Expression And Functional Studies Of Mitochondria Marker Molecular Cox Ⅳ And HSP60in HCC

Hepatocellular carcinoma (HCC) accounts for the third most cancer-related deathsworldwide and second most cancer-related deaths in China, thus HCC makes a greatchallenge 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 ofpatients. Therefore, screening novel biomarkers is important to well understand themolecular mechanisms of hepatocarcinogenesis, improve prognostic prediction anddecision 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 oxygenspecies (ROS) generation and calcium homeostasis regulation. Recently, the relationshipbetween mitochondrial abnormalty and cancer development has been becoming a hot spotof cancer research. It is generally considered that abnormal alterations of mitochondriawould affect multiple biological functions of cancer cells. Cytochrome c oxidase (COX) isthe terminal step of electron transport of oxidating repiratory chain in the mitochondria.There are13subunits in one COX complex, in which subunit IV (COX IV) play the ratelimiting role in the assembly of COX. Therefore, COX IV can serve as a molecularbiomarker of mitochondria. Previous studies have demonstrated that the abnormal expression of several COX subunits are associated with the proliferation, invasion andchemoresistance of cancer cells. However, the expression of COX IV in HCC and itsclinical significance have never been reported. Heat shock protein60(HSP60) is anuclear-encoded mitochondrial chaperon, which play important roles in the transport andfolding of mitochondrial proteins. Therefore, HSP60may serve as another biomarker ofmitochondria. A number of studies have demonstrated expression alterations of HSP60inmany types of malignancies. Moreover, the expression level of HSP60is closelyassociated with the clinical characteristics of HCC and the prognosis of patients. However,the expression of HSP60and its clinical correlation are unknown, and the impact ofHSP60expression on the biological characteristics of HCC cells has not been investigated.【Objectives】This study aimed to examine the expression of two mitochondrial biomarkers, COXIV and HSP60, in HCC tissues and its clinical significance. We also investigated theimpact of HSP60expression alterations on the biological characteristics of HCC cell lines.【Methods】1. Examination of COX IV expression in HCC: Real-time PCR was used to examinethe expression of COX IV mRNA in30HCC tissues and corresponding adjacentpericancer tissues. Immunohistochemistry （IHC） was used to examined the proteinexpression of COX IV in323HCC tissues and paired noncancerous liver tissues. Theassociation of COX IV expression with clinicopathological characteristics and clinicaloutcome of HCC was analyzed.2. Examination of HSP60expression in HCC: Real-time PCR and western blot wereused to examine the expression of HSP60mRNA in30HCC tissues and correspondingadjacent pericancer tissues. Immunohistochemistry (IHC) was used to examined theprotein expression of HSP60in331HCC tissues and paired noncancerous liver tissues.The association of HSP60expression with clinicopathological characteristics and clinicaloutcome of HCC was analyzed. Real-time PCR, western blot and immunofluorescencewere used to examine the expression of HSP60in HCC cell lines.3. pcDNA3.1-HSP60vectors and HSP60-specific siRNA were transfected into HCC cells by cationic liposomes. The proliferation, apoptosis, migration and invasion of HCC celllines were assessed after transfection with HSP60overexpression or silencing by RNAinterference.【Results】1. Expression of COX IV in HCC and its clinical significance:(1) real-time PCRresults demonstrated that the expression of COX IV mRNA was significantly lower thanthat in pericancer tissues (P <0.05). IHC further confirmed that the expression of COX IVprotein was significantly reduced in HCC tissues (P<0.001).(2) Low expression of COXIV was significantly associated with positive HBsAg (P=0.045)， high serum α-fetalprotein (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 significantlyassociated with poor relapse-free survival of HCC patients (P=0.024). Cox analysisfurther confirmed that low COX IV expression was significantly associated with increasedrelapse risk of HCC（OR=0.774；95%CI，(0.563-0.982)；P=0.037）.(4) Low COX IVexpression 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 HSP60in HCC and its clinical significance:(1) real-time PCRresults demonstrated that the expression of HSP60mRNA was significantly lower thanthat in pericancer tissues (P=0.006). IHC further confirmed that the expression of HSP60protein was significantly reduced in HCC tissues (P=0.0003). Western blot analysisfurther demonstrated that the expression of HSP60was remarkably reduced in HCC cells.(2) Immunofluorescence demonstrated that HSP60was mainly expressed in the cytoplasmof HCC cells. Real-time PCR and Western blot analyses showed that HSP60was highlyexpressed in BEL-7402and HepG2cell lines，and lowly expressed in SMMC7721、Huh7and SK-HEP1cell lines.(3) Low expression of HSP60was significantly associated withhigh serum AFP（P<0.001）and low differentiation grade (P=0.006) of HCC.(4) Therewas no significant association between HSP60expression and clinical outcome of HCC.3. Impact of HSP60expression on the biological characteristics of HCC cells:(1)HSP60overexpression significantly promoted the apoptosis of SK-HEP1cells（P <0.05）, whereas interference of HSP60by siRNA significantly inhibited the apoptosis ofBEL-7402cells (P <0.05).(2) HSP60overexpression significantly inhibited the migration(P<0.05) and invasion (P<0.05) of SK-HEP1cells, while interference of HSP60significantly promoted the migration (P<0.05) and invasion (P <0.05) of BEL-7402cells.【Conclusions】1. The reduced expression of the two mitochondrial biomarkers, COX IV and HSP60,indicates the potential abnormal alterations of mitochondrial biogenesis duringhepatocarcinogenesis.2. Downregulation of COX IV expression may play an important role in thedevelopment of HCC. The expression of COX IV may serve as an biomarker to predict theRFS of HCC patients.3. Downregulation of HSP60may inhibit the apoptosis, and promote the migrationand invasion of HCC cells.