| BackgroundAmong primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70-85% of total liver cancers worldwide, and it is the sixth most common cancer worldwide and the third most common cause of cancer mortality. Despite significant improvements in HCC management over the past 30 years, there are no effective chemoprevention strategies and only one systemic therapy has been approved for patients with advanced tumors. Until now, hepatic resection remains one of the major curative treatments for HCC; however, its prognosis remains extremely dismal, which is mainly attributed to the high frequency of intrahepatic metastatic recurrence. Unfortunately, the identification of patients who are at a greater risk for tumor recurrence after curative treatment for HCC remains a great challenge, particularly those with early stage disease who do not have significant vascular invasion or regional lymph node or distant metastasis.Chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections resulting in hepatic fibrosis and cirrhosis are major risk factors for HCC. It is estimated that 70-85% of HCC found worldwide are due to persistent viral infections with either HBV or HCV. The high HCC rates in parts of Asia and sub-Saharan Africa largely reflect the elevated prevalence of HBV infection, with over 8% of the populations in these regions chronically infected with the virus. HBV infection accounts for about 60% of the total incidence of liver cancer in developing countries and for about 23% in developed countries, whereas the corresponding percentages for HCV infection are 33% in developing countries and 20% in developed countries. In China, HBV infection is also the major risk factor for HCC. Sustained chronic inflammation due to HBV and HCV infection leads to liver cirrhosis and ultimately HCC.Accumulating evidences indicate that the liver is the key organ for the metabolism of lipids, lipoproteins and apolipoproteins, and in humans most apolipoproteins are produced by the liver. It has been demonstrated that plasma lipid profiles undergo changes in HCC patients, as hepatic cellular damage and HCC impairs these processes, leading to alterations in plasma lipid and lipoprotein patterns. The degree of liver cell injury can be reflected by analyzing the serum levels of lipids, lipoproteins and/or apolipoproteins in patients suffering from chronic liver diseases and HCC. Apolipoprotein M (apoM) is a recently discovered apolipoprotein that is mainly associated with high-density lipoprotein (HDL) in human plasma, with a small proportion being present in triglyceride-rich lipoproteins and low-density lipoproteins (LDL). ApoM is mainly expressed in hepatocytes and kidney tubular cells, but also expressed to a limited degree in the fetal liver and kidney. Jiang et al. have demonstrated that apoM plasma levels in HCC patients were significantly higher than those in normal subjects, but apoM mRNA and protein levels in the HCC tissues were significantly lower than those in the adjacent tissue. However, the relationship between apoM and HCC development and the underlying mechanisms by which apoM may contribute to HCC are still unknown. In the present study, we demonstrated that apoM had significant anti-cancer properties via the inhibition of invasion, migration, and proliferation, and most importantly via promoting cellular apoptosis by inhibition of Bcl2A1 expression. Moreover, hsa-miR-573 markedly promoted growth of xenograft tumors in nude mice and downregulated apoM expression with an accompanying reduction in cell apoptosis and induction of Bcl2A1 expression.Aims1.To study the effect of ApoM on migration, invasion, proliferation and promoted apoptosis of HepG2 cell in vitro.2. To study the effect of Hsa-miR-573 on apoM expression.3. To study the effect of Hsa-miR-573 on invasion, migration, proliferation and promoted apoptosis of HepG2 cell in vitro.4. To study the effect of ApoM and Hsa-miR-573 on apoptosis of HepG2 cells.5. To study the effect of ApoM and Hsa-miR-573 on migration, invasion, proliferation and promoted apoptosis of Hep3B cell and SK-Hep-1 cell in vitro.6. To study the effect of ApoM and hsa-miR-573 on tumor growth in nude mice.7. Whether Serum apoM concentration show a correlation with clinical and biochemical characteristics in HCC patients.Methods1. Effect of ApoM on migration, invasion, proliferation and promoted apoptosis of HepG2 cell in vitro.1.1 Human HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS with streptomycin (100μg/mL) and penicillin (100 U/ml). For long-term culturing, mycoplasma test was performed every month for all cell lines. All cells were incubated at 37℃,5% CO2. Cells were seeded in 6-or 12-well plates or 60-mm dishes and grown to 80-90% confluence before use.1.2 Human HepG2 cells were cultured in 25-cm2 vented flasks containing Dulbecco’s modified Eagle’s medium with 10% fetal calf serum under standard culture conditions (5% CO2,37℃). Packed empty LV vectors with green fluorescent protein (GFP; LV-Mock) and LV-mediated human apoM overexpression vector (LV-apoM) with GFP were prepared as described previously. The cells were infected with the LV stock at a multiplicity of infection of 20 transducing units per cell in the presence of 8 mg/mL of polybrene. Then cells were washed with fresh complete media after 24 h of incubation. The GFP-positive cells were counted 96 h post-transduction.1.3 Human HepG2 cells infected with the LV stock and infected with the LV-apoM were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10%sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to western blot analyses using rabbit polyclonal anti-apoM and rabbit polyclonal anti-β-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amersham Biosciences, Foster City, CA, USA).1.4 Transwell chambers (Millipore-Chemicon, Billerica, MA) were used for the invasion and migration assays. For the invasion assays, matrigel-coated chambers were placed in empty wells of 24-well tissue culture plates. Human HepG2 cells infected with the LV stock or infected with the LV-apoM were cultured in a serum-free medium for 24 h and then trypsinized and suspended with a culture medium containing 0.1% bovine serum albumin at a concentration of 5×104 cells/mL. Then,500 μL of suspension of Human HepG2 cells infected with the LV stock or infected with the LV-apoM was added into each chamber and 750 μL of complete medium was added into each well. After incubation for 36 h, the non-invading cells were removed from the upper surface of the chamber membrane using cotton-tipped swabs. The cells on the lower surface of the chamber membrane were fixed in 4% paraformaldehyde for 10 min, stained with crystal violet, and then counted after photographing the membrane through a microscope. For the migration assays, non-coated chambers were used and migrated Human HepG2 cells infected with the LV stock or infected with the LV-apoM on the lower surface of the chamber membrane were fixed after a 24-h incubation, stained, and then photographed for further analysis.1.5 The Cell Counting Kit-8 (CCK8; Wako Pure Chemical Industries, Osaka, Japan) was used to measure cell viability in order to evaluate cell proliferation in Human HepG2 cells infected with the LV stock or infected with the LV-apoM. Cells treated with LV-Mock and LV-apoM, respectively, seeded into 96-well culture plates in triplicate. At various time points (0,12,24,36, and 48 h),10 μL of CCK-8 solution were added to each well in an assay plate and incubated in a CO2 incubator for 1 h. Then,10 μL of 1%(w/v) SDS solution was added to each well to stop the reaction and the plate was read by a microplate reader for optical density at 450 nm (Thermo Electron Corporation, Marietta, OH, USA).1.6 Human HepG2 cells infected with the LV stock or infected with the LV-apoM were harvested and cell cycle analysis and apoptosis quantification were performed by flow cytometry using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) equipped with an argon-ion laser (488 nm). Cell cycle analysis was performed by flow cytometry using a propidium iodide (PI) cell cycle detection kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instructions. Cell apoptosis analysis was performed by flow cytometry using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I (BD Bioscience, MA) according to the manufacturer’s instructions.2. Effect of Hsa-miR-573 on apoM expression in vitro.2.1 Total RNA from liver tissues or cultured cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Total RNA from 200 μL of serum was isolated using the miRVana PARIS kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Target prediction algorithms such as miRBase, PicTar, TargetScan, and RNAhybrid predicted that hsa-miR-145-5p, hsa-miR-145-3p, hsa-miR-221-5p, hsa-miR-221-3p, hsa-miR-222-5p, hsa-miR-222-3p, hsa-miR-766-5p, hsa-miR-766-3p, and hsa-miR-573 