Introduction

Hepatocellular carcinoma (HCC) is a kind of clinical common malignant tumour with an insidious onset, which is invasively fast-growing and has a poor prognosis [1]. Although surgical excision was demonstrated to be the first choice for HCC treatment, most HCC is not diagnosed until the advanced stage of the disease, when surgical treatments are not suitable for treating the disease [2]. Therefore, early detection and treatment are key to improving therapeutic outcomes, reducing mortality, and increasing the long-term survival rate in HCC patients.

Alpha-fetoprotein (AFP) was the only widely accepted and applied biomarker in clinical practice because of its practical value for the diagnosis and monitoring of the development of HCC. However, the AFP method experienced insufficient sensitivity and specificity in the early diagnosis of HCC. Meanwhile, the AFP level is also easily affected by other diseases, such as hepatitis during pregnancy and liver regeneration after damage, which increases the inaccuracy of clinical diagnoses [35]. There is, therefore, an urgent need to identify new biomarkers with high sensitivity and high specificity for the early diagnosis of HCC.

Proteomics technology platforms are an extremely useful tool for the discovery of new cancer biomarkers. A highly desirable biomarker for cancer screening and monitoring would be a biomarker that can be measured in body fluid samples [6]. Accordingly, blood samples such as serum and plasma have been the ideal targets of proteomics studies aimed at identifying cancer diagnostic and prognostic biomarkers [7, 8]. However, several challenges have hindered the progress of these studies. The main 2 reasons include the complex nature of serum and plasma samples and the large dynamic range between the concentrations of different proteins.

Secreted proteins play important roles in signal transduction, cellular growth, proliferation, differentiation, and apoptosis. They are also important in tumourigenesis, development, invasion, and metastasis of HCC [9]. Therefore, the secretomes of cell lines are also performed during screening. Many researchers have reported the application of secretomes in the screening of diagnostic and prognostic protein biomarkers [1012]. Essentially, it is well established that any potential biomarker candidates screened from HCC cell lines should be ultimately validated in clinical tissue samples that are closer to tumours than any of the model systems. As a result, it is more direct and convincing to utilise the primary culture of tumour tissues and the proteomic analysis of serum-free conditioned media to search the diagnostic or prognostic biomarkers [13, 14].

We, thus, conducted this study to investigate the molecular signatures of HCC by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Material and methods

Sample collection and tissue culture in vitro

In our study, the HCC tissue group, the adjacent noncancerous tissue (AN) group, and the distal noncancerous tissue (DN) group were obtained from 2 primary HCC patients who were diagnosed with HCC by post-operative pathological examinations and subjected to standard radical resection. The fresh tissues were collected at the time of surgery from the HCC patients and immediately washed with phosphate-buffered saline in a sterile environment. Subsequently, the tissues were cut into 2 mm3 pieces, washed several times until the tissues became colourless, and then cultured in a Dulbecco’s modified eagle serum-free medium at 5% CO2 for 24 h. Thereafter, the supernatants were collected for protein extraction. This study was approved by the Ethics Committee of our hospital, and the 2 patients signed informed consent forms.

Protein extraction and digestion

The collected culture supernatant was centrifuged at a low speed (200 g) to remove the cells and tissue debris and then filtered with a 0.22 µm filter membrane to remove the residual cells. Thereafter, the filtrate was concentrated with 3K ultrafiltration until the phenolic red colour was completely removed. The proteins were precipitated by ice-cold acetone, and the protein concentration of the supernatant was determined by bicinchoninic acid assay following the manufacturer’s protocol. Subsequently, 4 µl of a reducing reagent was added to each sample tube and vortex to mix and incubate the tubes at 60°C for 1 h, and 2 µl of a cysteine blocking reagent was added to each tube and vortex to mix and incubate the tubes at room temperature for 10 min. Finally, the proteins were digested by sequence-grade modified trypsin through filter-aided sample preparation.

Isobaric tags for relative and absolute quantification labelling

The peptides from 100 µg proteins per group were labelled according to the Applied Biosystems iTRAQ™ reagent chemistry reference guide. The peptides were labelled as follows: 2 HCC groups were labelled 113 and 114, 2 AN groups were labelled 114 and 115, 2 DN groups were labelled 116 and 117, and the mixed DN group samples with an equal amount were labelled 119 and 121. The labeled peptides were mixed with an equal amount and dried in a vacuum centrifuge for further usage.

High pH reversed-phase separation

The dried peptide mixture was fractionated by high pH separation using ekspertTM ultraLC 100 pump. Mobile phase A: 20 mM ammonium formate in water, mobile phase B: 20 mM ammonium formate in 80% ACN, the pH was adjusted to 10.0 with ammonium hydroxide. High pH (pH = 10) separation was performed using a 65-min linear gradient as follows: 0–5 min, 0–5% B; 5–30 min, 5–15% B; 30–45 min, 15–38% B; 45–46 min, 38–90% B, 46–54.5 min, 90–90% B; 54.5–55 min, 90–5% B; 55–65 min, 5–5% B. Finally, 40 fractions were collected, and 4 fractions with the same time interval were pooled together to reduce the fraction numbers, such as 1, 2 and 21, 22 and 3, 4 and 23, 24, and so on [15]. Ten fractions at the end were dried in a vacuum concentrator for further usage.

The Nano-LC-MS/MS analysis

The fractions were re-suspended with 30 µl solution A (solution A: 0.1% FA and 2% ACN in water) and 8 µl was loaded on an exigent nano LC-UltraTM system nano-LC with a trap column (ChromXP C18-CL-3 µm, 120A, 350 µm × 0.5 µm) with a flow of 2 µl/min. The column flow rate was maintained at 300 nl/min with a 101 min linear gradient as follows: 0–0.1 min, 5–10% B; 0.1–60 min, 10–25% B; 60–85 min, 25–48% B; 85–86 min, 48–80% B, 86–90 min, 80–80% B; 90–91 min, 80–5% B; 91–101 min, 5–5% B (solution B: 0.1% FA and 2% ACN in water). The MS data were collected by the Triple TOF 5600 system. The electrospray voltage of 2.3 kV and 150°C heating at the inlet of the mass spectrometer was used. The resolution was set at 30,000 with the scan range of 300–1500 m/z. The cumulative scanning time was 250 ms in the high-resolution scanning mode, and up to 40 sub-ion scans could be performed each time. Each Fraction was repeated three times with instrumental analysis, and all parent ions were collision-induced dissociation using fluctuating collision energy.

