (3) Low expression of HSP60 was significantly associated with high serum AFP（P<0.001）and low differentiation grade (P = 0.006) of HCC. (4) There was no significant association between HSP60 expression and clinical outcome of HCC.
3. Impact of HSP60 expression on the biological characteristics of HCC cells: (1) HSP60 overexpression significantly promoted the apoptosis of SK-HEP1 cells （P < 0.05）,
whereas interference of HSP60 by siRNA significantly inhibited the apoptosis of BEL-7402 cells (P <0.05 ). (2) HSP60 overexpression significantly inhibited the migration (P<0.05) and invasion (P<0.05) of SK-HEP1 cells, while interference of HSP60 significantly promoted the migration (P<0.05) and invasion (P < 0.05) of BEL-7402 cells. 【Conclusions】
1. The reduced expression of the two mitochondrial biomarkers, COX IV and HSP60, indicates the pot
ential abnormal alterations of mitochondrial biogenesis during hepatocarcinogenesis.
2. Downregulation of COX IV expression may play an important role in the development of HCC. The expression of COX IV may serve as an biomarker to predict the RFS of HCC patients.
3. Downregulation of HSP60 may inhibit the apoptosis, and promote the migration and invasion of HCC cells.
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