are putative regulators of apoM. The nine microRNAs (miRNAs) putatively regulating apoM were detected with the All-in-OneTM miRNA qPCR Kit (GeneCopoeia, Rockville, MD, USA) according to the instruction manual in 20-μL reaction volumes. Real-time PCR was performed on a real-time PCR ABI 7500 Fast system (Applied Biosy stems, Foster City, CA, USA). The expression of U6 RNA was used as an endogenous control. For serum hsa-miR-573 analysis, synthetic spiked-in Caenorhabditis elegans miR-39 was added to the serum samples prior to RNA extraction as an internal control[19]. The mRNA levels were evaluated by real-time quantitative PCR using an ABI 7500 Fast Real Time PCR system with SYBR Green detection chemistry (TaKaRa Bio, Inc., Shiga, Japan). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as the internal control. Quantitative measurements were determined using the △△Ct method. All samples were measured in triplicate and the mean value was considered for comparative analysis.2.2 HepG2 cells were transfected with 50 nM miRNA mimic (hsa-miR-145-5p, hsa-miR-145-3p, hsa-miR-221-5p, hsa-miR-221-3p, hsa-miR-222-5p, hsa-miR-222-3p, hsa-miR-766-5p, hsa-miR-766-3p, and hsa-miR-573, respectively) utilizing Lipofectamine 2000 transfection reagent for 48 h according to the manufacturer’s instructions. All experimental control samples were treated with an equal concentration of a non-targeting control mimic sequence (negative controls).2.3 Human apoM cDNA, containing putative and mutant target sites for hsa-miR-573, was chemically synthesized and inserted into a pMIR-REPORTM vector (Ambion). The pMIR-REPORTM beta-galactosidase control vector (Ambion) was used as a reference. For the luciferase assay,293T cells (human embryonic kidney 293 cells) were co-transfected with wild-(pMIR-apoM-wt) or mutant-type (pMIR-apoM-mt) reporter vectors and hsa-miR-573 mimics using Lipofectamine 2000 transfection reagent. Luciferase activity was measured 48 h post-transfection using a dual-luciferase assay kit (Promega, Madison, WI, USA)3. Effect of Hsa-miR-573 on invasion, migration, proliferation and promoted apoptosis of HepG2 cell in vitro.3.1 Human HepG2 cells were obtained from ATCC and grown in DMEM containing 10% FCS with streptomycin (100μg/mL) and penicillin (100 U/ml). For long-term culturing, mycoplasma test was performed every month for all cell lines. The cells were incubated at 37℃,5% CO2. Cells were seeded in 6-or 12-well plates or 60-mm dishes and grown to 80-90% confluence before use.3.2 Human HepG2 cells were cultured in 25-cm2 vented flasks containing Dulbecco’s modified Eagle’s medium with 10% fetal calf serum under (5% CO2,37℃)standard culture conditions. Packed empty LV vectors with green fluorescent protein (GFP; LV-Mock) and LV-mediated hsa-miR-573 overexpression vector (LV-miR-573) with GFP were prepared as described previously. Cells were infected with the LV stock at a multiplicity of infection of 20 transducing units per cell in the presence of 8 mg/mL of polybrene. Then cells were washed with fresh complete media after 24 h of incubation. The GFP-positive cells were counted 96 h after transduction.3.3 Human HepG2 cells infected with the LV stock or infected with the LV-miR-573 were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to western blot analyses using rabbit polyclonal anti-apoM or rabbit polyclonal anti-β-actin antibody. The proteins were visualized using a chemiluminescence method.3.4 Transwell chambers were used for the invasion or migration assays. For the invasion assays, matrigel-coated chambers were placed in empty wells of 24-well tissue culture plates. Human HepG2 cells infected with the LV stock or infected with the LV-miR-573 were cultured in a serum-free medium for 24 h, then trypsinized and suspended with a culture medium containing 0.1% bovine serum albumin at a concentration of 5×104 cells/mL. Then,500 μL of suspension of Human HepG2 cells infected with the LV stock or infected with the LV-miR-573 was added into each chamber and 750 μL of complete medium was added into each well. After incubation for 36 h, the non-invading cells were removed from the upper surface of the chamber membrane using cotton-tipped swabs. The cells on the lower surface of the chamber membrane were fixed in 4% paraformaldehyde for 10 min, stained with crystal violet, then counted after photographing the membrane through a microscope. For the migration assays, non-coated chambers