Data analysis

The MS data were processed using ProteinPilot 4.5 (AB SCIX, Foster City, CA, USA) and then searched using Mascot (version 2.2; Matrix Science, London, United Kingdom) search algorithms against the UniProt human database. The enzyme specificity of trypsin was used and up to a maximum of 2 missed cleavages were allowed for protease digestion. Mascot was searched with a parent ion tolerance of 10 parts per million (ppm) and a fragment ion mass tolerance of 0.05 Da. Carbamidomethylation of cysteine, as well as iTRAQ modification of peptide N-terminus and lysine residues, were set as a fixed modification; oxidation of methionine and iTRAQ 8-plex labelling of tyrosine were specified as variable modifications. The proteins were accepted if the protein FDR was < 1%. To identify proteins whose expression was significantly altered in the 2 different groups, a threshold of the iTRAQ ratios were used to define differentially expressed proteins. The proteins were considered to be differentially expressed if the iTRAQ ratio was > 1.5 or < 0.67 in the 2 different groups with the p-value of < 0.05, which were statistically analysed by a paired T-test. The gene ontology (GO) annotation and pathway enrichment analysis of the differentially expressed proteins were carried out using the online tool DAVID (http://david.abcc.ncifcrf.gov/). The gene ontology annotation contains biological processes, cell components, and molecular functions. The pathway analysis was based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The gene ontology annotations and signalling pathways were ranked in terms of the enrichment or number of the differentially expressed proteins. The protein and protein interaction was performed using the online String database (https://string-db.org/).

Results

The relative quantification of the secretome of the primary hepatocellular carcinoma patients

In this study, total proteins were extracted from the collected tumours, their adjacent noncancerous tissues and their distal noncancerous tissues were taken from patients and analysed using iTRAQ 2D LC-MS/MS, and the workflow as described in Figure 1. In total, we quantified 5214 proteins, of which 190 and 44 proteins were classified as differentially expressed in the HCC tissues/distal noncancerous tissues (HCC/DN) group and the adjacent noncancerous tissues/distal noncancerous tissues (AN/DN) group (Tables I and II). As is evident in Figure 2A, the number of differentially expressed proteins identified in the HCC/DN group was much higher than that in the AN/DN group.

Table I

Differentially expressed proteins identified between hepatocellular carcinoma tissues and distal noncancerous tissues

No.AccessionNameFCP-value
1sp|P08670|VIME_HUMANVimentin OS = Homo sapiens GN = VIM PE = 1 SV = 44.8752850.00000000486
2sp|P16615|AT2A2_HUMANSarcoplasmic/endoplasmic reticulum calcium ATPase 2 OS = Homo sapiens GN = ATP2A2 PE = 1 SV = 14.6558610.000000146
3sp|P07602|SAP_HUMANProsaposin OS = Homo sapiens GN = PSAP PE = 1 SV = 24.6131760.00058
4sp|P10809|CH60_HUMAN60 kDa heat shock protein, mitochondrial OS = Homo sapiens GN = HSPD1 PE = 1 SV = 24.4874540.0000000000382
5sp|P14314|GLU2B_HUMANGlucosidase 2 subunit beta OS = Homo sapiens GN = PRKCSH PE = 1 SV = 23.8725760.000281
6sp|P31327|CPSM_HUMANCarbamoyl-phosphate synthase [ammonia], mitochondrial OS = Homo sapiens GN = CPS1 PE = 1 SV = 23.5318320.0000000000162
7sp|Q10471|GALT2_HUMANPolypeptide N-acetylgalactosaminyltransferase 2 OS = Homo sapiens GN = GALNT2 PE = 1 SV = 13.435580.00000579
8sp|Q04695|K1C17_HUMANKeratin, type I cytoskeletal 17 OS = Homo sapiens GN = KRT17 PE = 1 SV = 23.3113110.000898
9sp|P05783|K1C18_HUMANKeratin, type I cytoskeletal 18 OS = Homo sapiens GN = KRT18 PE = 1 SV = 23.1622780.0000367
10sp|P32004|L1CAM_HUMANNeural cell adhesion molecule L1 OS = Homo sapiens GN = L1CAM PE = 1 SV = 23.1332860.00000598
11sp|P27797|CALR_HUMANCalreticulin OS = Homo sapiens GN = CALR PE = 1 SV = 13.0760970.00000882
12sp|P07237|PDIA1_HUMANProtein disulphide-isomerase OS = Homo sapiens GN = P4HB PE = 1 SV = 33.0199520.000287
13sp|P55084|ECHB_HUMANTrifunctional enzyme subunit beta, mitochondrial OS = Homo sapiens GN = HADHB PE = 1 SV = 32.857590.000323
14sp|P80723|BASP1_HUMANBrain acid soluble protein 1 OS = Homo sapiens GN = BASP1 PE = 1 SV = 22.857590.0000445
15sp|Q14697|GANAB_HUMANNeutral alpha-glucosidase AB OS = Homo sapiens GN = GANAB PE = 1 SV = 32.8054340.00000569
16sp|Q00839|HNRPU_HUMANHeterogeneous nuclear ribonucleoprotein U OS = Homo sapiens GN = HNRNPU PE = 1 SV = 62.7289780.000519
17sp|Q12931|TRAP1_HUMANHeat shock protein 75 kDa, mitochondrial OS = Homo sapiens GN = TRAP1 PE = 1SV = 32.6791680.000193
18sp|P14625|ENPL_HUMANEndoplasmin OS = Homo sapiens GN = HSP90B1 PE = 1 SV = 12.6302680.0000362
19sp|P40939|ECHA_HUMANTrifunctional enzyme subunit alpha, mitochondrial OS = Homo sapiens GN = HADHA PE = 1 SV = 22.6302680.000143
20sp|P27824|CALX_HUMANCalnexin OS = Homo sapiens GN = CANX PE = 1 SV = 22.6302680.0000476
21sp|P42704|LPPRC_HUMANLeucine-rich PPR motif-containing protein, mitochondrial OS = Homo sapiens GN = LRPPRC PE = 1 SV = 32.5585860.0000000421
22sp|P06576|ATPB_HUMANATP synthase subunit beta, mitochondrial OS = Homo sapiens GN = ATP5B PE = 1 SV = 32.5585860.00071
23sp|Q8TEM1|PO210_HUMANNuclear pore membrane glycoprotein 210 OS = Homo sapiens GN = NUP210 PE = 1 SV = 32.5585860.000049
24sp|P05023|AT1A1_HUMANSodium/potassium-transporting ATPase subunit alpha-1 OS = Homo sapiens GN = ATP1A1 PE = 1 SV = 12.5351290.000341
25sp|P02545|LMNA_HUMANPrelamin-A/C OS = Homo sapiens GN = LMNA PE = 1 SV = 12.5118860.000397
26sp|Q9NR30|DDX21_HUMANNucleolar RNA helicase 2 OS = Homo sapiens GN = DDX21 PE = 1 SV = 52.5118860.000515
27sp|P02786|TFR1_HUMANTransferrin receptor protein 1 OS = Homo sapiens GN = TFRC PE = 1 SV = 22.4660390.000549
28sp|P49792|RBP2_HUMANE3 SUMO-protein ligase RanBP2 OS = Homo sapiens GN = RANBP2 PE = 1 SV = 22.4434310.000679
29sp|Q86UP2|KTN1_HUMANKinectin OS = Homo sapiens GN = KTN1 PE = 1 SV = 12.4210290.000000491
30sp|Q07065|CKAP4_HUMANCytoskeleton-associated protein 4 OS = Homo sapiens GN = CKAP4 PE = 1 SV = 22.4210290.00000087
31sp|P11021|GRP78_HUMAN78 kDa glucose-regulated protein OS = Homo sapiens GN = HSPA5 PE = 1 SV = 22.3988330.0000000146
32sp|Q9P2E9|RRBP1_HUMANRibosome-binding protein 1 OS = Homo sapiens GN = RRBP1 PE = 1 SV = 42.3988330.000000948
33sp|P52272|HNRPM_HUMANHeterogeneous nuclear ribonucleoprotein M OS = Homo sapiens GN = HNRNPM PE = 1 SV = 32.2080050.000000866
34sp|P04843|RPN1_HUMANDolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 OS = Homo sapiens GN = RPN1 PE = 1 SV = 12.2080050.00056
35sp|Q9UQ35|SRRM2_HUMANSerine/arginine repetitive matrix protein 2 OS = Homo sapiens GN = SRRM2 PE = 1 SV = 22.1677040.0000419
36sp|P08195|4F2_HUMAN4F2 cell-surface antigen heavy chain OS = Homo sapiens GN = SLC3A2 PE = 1 SV = 32.1086280.000497
37sp|Q16891|MIC60_HUMANMICOS complex subunit MIC60 OS = Homo sapiens GN = IMMT PE = 1 SV = 12.1086280.00000733
38sp|Q96RP9|EFGM_HUMANElongation factor G, mitochondrial OS = Homo sapiens GN = GFM1 PE = 1 SV = 22.1086280.000905
39sp|P78527|PRKDC_HUMANDNA-dependent protein kinase catalytic subunit OS = Homo sapiens GN = PRKDC PE = 1 SV = 32.0892960.000000377
40sp|Q9NSE4|SYIM_HUMANIsoleucine--tRNA ligase, mitochondrial OS = Homo sapiens GN = IARS2 PE = 1 SV = 22.0701410.000341
41sp|Q9H0D6|XRN2_HUMAN5'-3' exoribonuclease 2 OS = Homo sapiens GN = XRN2 PE = 1 SV = 12.0511620.000538
42sp|Q9NZM1|MYOF_HUMANMyoferlin OS = Homo sapiens GN = MYOF PE = 1 SV = 12.0137240.000021
43sp|Q08211|DHX9_HUMANATP-dependent RNA helicase A OS = Homo sapiens GN = DHX9 PE = 1 SV = 42.0137240.000672
44sp|P38646|GRP75_HUMANStress-70 protein, mitochondrial OS = Homo sapiens GN = HSPA9 PE = 1 SV = 21.9952620.00000556
45sp|P25705|ATPA_HUMANATP synthase subunit alpha, mitochondrial OS = Homo sapiens GN = ATP5A1 PE = 1 SV = 11.9952620.0000861
46sp|P13667|PDIA4_HUMANProtein disulphide-isomerase A4 OS = Homo sapiens GN = PDIA4 PE = 1 SV = 21.9588450.00000668