were used and migrated Human HepG2 cells infected with the LV stock or infected with the LV-miR-573 on the lower surface of the chamber membrane were fixed after a 24-h incubation, stained, then photographed for further analysis.3.5 The CCK8 was used to measure cell viability in order to evaluate cell proliferation in Human HepG2 cells infected with the LV stock or infected with the LV-miR-573. Cells treated with LV-Mock and LV-miR-573, respectively, seeded into 96-well culture plates in triplicate. At various time points (0,12,24,36, or 48 h),10 μL of CCK-8 solution were added to each well in an assay plate and incubated in a CO2 incubator for 1 h. And then,10 μL of 1% (w/v) SDS solution was added to each well to stop the reaction and the plate was read by a microplate reader for optical density at 450 nm (Thermo Electron Corporation, Marietta, OH, USA).3.6 Human HepG2 cells infected with the LV stock or infected with the LV-miR-573 were harvested. Cell cycle analysis and apoptosis quantification were performed by flow cytometry using a FACScan flow cytometer equipped with an argon-ion laser (488 nm). Cell cycle analysis was performed by flow cytometry using a propidium iodide (PI) cell cycle detection kit according to the manufacturer’s instructions. Cell apoptosis analysis was performed by flow cytometry using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.4. Effect of Hsa-miR-573 and ApoM on apoptotic mechanism of HepG2 cells in vitro.4.1 Human HepG2 cells were obtained from ATCC and grown in DMEM containing 10% FCS with streptomycin (100μg/mL) and penicillin (100 U/ml). For long-term culturing, mycoplasma test was performed every month for all cell lines. Cells were incubated at 37℃,5% CO2.The cells were seeded in 6-or 12-well plates or 60-mm dishes and grown to 80-90% confluence before use.4.2 The cells were cultured in 25-cm2 vented flasks containing Dulbecco’s modified Eagle’s medium with 10% fetal calf serum under standard culture conditions (5% CO2,37℃). Packed empty LV vectors with green fluorescent protein (GFP; LV-Mock), LV-mediated human apoM overexpression vector (LV-apoM), or LV-mediated hsa-miR-573 overexpression vector (LV-miR-573) with GFP were prepared as described previously. The cells were infected with the LV stock at a multiplicity of infection of 20 transducing units per cell in the presence of 8 mg/mL of polybrene. Cells were washed with fresh complete media after 24 h of incubation. The GFP-positive cells were counted 96 h post-transduction.4.3 Total RNA from Human HepG2 cells infected with the LV stock or infected with the LV-Mock or infected with the LV-miR-573 was extracted using TRIzol reagent in accordance with the manufacturer’s instructions. The mRNA levels were evaluated by real-time quantitative PCR using an ABI 7500 Fast Real Time PCR system with SYBR Green detection chemistry (TaKaRa Bio, Inc., Shiga, Japan). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as the internal control. Quantitative measurements were determined using the △△Ct method. All samples were measured in triplicate and the mean value was considered for comparative analysis. Then, we analyzed gene expression of caspasel (CASPl), CASP2, CASP3, CASP4, CASP5, CASP6, CASP8, CASP9, CASP10, P53, P63, P73, B-cell lymphoma (Bcl)-2, Bcl2-A1, Bcl2-L1, Bcl2-L2, Bcl-XL, Bcl-2 homologous antagonist/killer (Bak), Bakl, Bcl-2-like protein 4 (Bax), bicaudal homolog (Bik), myelocytomatosis viral oncogene homolog (c-myc), survivin, and induced myeloid leukemia cell differentiation (mcl-1) in HepG2 cells.4.4 The PIRES2-EGFP and PCR-XL-TOPO vectors (containing Bcl2A1, which was assembled by the chemically synthesized oligos via PCR) were purchased from Invitrogen. Segments of Eco-RI-Bcl2A1 and IRES-EGFP-XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. Eco-RI-Bcl2A1-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested using the EcoRI/XhoI endonucleases, incorporated into the pcDNA3.1(+) vector, and then transformed into competent Escherichia coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-Bcl2A1. The plasmid transfection process was performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.4.5 HepG2 cells were treated with:(1) LV-apoM, (2) LV-apoM+ pcDNA3.1-Bcl2Al,(3) LV-miR-573, (4) LV-miR-573+pcDNA3.1-Bcl2A1.Then, Cells were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to western blot analyses using rabbit polyclonal anti-apoM and anti-Bcl2A1 