47sp|P06748|NPM_HUMANNucleophosmin OS = Homo sapiens GN = NPM1 PE = 1 SV = 21.9408860.0000862
48sp|P55265|DSRAD_HUMANDouble-stranded RNA-specific adenosine deaminase OS = Homo sapiens GN = ADAR PE = 1 SV = 41.9230920.000118
49sp|Q9UHB6|LIMA1_HUMANLIM domain and actin-binding protein 1 OS = Homo sapiens GN = LIMA1 PE = 1 SV = 11.9230920.000813
50sp|Q13423|NNTM_HUMANNAD(P) transhydrogenase, mitochondrial OS = Homo sapiens GN = NNT PE = 1 SV = 31.8535320.0000358
51sp|Q9Y2W1|TR150_HUMANThyroid hormone receptor-associated protein 3 OS = Homo sapiens GN = THRAP3 PE = 1 SV = 21.8365380.000124
52sp|Q13263|TIF1B_HUMANTranscription intermediary factor 1-beta OS = Homo sapiens GN = TRIM28 PE = 1 SV = 51.7864880.000475
53sp|Q15149|PLEC_HUMANPlectin OS = Homo sapiens GN = PLEC PE = 1 SV = 31.5848930.000000000695
54sp|Q13813|SPTN1_HUMANSpectrin alpha chain, non-erythrocytic 1 OS = Homo sapiens GN = SPTAN1 PE = 1 SV = 31.5559660.0000874
55sp|Q9Y490|TLN1_HUMANTalin-1 OS = Homo sapiens GN = TLN1 PE = 1 SV = 30.602560.0000353
56sp|Q14315|FLNC_HUMANFilamin-C OS = Homo sapiens GN = FLNC PE = 1 SV = 30.5915620.00000147
57sp|Q5T4S7|UBR4_HUMANE3 ubiquitin-protein ligase UBR4 OS = Homo sapiens GN = UBR4 PE = 1 SV = 10.5597580.000653
58sp|Q14152|EIF3A_HUMANEukaryotic translation initiation factor 3 subunit A OS = Homo sapiens GN = EIF3A PE = 1 SV = 10.5445030.000563
59sp|Q14974|IMB1_HUMANImportin subunit beta-1 OS = Homo sapiens GN = KPNB1 PE = 1 SV = 20.5152290.000226
60sp|P41091|IF2G_HUMANEukaryotic translation initiation factor 2 subunit 3 OS = Homo sapiens GN = EIF2S3 PE = 1 SV = 30.5105050.000686
61sp|P53621|COPA_HUMANCoatomer subunit alpha OS = Homo sapiens GN = COPA PE = 1 SV = 20.5011870.000472
62sp|Q16851|UGPA_HUMANUTP--glucose-1-phosphate uridylyltransferase OS = Homo sapiens GN = UGP2 PE = 1 SV = 50.5011870.000532
63sp|Q96P70|IPO9_HUMANImportin-9 OS = Homo sapiens GN = IPO9 PE = 1 SV = 30.5011870.000168
64sp|Q8WUM4|PDC6I_HUMANProgrammed cell death 6-interacting protein OS = Homo sapiens GN = PDCD6IP PE = 1 SV = 10.492040.0000198
65sp|P46940|IQGA1_HUMANRas GTPase-activating-like protein IQGAP1 OS = Homo sapiens GN = IQGAP1 PE = 1 SV = 10.4875290.000000907
66sp|Q92973|TNPO1_HUMANTransportin-1 OS = Homo sapiens GN = TNPO1 PE = 1 SV = 20.4830590.000241
67sp|Q92598|HS105_HUMANHeat shock protein 105 kDa OS = Homo sapiens GN = HSPH1 PE = 1 SV = 10.478630.000511
68sp|Q14204|DYHC1_HUMANCytoplasmic dynein 1 heavy chain 1 OS = Homo sapiens GN = DYNC1H1 PE = 1 SV = 50.4698940.000000000000591
69sp|Q92616|GCN1_HUMANeIF-2-alpha kinase activator GCN1 OS = Homo sapiens GN = GCN1 PE = 1 SV = 60.4655860.00000189
70sp|P35579|MYH9_HUMANMyosin-9 OS = Homo sapiens GN = MYH9 PE = 1 SV = 40.4613180.00000467
71sp|P35606|COPB2_HUMANCoatomer subunit beta' OS = Homo sapiens GN = COPB2 PE = 1 SV = 20.4613180.000382
72sp|P27708|PYR1_HUMANCAD protein OS = Homo sapiens GN = CAD PE = 1 SV = 30.4528980.00000122
73sp|Q86VP6|CAND1_HUMANCullin-associated NEDD8-dissociated protein 1 OS = Homo sapiens GN = CAND1 PE = 1 SV = 20.4446310.00042
74sp|P20073|ANXA7_HUMANAnnexin A7 OS = Homo sapiens GN = ANXA7 PE = 1 SV = 30.424620.000415
75sp|Q13228|SBP1_HUMANSelenium-binding protein 1 OS = Homo sapiens GN = SELENBP1 PE = 1 SV = 20.4130470.00023
76sp|Q96AC1|FERM2_HUMANFermitin family homolog 2 OS = Homo sapiens GN = FERMT2 PE = 1 SV = 10.4055080.0000768
77sp|Q9UQ80|PA2G4_HUMANProliferation-associated protein 2G4 OS = Homo sapiens GN = PA2G4 PE = 1 SV = 30.4055080.000957
78sp|P46821|MAP1B_HUMANMicrotubule-associated protein 1B OS = Homo sapiens GN = MAP1B PE = 1 SV = 20.4017910.0000993
79sp|P40763|STAT3_HUMANSignal transducer and activator of transcription 3 OS = Homo sapiens GN = STAT3 PE = 1 SV = 20.3908410.000108
80sp|P22314|UBA1_HUMANUbiquitin-like modifier-activating enzyme 1 OS = Homo sapiens GN = UBA1 PE = 1 SV = 30.3872580.00000000036
81sp|P34932|HSP74_HUMANHeat shock 70 kDa protein 4 OS = Homo sapiens GN = HSPA4 PE = 1 SV = 40.3872580.000133
82sp|P62826|RAN_HUMANGTP-binding nuclear protein Ran OS = Homo sapiens GN = RAN PE = 1 SV = 30.3837070.000165
83sp|P23526|SAHH_HUMANAdenosylhomocysteinase OS = Homo sapiens GN = AHCY PE = 1 SV = 40.3837070.00032
84sp|P27816|MAP4_HUMANMicrotubule-associated protein 4 OS = Homo sapiens GN = MAP4 PE = 1 SV = 30.3801890.0000000706
85sp|P0DMV9|HS71B_HUMANHeat shock 70 kDa protein 1B OS = Homo sapiens GN = HSPA1B PE = 1 SV = 10.3664380.00000206
86sp|Q99832|TCPH_HUMANT-complex protein 1 subunit eta OS = Homo sapiens GN = CCT7 PE = 1 SV = 20.3630780.000659
87sp|Q99613|EIF3C_HUMANEukaryotic translation initiation factor 3 subunit C OS = Homo sapiens GN = EIF3C PE = 1 SV = 10.3564510.000267
88sp|P48643|TCPE_HUMANT-complex protein 1 subunit epsilon OS = Homo sapiens GN = CCT5 PE = 1 SV = 10.3531830.000000384
89sp|P78344|IF4G2_HUMANEukaryotic translation initiation factor 4 gamma 2 OS = Homo sapiens GN = EIF4G2 PE = 1 SV = 10.3531830.000055
90sp|P23921|RIR1_HUMANRibonucleoside-diphosphate reductase large subunit OS = Homo sapiens GN = RRM1 PE = 1 SV = 10.3435580.0000000196
91sp|Q06210|GFPT1_HUMANGlutamine--fructose-6-phosphate aminotransferase [isomerizing] 1 OS = Homo sapiens GN = GFPT1 PE = 1 SV = 30.3372870.000328
92sp|Q9BXJ9|NAA15_HUMANN-alpha-acetyltransferase 15, NatA auxiliary subunit OS = Homo sapiens GN = NAA15 PE = 1 SV = 10.3311310.0000275
93sp|P49368|TCPG_HUMANT-complex protein 1 subunit gamma OS = Homo sapiens GN = CCT3 PE = 1 SV = 40.3280950.0000143
94sp|O75083|WDR1_HUMANWD repeat-containing protein 1 OS = Homo sapiens GN = WDR1 PE = 1 SV = 40.3221070.00061
95sp|P55786|PSA_HUMANPuromycin-sensitive aminopeptidase OS = Homo sapiens GN = NPEPPS PE = 1 SV = 20.3162280.000026
96sp|P09960|LKHA4_HUMANLeukotriene A-4 hydrolase OS = Homo sapiens GN = LTA4H PE = 1 SV = 20.3162280.0000935
97sp|P30520|PURA2_HUMANAdenylosuccinate synthetase isozyme 2 OS = Homo sapiens GN = ADSS PE = 1 SV = 30.3162280.0000165
98sp|Q15691|MARE1_HUMANMicrotubule-associated protein RP/EB family member 1 OS = Homo sapiens GN = MAPRE1 PE = 1 SV = 30.3162280.000408
99sp|P12814|ACTN1_HUMANAlpha-actinin-1 OS = Homo sapiens GN = ACTN1 PE = 1 SV = 20.3133290.000176
100sp|P30044|PRDX5_HUMANPeroxiredoxin-5, mitochondrial OS = Homo sapiens GN = PRDX5 PE = 1 SV = 40.3133290.000749
101sp|P78371|TCPB_HUMANT-complex protein 1 subunit beta OS = Homo sapiens GN = CCT2 PE = 1 SV = 40.304790.000000242
102sp|P54136|SYRC_HUMANArginine--tRNA ligase, cytoplasmic OS = Homo sapiens GN = RARS PE = 1 SV = 20.304790.000287
103sp|P50991|TCPD_HUMANT-complex protein 1 subunit delta OS = Homo sapiens GN = CCT4 PE = 1 SV = 40.304790.0000303
104sp|P23588|IF4B_HUMANEukaryotic translation initiation factor 4B OS = Homo sapiens GN = EIF4B PE = 1 SV = 20.304790.00000105
105sp|P13489|RINI_HUMANRibonuclease inhibitor OS = Homo sapiens GN = RNH1 PE = 1 SV = 20.304790.000529
106sp|P55263|ADK_HUMANAdenosine kinase OS = Homo sapiens GN = ADK PE = 1 SV = 20.3019950.000591
107sp|P40227|TCPZ_HUMANT-complex protein 1 subunit zeta OS = Homo sapiens GN = CCT6A PE = 1 SV = 30.2964830.000029
108sp|P15559|NQO1_HUMANNAD(P)H dehydrogenase [quinone] 1 OS = Homo sapiens GN = NQO1 PE = 1 SV = 10.2964830.000441
109sp|P50990|TCPQ_HUMANT-complex protein 1 subunit theta OS = Homo sapiens GN = CCT8 PE = 1 SV = 40.2910720.000000345
110sp|P31947|1433S_HUMAN14-3-3 protein sigma OS = Homo sapiens GN = SFN PE = 1 SV = 10.2857590.000365
111sp|Q16658|FSCN1_HUMANFascin OS = Homo sapiens GN = FSCN1 PE = 1 SV = 30.2805430.00000154
112sp|Q01518|CAP1_HUMANAdenylyl cyclase-associated protein 1 OS = Homo sapiens GN = CAP1 PE = 1 SV = 50.2754230.0000322
113sp|P00966|ASSY_HUMANArgininosuccinate synthase OS = Homo sapiens GN = ASS1 PE = 1 SV = 20.2728980.0000194
114sp|P60981|DEST_HUMANDestrin OS = Homo sapiens GN = DSTN PE = 1 SV = 30.2728980.0000395
115sp|P52209|6PGD_HUMAN6-phosphogluconate dehydrogenase, decarboxylating OS = Homo sapiens GN = PGD PE = 1 SV = 30.2703960.0000138
116sp|Q9Y2T3|GUAD_HUMANGuanine deaminase OS = Homo sapiens GN = GDA PE = 1 SV = 10.2703960.000249
117sp|Q7L1Q6|BZW1_HUMANBasic leucine zipper and W2 domain-containing protein 1 OS = Homo sapiens GN = BZW1 PE = 1 SV = 10.2654610.000453
118sp|Q16401|PSMD5_HUMAN26S proteasome non-ATPase regulatory subunit 5 OS = Homo sapiens GN = PSMD5 PE = 1 SV = 30.2606150.00000517
119sp|P16152|CBR1_HUMANCarbonyl reductase [NADPH] 1 OS = Homo sapiens GN = CBR1 PE = 1 SV = 30.2606150.00017
120sp|Q9NTK5|OLA1_HUMANObg-like ATPase 1 OS = Homo sapiens GN = OLA1 PE = 1 SV = 20.2582260.000491
121sp|P11586|C1TC_HUMANC-1-tetrahydrofolate synthase, cytoplasmic OS = Homo sapiens GN = MTHFD1 PE = 1 SV = 30.2535130.00000226
122sp|P40925|MDHC_HUMANMalate dehydrogenase, cytoplasmic OS = Homo sapiens GN = MDH1 PE = 1 SV = 40.2511890.00000186
123sp|O95373|IPO7_HUMANImportin-7 OS = Homo sapiens GN = IPO7 PE = 1 SV = 10.2443430.000934
124sp|Q9Y617|SERC_HUMANPhosphoserine aminotransferase OS = Homo sapiens GN = PSAT1 PE = 1 SV = 20.2443430.0000026
125sp|P54578|UBP14_HUMANUbiquitin carboxyl-terminal hydrolase 14 OS = Homo sapiens GN = USP14 PE = 1 SV = 30.2355050.000113
126sp|P36952|SPB5_HUMANSerpin B5 OS = Homo sapiens GN = SERPINB5 PE = 1 SV = 20.2355050.0000299
127sp|Q9UGI8|TES_HUMANTestin OS = Homo sapiens GN = TES PE = 1 SV = 10.2355050.000587
128sp|P49588|SYAC_HUMANAlanine--tRNA ligase, cytoplasmic OS = Homo sapiens GN = AARS PE = 1 SV = 20.2333460.000000015
129sp|P54577|SYYC_HUMANTyrosine--tRNA ligase, cytoplasmic OS = Homo sapiens GN = YARS PE = 1 SV = 40.2312070.000000451
130sp|Q16719|KYNU_HUMANKynureninase OS = Homo sapiens GN = KYNU PE = 1 SV = 10.2290870.000151
131sp|P07900|HS90A_HUMANHeat shock protein HSP 90-alpha OS = Homo sapiens GN = HSP90AA1 PE = 1 SV = 50.2269860.00000039
132sp|P23381|SYWC_HUMANTryptophan--tRNA ligase, cytoplasmic OS = Homo sapiens GN = WARS PE = 1 SV = 20.2249060.0000214
133sp|P50395|GDIB_HUMANRab GDP dissociation inhibitor beta OS = Homo sapiens GN = GDI2 PE = 1 SV = 20.2187760.000381
134sp|P21266|GSTM3_HUMANGlutathione S-transferase Mu 3 OS = Homo sapiens GN = GSTM3 PE = 1 SV = 30.2187760.0000181
135sp|Q01813|PFKAP_HUMANATP-dependent 6-phosphofructokinase, platelet type OS = Homo sapiens GN = PFKP PE = 1 SV = 20.216770.0000109
136sp|P29401|TKT_HUMANTransketolase OS = Homo sapiens GN = TKT PE = 1 SV = 30.2147830.00000818
137sp|O14980|XPO1_HUMANExportin-1 OS = Homo sapiens GN = XPO1 PE = 1 SV = 10.2147830.0000144
138sp|P35237|SPB6_HUMANSerpin B6 OS = Homo sapiens GN = SERPINB6 PE = 1 SV = 30.2147830.000441
139sp|P26038|MOES_HUMANMoesin OS = Homo sapiens GN = MSN PE = 1 SV = 30.2128140.000000083
140sp|P60174|TPIS_HUMANTriosephosphate isomerase OS = Homo sapiens GN = TPI1 PE = 1 SV = 30.2108630.000000565
141sp|P17987|TCPA_HUMANT-complex protein 1 subunit alpha OS = Homo sapiens GN = TCP1 PE = 1 SV = 10.2108630.00000355