antibodies (Proteintech Group, Inc., Chicago, IL, USA), and rabbit polyclonal anti-β-actin antibody. The proteins were visualized using a chemiluminescence method.4.6 HepG2 cells were treated with:(1) LV-apoM, (2) LV-apoM+ pcDNA3.1-Bcl2A1,(3) LV-miR-573, (4) LV-miR-573+pcDNA3.1-Bcl2A1.Then, Cells were harvested and cell cycle analysis and apoptosis quantification were performed by flow cytometry using a FACScan flow cytometer equipped with an argon-ion laser (488 nm). Cell cycle analysis was performed by flow cytometry using a propidium iodide (PI) cell cycle detection kit according to the manufacturer’s instructions. Cell apoptosis analysis was performed by flow cytometry using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.5. Effect of ApoM and Hsa-miR-573 on migration, invasion, proliferation and promoted apoptosis of Hep3B cell and SK-Hep-1 cell in vitro.5.1 Hep3B cells and SK-Hep-1 cells were obtained from ATCC and grown in DMEM containing 10% FCS with streptomycin (100μg/mL) and penicillin (100 U/ml). For long-term culturing, mycoplasma test was performed every month for all cell lines. The cells were incubated at 37℃,5%CO2. Cells were seeded in 6- or 12-well plates or 60-mm dishes and grown to 80-90% confluence before use.5.2 Hep3B cells and SK-Hep-1 cells were cultured in 25-cm2 vented flasks containing Dulbecco’s modified Eagle’s medium with 10% fetal calf serum under standard culture conditions (5% CO2,37℃). Packed empty LV vectors with green fluorescent protein (GFP; LV-Mock), LV-mediated human apoM overexpression vector (LV-apoM), or LV-mediated hsa-miR-573 overexpression vector (LV-miR-573) with GFP were prepared as described previously. Cells were infected with the LV stock at a multiplicity of infection of 20 transducing units per cell in the presence of 8 mg/mL of polybrene.Cells were washed with fresh complete media after 24 h of incubation. The GFP-positive cells were counted 96 h post-transduction.5.3 The cells were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to western blot analyses using rabbit polyclonal anti-apoM or anti-Bcl2A1 antibodies, and rabbit polyclonal anti-β-actin antibody. The proteins were visualized using a chemiluminescence method.5.4 Transwell chambers were used for the invasion and migration assays. For the invasion assays, matrigel-coated chambers were placed in empty wells of 24-well tissue culture plates. Hep3B cells and SK-Hep-1 cells infected with the LV stock or infected with the LV-apoM or infected with the LV-miR-573 were cultured in a serum-free medium for 24 h, then trypsinized and suspended with a culture medium containing 0.1% bovine serum albumin at a concentration of 5×104 cells/mL. And then,500 μL of suspension of Hep3B cells or SK-Hep-1 cells infected with the LV stock or infected with the LV-apoM or infected with the LV-miR-573 was added into each chamber and 750 μL of complete medium was added into each well. After incubation for 36 h, Non-invading cells were removed from the upper surface of the chamber membrane using cotton-tipped swabs. Cells on the lower surface of the chamber membrane were fixed in 4% paraformaldehyde for 10 min, stained with crystal violet, Then counted after photographing the membrane through a microscope. For the migration assays, non-coated chambers were used and migrated Hep3B cells and SK-Hep-1 cells infected with the LV stock or infected with the LV-apoM or infected with the LV-miR-573 on the lower surface of the chamber membrane were fixed after a 24-h incubation, stained, then photographed for further analysis.5.5 The Cell Counting Kit-8 was used to measure cell viability in order to evaluate cell proliferation in Hep3B cells and SK-Hep-1 cells infected with the LV stock or infected with the LV-apoM or infected with the LV-miR-573. The cells treated with LV-Mock or LV-apoM or LV-miR-573, respectively, seeded into 96-well culture plates in triplicate. At various time points (0,12,24,36, and 48 h),10 μL of CCK-8 solution were added to each well in an assay plate and incubated in a CO2 incubator for 1 h. And then,10 μL of 1%(w/v) SDS solution was added to each well to stop the reaction and the plate was read by a microplate reader for optical density at 450 nm.5.6 Hep3B cells and SK-Hep-1 cells infected with the LV stock or infected with the LV-apoM or infected with the LV-miR-573 were harvested and cell cycle analysis and apoptosis quantification were performed by flow cytometry