142sp|P37837|TALDO_HUMANTransaldolase OS = Homo sapiens GN = TALDO1 PE = 1 SV = 20.2108630.00002
143sp|P00491|PNPH_HUMANPurine nucleoside phosphorylase OS = Homo sapiens GN = PNP PE = 1 SV = 20.2108630.000182
144sp|P12429|ANXA3_HUMANAnnexin A3 OS = Homo sapiens GN = ANXA3 PE = 1 SV = 30.2070140.000102
145sp|P60842|IF4A1_HUMANEukaryotic initiation factor 4A-I OS = Homo sapiens GN = EIF4A1 PE = 1 SV = 10.2051160.00039
146sp|P08133|ANXA6_HUMANAnnexin A6 OS = Homo sapiens GN = ANXA6 PE = 1 SV = 30.2032360.0000209
147sp|P22102|PUR2_HUMANTrifunctional purine biosynthetic protein adenosine-3 OS = Homo sapiens GN = GART PE = 1 SV = 10.2013720.00000215
148sp|Q16881|TRXR1_HUMANThioredoxin reductase 1, cytoplasmic OS = Homo sapiens GN = TXNRD1 PE = 1 SV = 30.1995260.00000000739
149sp|P35241|RADI_HUMANRadixin OS = Homo sapiens GN = RDX PE = 1 SV = 10.1995260.0000185
150sp|P30085|KCY_HUMANUMP-CMP kinase OS = Homo sapiens GN = CMPK1 PE = 1 SV = 30.1923090.000245
151sp|P17812|PYRG1_HUMANCTP synthase 1 OS = Homo sapiens GN = CTPS1 PE = 1 SV = 20.1887990.000024
152sp|P49327|FAS_HUMANFatty acid synthase OS = Homo sapiens GN = FASN PE = 1 SV = 30.1836540,00
153sp|P08238|HS90B_HUMANHeat shock protein HSP 90-beta OS = Homo sapiens GN = HSP90AB1 PE = 1 SV = 40.1836540.0000176
154sp|P31939|PUR9_HUMANBifunctional purine biosynthesis protein PURH OS = Homo sapiens GN = ATIC PE = 1 SV = 30.1836540.000000181
155sp|P36871|PGM1_HUMANPhosphoglucomutase-1 OS = Homo sapiens GN = PGM1 PE = 1 SV = 30.1836540.00000167
156sp|P18669|PGAM1_HUMANPhosphoglycerate mutase 1 OS = Homo sapiens GN = PGAM1 PE = 1 SV = 20.1836540.000112
157sp|P11413|G6PD_HUMANGlucose-6-phosphate 1-dehydrogenase OS = Homo sapiens GN = G6PD PE = 1 SV = 40.1770110.00000103
158sp|P17655|CAN2_HUMANCalpain-2 catalytic subunit OS = Homo sapiens GN = CAPN2 PE = 1 SV = 60.1770110.00000121
159sp|O43175|SERA_HUMAND-3-phosphoglycerate dehydrogenase OS = Homo sapiens GN = PHGDH PE = 1 SV = 40.1753880.0000184
160sp|P04075|ALDOA_HUMANFructose-bisphosphate aldolase A OS = Homo sapiens GN = ALDOA PE = 1 SV = 20.173780.0000134
161sp|P41250|SYG_HUMANGlycine--tRNA ligase OS = Homo sapiens GN = GARS PE = 1 SV = 30.173780.000000134
162sp|O75874|IDHC_HUMANIsocitrate dehydrogenase [NADP] cytoplasmic OS = Homo sapiens GN = IDH1 PE = 1 SV = 20.1721870.000000103
163sp|P18206|VINC_HUMANVinculin OS = Homo sapiens GN = VCL PE = 1 SV = 40.1706080,00
164sp|P31948|STIP1_HUMANStress-induced-phosphoprotein 1 OS = Homo sapiens GN = STIP1 PE = 1 SV = 10.1584890.0000000108
165sp|P53396|ACLY_HUMANATP-citrate synthase OS = Homo sapiens GN = ACLY PE = 1 SV = 30.1570360.000000000426
166sp|Q9Y266|NUDC_HUMANNuclear migration protein nudC OS = Homo sapiens GN = NUDC PE = 1 SV = 10.1570360.000000195
167sp|P55060|XPO2_HUMANExportin-2 OS = Homo sapiens GN = CSE1L PE = 1 SV = 30.1555970.00000000656
168sp|O43776|SYNC_HUMANAsparagine--tRNA ligase, cytoplasmic OS = Homo sapiens GN = NARS PE = 1 SV = 10.1555970.0000275
169sp|P13797|PLST_HUMANPlastin-3 OS = Homo sapiens GN = PLS3 PE = 1 SV = 40.1485940.000000327
170sp|Q14914|PTGR1_HUMANProstaglandin reductase 1 OS = Homo sapiens GN = PTGR1 PE = 1 SV = 20.1432190.000000538
171sp|P62258|1433E_HUMAN14-3-3 protein epsilon OS = Homo sapiens GN = YWHAE PE = 1 SV = 10.1380380.0000521
172sp|P26639|SYTC_HUMANThreonine--tRNA ligase, cytoplasmic OS = Homo sapiens GN = TARS PE = 1 SV = 30.1367730.000000000522
173sp|P27348|1433T_HUMAN14-3-3 protein theta OS = Homo sapiens GN = YWHAQ PE = 1 SV = 10.1318260.0000612
174sp|Q15185|TEBP_HUMANProstaglandin E synthase 3 OS = Homo sapiens GN = PTGES3 PE = 1 SV = 10.129420.000842
175sp|P00338|LDHA_HUMANL-lactate dehydrogenase A chain OS = Homo sapiens GN = LDHA PE = 1 SV = 20.1282330.00000000672
176sp|P08758|ANXA5_HUMANAnnexin A5 OS = Homo sapiens GN = ANXA5 PE = 1 SV = 20.1282330.000000000417
177sp|Q15181|IPYR_HUMANInorganic pyrophosphatase OS = Homo sapiens GN = PPA1 PE = 1 SV = 20.1282330.0000608
178sp|P07195|LDHB_HUMANL-lactate dehydrogenase B chain OS = Homo sapiens GN = LDHB PE = 1 SV = 20.1224620.0000861
179sp|P06733|ENOA_HUMANAlpha-enolase OS = Homo sapiens GN = ENO1 PE = 1 SV = 20.1158780.0000000119
180sp|Q06830|PRDX1_HUMANPeroxiredoxin-1 OS = Homo sapiens GN = PRDX1 PE = 1 SV = 10.1047130.000282
181sp|P13639|EF2_HUMANElongation factor 2 OS = Homo sapiens GN = EEF2 PE = 1 SV = 40.1028020.0000215
182sp|O00299|CLIC1_HUMANChloride intracellular channel protein 1 OS = Homo sapiens GN = CLIC1 PE = 1 SV = 40.1028020.000245
183sp|P15311|EZRI_HUMANEzrin OS = Homo sapiens GN = EZR PE = 1 SV = 40.10.0000000224
184sp|P15121|ALDR_HUMANAldose reductase OS = Homo sapiens GN = AKR1B1 PE = 1 SV = 30.0990830.00000144
185sp|P37802|TAGL2_HUMANTransgelin-2 OS = Homo sapiens GN = TAGLN2 PE = 1 SV = 30.0954990.0000496
186sp|P06744|G6PI_HUMANGlucose-6-phosphate isomerase OS = Homo sapiens GN = GPI PE = 1 SV = 40.0912010.0000000011
187sp|P00558|PGK1_HUMANPhosphoglycerate kinase 1 OS = Homo sapiens GN = PGK1 PE = 1 SV = 30.0839460.00000202
188sp|P30041|PRDX6_HUMANPeroxiredoxin-6 OS = Homo sapiens GN = PRDX6 PE = 1 SV = 30.0794330.000000231
189sp|Q01581|HMCS1_HUMANHydroxymethylglutaryl-CoA synthase, cytoplasmic OS = Homo sapiens GN = HMGCS1 PE = 1 SV = 20.067920.0000000145
190sp|P12725|A1AT_SHEEPAlpha-1-antiproteinase OS = Ovis aries PE = 1 SV = 10.0642690.0000536
Table II

Differentially expressed proteins identified between adjacent noncancerous tissues and distal noncancerous tissues of hepatocellular carcinoma

No.AccessionNameFCP-value
1sp|P12763|FETUA_BOVINAlpha-2-HS-glycoprotein OS = Bos taurus GN = AHSG PE = 1 SV = 24.370.000000647
2sp|O00299|CLIC1_HUMANChloride intracellular channel protein 1 OS = Homo sapiens GN = CLIC1 PE = 1 SV = 43.499452110.00064666
3sp|P78417|GSTO1_HUMANGlutathione S-transferase omega-1 OS = Homo sapiens GN = GSTO1 PE = 1 SV = 23.467369080.00088023
4sp|P00558|PGK1_HUMANPhosphoglycerate kinase 1 OS = Homo sapiens GN = PGK1 PE = 1 SV = 33.340.000000494
5sp|P17812|PYRG1_HUMANCTP synthase 1 OS = Homo sapiens GN = CTPS1 PE = 1 SV = 23.280952930.00090827
6sp|Q01813|PFKAP_HUMANATP-dependent 6-phosphofructokinase. platelet type OS = Homo sapiens GN = PFKP PE = 1 SV = 23.22106910.00021282
7sp|P06744|G6PI_HUMANGlucose-6-phosphate isomerase OS = Homo sapiens GN = GPI PE = 1 SV = 43.160.00000522
8sp|P04264|K2C1_HUMANKeratin. type II cytoskeletal 1 OS = Homo sapiens GN = KRT1 PE = 1 SV = 63.1332860.00012301
9sp|P21266|GSTM3_HUMANGlutathione S-transferase Mu 3 OS = Homo sapiens GN = GSTM3 PE = 1 SV = 32.880.0000379
10sp|P30041|PRDX6_HUMANPeroxiredoxin-6 OS = Homo sapiens GN = PRDX6 PE = 1 SV = 32.831392050.00092527
11sp|P08133|ANXA6_HUMANAnnexin A6 OS = Homo sapiens GN = ANXA6 PE = 1 SV = 32.831392050.00077018
12sp|P31939|PUR9_HUMANBifunctional purine biosynthesis protein PURH OS = Homo sapiens GN = ATIC PE = 1 SV = 32.560.0000395
13sp|P36871|PGM1_HUMANPhosphoglucomutase-1 OS = Homo sapiens GN = PGM1 PE = 1 SV = 32.490.00000426
14sp|Q15181|IPYR_HUMANInorganic pyrophosphatase OS = Homo sapiens GN = PPA1 PE = 1 SV = 22.488857030.00020884
15sp|Q96P70|IPO9_HUMANImportin-9 OS = Homo sapiens GN = IPO9 PE = 1 SV = 32.466038940.00095205
16sp|P50395|GDIB_HUMANRab GDP dissociation inhibitor beta OS = Homo sapiens GN = GDI2 PE = 1 SV = 22.380.0000832
17sp|P00491|PNPH_HUMANPurine nucleoside phosphorylase OS = Homo sapiens GN = PNP PE = 1 SV = 22.376840110.00012449
18sp|Q9Y617|SERC_HUMANPhosphoserine aminotransferase OS = Homo sapiens GN = PSAT1 PE = 1 SV = 22.269865040.00016311
19sp|P55060|XPO2_HUMANExportin-2 OS = Homo sapiens GN = CSE1L PE = 1 SV = 32.250.00000809
20sp|P13797|PLST_HUMANPlastin-3 OS = Homo sapiens GN = PLS3 PE = 1 SV = 42.0511620.00014754
21sp|Q01581|HMCS1_HUMANHydroxymethylglutaryl-CoA synthase. cytoplasmic OS = Homo sapiens GN = HMGCS1 PE = 1 SV = 21.958845020.00053098
22sp|P18206|VINC_HUMANVinculin OS = Homo sapiens GN = VCL PE = 1 SV = 41.910.00000266
23sp|P49588|SYAC_HUMANAlanine-tRNA ligase. cytoplasmic OS = Homo sapiens GN = AARS PE = 1 SV = 21.870.0000459
24sp|P49327|FAS_HUMANFatty acid synthase OS = Homo sapiens GN = FASN PE = 1 SV = 31.850.000000763
25sp|P11413|G6PD_HUMANGlucose-6-phosphate 1-dehydrogenase OS = Homo sapiens GN = G6PD PE = 1 SV = 41.803017970.00072658
26sp|Q86UP2|KTN1_HUMANKinectin OS = Homo sapiens GN = KTN1 PE = 1 SV = 10.602559630.00045493
27sp|P13667|PDIA4_HUMANProtein disulfide-isomerase A4 OS = Homo sapiens GN = PDIA4 PE = 1 SV = 20.6030.0000523
28sp|P25705|ATPA_HUMANATP synthase subunit alpha. mitochondrial OS = Homo sapiens GN = ATP5A1 PE = 1 SV = 10.539510610.00031264
29sp|O75369|FLNB_HUMANFilamin-B OS = Homo sapiens GN = FLNB PE = 1 SV = 20.5350.000067
30sp|P31327|CPSM_HUMANCarbamoyl-phosphate synthase [ammonia]. mitochondrial OS = Homo sapiens GN = CPS1 PE = 1 SV = 20.4920.00000077
31sp|Q9P2E9|RRBP1_HUMANRibosome-binding protein 1 OS = Homo sapiens GN = RRBP1 PE = 1 SV = 40.4880.0000851
32sp|Q14126|DSG2_HUMANDesmoglein-2 OS = Homo sapiens GN = DSG2 PE = 1 SV = 20.405508490.00097142
33sp|P30050|RL12_HUMAN60S ribosomal protein L12 OS = Homo sapiens GN = RPL12 PE = 1 SV = 10.387257610.00062865
34sp|P40926|MDHM_HUMANMalate dehydrogenase. mitochondrial OS = Homo sapiens GN = MDH2 PE = 1 SV = 30.373250190.00081137
35sp|Q07065|CKAP4_HUMANCytoskeleton-associated protein 4 OS = Homo sapiens GN = CKAP4 PE = 1 SV = 20.3660.00000472
36sp|P23246|SFPQ_HUMANSplicing factor. proline- and glutamine-rich OS = Homo sapiens GN = SFPQ PE = 1 SV = 20.363078090.00011904
37sp|Q8IVF2|AHNK2_HUMANProtein AHNAK2 OS = Homo sapiens GN = AHNAK2 PE = 1 SV = 20.360.0000000183
38sp|Q13813|SPTN1_HUMANSpectrin alpha chain. non-erythrocytic 1 OS = Homo sapiens GN = SPTAN1 PE = 1 SV = 30.3440.0000000289
39sp|P19338|NUCL_HUMANNucleolin OS = Homo sapiens GN = NCL PE = 1 SV = 30.3160.000000799
40sp|P11021|GRP78_HUMAN78 kDa glucose-regulated protein OS = Homo sapiens GN = HSPA5 PE = 1 SV = 20.270.000000000617
41sp|P27824|CALX_HUMANCalnexin OS = Homo sapiens GN = CANX PE = 1 SV = 20.25585860.0005111
42sp|P46779|RL28_HUMAN60S ribosomal protein L28 OS = Homo sapiens GN = RPL28 PE = 1 SV = 30.2376840.00034004
43sp|P10809|CH60_HUMAN60 kDa heat shock protein. mitochondrial OS = Homo sapiens GN = HSPD1 PE = 1 SV = 20.2150.000000000379
44sp|P08670|VIME_HUMANVimentin OS = Homo sapiens GN = VIM PE = 1 SV = 40.1750.00000297
Figure 1