using a FACScan flow cytometer equipped with an argon-ion laser (488 nm). Cell cycle analysis was performed by flow cytometry using a propidium iodide (PI) cell cycle detection kit according to the manufacturer’s instructions. Cell apoptosis analysis was performed by flow cytometry using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.6. Effect of hsa-miR-573 and apoM on tumor growth in nude mice.6.1 All studies were performed in accordance with protocols approved by the Animal Use and Care Committee of Nanfang Hospital. Athymic nude mice (BALB/c, specific pathogen-free grade, male, about 16 g,4 weeks old) were randomized into three groups (LV-Mock (n=15), LV-apoM group (n= 15), and LV-miR-573(n=15)) and housed five per cage at 25℃ under a 12-h light/dark cycle. All mice were fed with autoclaved mouse chow. The inoculation dosage was 1×107HepG2 cells in 200 μL of phosphate-buffered saline per mouse. Mice in the LV-Mock group were subcutaneously injected with control LV (LV-Mock), those in the LV-apoM group were subcutaneously injected with LVs overexpressing apoM (LV-apoM), and mice in the LV-miR-573 group were subcutaneously injected with LVs overexpressing hsa-miR-573 (LV-miR-573). The body weights of all of nude mice and metastatic tumors were measured every 3 days. All mice were sacrificed and the formed tumors were removed, fixed, and embedded in paraffin.6.2 Xenograft tumors were harvested and protein extracts prepared according to established methods. Extracts were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then subjected to western blot analyses using rabbit polyclonal anti-apoM and anti-Bcl2A1 antibodies, and rabbit polyclonal anti-β-actin antibody. The proteins were visualized using a chemiluminescence method.6.3 The hepatocytes isolated from the xenograft tumors were harvested and cell cycle analysis and apoptosis quantification were performed by flow cytometry using a FACScan flow cytometer equipped with an argon-ion laser (488 nm). Cell cycle analysis was performed by flow cytometry using a propidium iodide (PI) cell cycle detection kit according to the manufacturer’s instructions. Cell apoptosis analysis was performed by flow cytometry using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.7. Whether Serum apoM concentration show a correlation with clinical and biochemical characteristics in HCC patients.7.1 A total of 655 serum samples were selected from Laboratory Medicine Center of Nanfang Hospital between October 2012 and April 2013. Based on clinical diagnosis results,655 serum samples were divided into four subject groups:181 HCC patients, 182 chronic hepatitis B patients,63 chronic hepatitis C patients, and 89 liver cirrhosis patients, and 140 normal subjects. All subjects in the control group had normal aminotransferase activities, no history of liver disease or alcohol abuse, and were negative for HBV, HCV, and HIV infections. The characteristics of all subjects are shown in Table 1.7.2 The serum apoM concentrations were quantitatively measured in duplicate using a commercial enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science Inc., Wuhan, China). The optical density at 450 nm was measured on a microtiter plate reader (Titertek Multiskan MC, Sterling, VA, USA) and the apoM concentrations were determined by linear regression from a standard curve using the apoM supplied with the kit to prepare the standards. The alanine transaminase (ALT), aspartate transaminase (AST), alpha-fetoprotein (AFP), HBsAg-T, and HBV-DNA concentrations were determined using an automated analyzer. We also analyzed the univariate associations between serum apoM concentration and age.7.3 Receiver operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated to evaluate specificity and sensitivity of predictive value or feasibility of using serum apoM as a marker for liver disease progression.7.4 We further validated hsa-miR-573 in a cohort of sera from healthy volunteers and subjects with CHB, CHC, cirrhosis, and HCC as indicated above. Real-time PCR was performed on a real-time PCR ABI 7500 Fast system. The expression of U6 RNA was used as an endogenous control. For serum hsa-miR-573 analysis, synthetic spiked-in Caenorhabditis elegans miR-39 was added to the serum samples prior to RNA extraction as an internal control.Results1. Effect of ApoM on migration, invasion, proliferation and promoted apoptosis of HepG2 cell in vitro.Jiang et al. have demonstrated that apoM mRNA and protein levels in the HCC tissues were significantly lower than those in the adjacent tissue. Since the apoM has been implicated in HCC pathogenesis, we first examined the effect of apoM on migration and invasive capacity in HepG2 cells. HepG2 cells were infected with empty LV vectors (LV-Mock) or human apoM overexpression LV vectors (LV-apoM). ApoM protein expression in HepG2 cells was assessed by western blot analyses. In comparison to the LV-Mock treated cells, apoM expression was markedly increased in LV-apoM treated cells. The transwell assay was employed to investigate the effect of apoM on HepG2 cell migration and invasion. HepG2 cells infected with LV-apoM displayed significantly reduced migration and invasion abilities compared to HepG2 cells infected with LV-Mock.Next, we determined whether apoM expression influenced HepG2 cells survival or proliferation in vitro using the CCK-8 assay, which showed that apoM LV constructs slightly decreased the proportion of living HepG2 cells. We further investigated the effects of apoM on the cell cycle via flow cytometry. There was an increase of the percentage of cells in the G1 phase, a decrease of those in the S phase, and no change in those in the G2M phase post-infection as compared to control cells. Apoptosis in HepG2 cells expressing the LV-Mock and LV-apoM vectors was evaluated using co-labeling with annexin V (AV) and propidium iodide (PI) via flow cytometry. Treatment with the LV-apoM vector significantly increased the percentage of apoptotic cells in comparison to treatment with the LV-Mock vector in HepG2 cells (86.48±2.33 vs.3.31±0.56, p=0.000). Together, these data strongly suggest important pathological roles of apoM in HCC.2. Effect of Hsa-miR-573 on apoM expression in vitro.Since it has been showed that both apoM mRNA and protein levels in the HCC tissues were significantly lower than those in the adjacent tissue and microRNAs (miRNAs) are small noncoding RNA molecules that inhibiting gene expression by interacting with the 3’untranslated region of target mRNAs. Thus, we presumed that miRNAs may involve in the regulation of apoM expression. We first used 4 target prediction programs, miRBase, PicTar, TargetScan, and RNAhybrid, to screen for miRNAs that target apoM. Our analysis predicted 9 potential apoM-targeting miRNAs, hsa-miR-145-5p, hsa-miR-145-3p, hsa-miR-221-5p, hsa-miR-221-3p, hsa-miR-222-5p, hsa-miR-222-3p, hsa-miR-766-5p, hsa-miR-766-3p, and hsa-miR-573. We next investigated the effects of mimics of the nine putative miRNAs on apoM expression in HepG2 cells. Mimics of hsa-miR-145-5p, hsa-miR-145-3p, hsa-miR-222-5p, hsa-miR-222-3p, hsa-miR-766-5p, hsa-miR-766-3p, and hsa-miR-573 downregulated apoM mRNA expression. In addition, mimics of hsa-miR-145-3p, hsa-miR-766-5p, hsa-miR-766-3p, and hsa-miR-573 downregulated apoM protein expression. Since both apoM mRNA and protein levels were significantly decreased by hsa-miR-573 mimic expression and the minimum free energy of hsa-miR-573 was-20.7 kcal/mol, we further explored whether apoM could be directly regulated by hsa-miR-573 using a luciferase assay. After co-transfection with hsa-miR-573 and pMIR-apoM-wild vectors, the luciferase activity was significantly decreased. This effect was completely abolished by substituting this vector with its mutant version. Taken together, these results suggest that hsa-miR-573 inhibits apoM expression by directly targeting its 3’-UTR.3. Effect of Hsa-miR-573 on invasion, migration, proliferation and promoted apoptosis of HepG2 cell in vitro.Since apoM is a target for hsa-miR-573 and takes part in regulating invasion, migration, proliferation and apoptosis of HepG2 cells, we then investigated the effects of hsa-miR-573 on invasion, migration, proliferation and apoptosis in HepG2 cells. HepG2 cells were infected with empty LV vectors (LV-Mock) or hsa-miR-573 overexpression LV vectors (LV-miR-573). ApoM protein expression in HepG2 cells was assessed by western blot analyses. In comparison to the LV-Mock-treated cells, apoM expression was markedly decreased in LV-miR-573-treated cells. Transwell assay revealed that LV-miR-573-infected hepG2 cells displayed a significantly increased ability to migrate and invade in comparison with LV-Mock-infected HepG2 cells. Also, the CCK-8 assay found that LV-miR-573 treatment significantly increased the pro... |
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