A schematic view of the experimental design and the isobaric tags for relative and absolute quantification (iTRAQ) 8 plex-labelling. Sample preparation procedures for shotgun mass spectrometry (MS/MS) analysis and important steps in the proteomic strategies were included. The tissues from the primary hepatocellular carcinoma (HCC) patients were divided into 3 groups: HCC group, adjacent noncancerous group, and dendritic cell group. Different tissues were cultured in a serum-free medium and the proteins were extracted from the cultural supernatant and then digested by trypsin and labelled by different iTRAQ reagents. The digested peptides were separated by high pH reversed-phase liquid chromatography (LC) and analysed by LC-MS/MS

https://www.archivesofmedicalscience.com/f/fulltexts/118871/AMS-21-4-118871-g001_min.jpg

When we compared the differences between the 2 groups, we found that among these differentially expressed proteins, 35 proteins altered their expression in both HCC types, while 155 proteins were dysregulated in the HCC/DN group only and 9 proteins were dysregulated in the AN/DN group only (Figure 2 B). We then analysed the biological functions and signalling pathways of these proteins, including the proteins differentially expressed in both groups and the proteins differentially expressed individually in 1 group.

Figure 2

Features of the hepatocellular carcinoma secretome dataset from the isobaric tags for relative and absolute quantification shotgun analysis. A – The distribution of differently abundant proteins in 2 groups. B – Venn diagrams show the numbers of the identified proteins and the overlaps of differently abundant proteins in the 2 groups

https://www.archivesofmedicalscience.com/f/fulltexts/118871/AMS-21-4-118871-g002_min.jpg

The gene ontology analysis of the differentially expressed proteins in primary hepatocellular carcinomas

The gene ontology annotation analysis showed that the cell components of the differentially expressed proteins either overlapped in the 2 groups or were unique in 1 group and were mostly located in the extracellular exosome (Figure 3). For the biological process analysis, the GO annotation analysis showed that the proteins overlapped in both groups and were the major participants in the protein folding, lipid metabolic process, gluconeogenesis, nucleobase-containing compound metabolic process, and canonical glycolysis (Figure 3 A).

Figure 3

The gene ontology (GO) analysis of the differently abundant proteins. A – The GO analysis of differently abundant proteins overlapped in the 2 groups. B – The GO analysis of differently abundant proteins only involved in the hepatocellular carcinoma tissues/distal noncancerous tissues group. C – The GO analysis of differently abundant proteins only involved in the adjacent noncancerous tissues/distal noncancerous tissues group

https://www.archivesofmedicalscience.com/f/fulltexts/118871/AMS-21-4-118871-g003_min.jpg

There were 155 dysregulated proteins in the HCC group compared to the distal noncancerous tissues (DN) group, but these proteins were not dysregulated in the adjacent noncancerous (AN) tissues group compared to the DN group. These dysregulated proteins were mainly involved in signal transduction, cell proliferation, protein stabilisation, and negative regulation of the apoptotic process (Figure 3 B).

Interestingly, there were 9 dysregulated proteins in the AN group compared to the DN group, but they were not dysregulated in the HCC group compared to the DN group. The gene ontology results also showed that these dysregulated proteins were mainly involved in desmosome organisation, positive regulation of sister chromatid cohesion, translation, rRNA processing, nuclear-transcribed mRNA catabolic process, translational initiation, and SRP-dependent co-translational protein targeting to the membrane (Figure 3 C).

The Kyoto Encyclopaedia of Genes and Genomes pathway analysis of the differentially expressed proteins

As shown in Figure 4, the pathway of metabolism, genetic information processing, environmental information processing, and cellular was analysed. According to the results of the analysis, the dysregulated proteins in HCC are mostly involved in the Janus kinase-signal transducer and activator of the transcription (JAK-STAT) pathway and mitogen-activated protein kinase (MAPK) pathway. However, the signalling pathway that was only enriched in the AN group comprised mainly basic metabolisms, such as biological oxidations, amino acids metabolism, trichloroacetic acid (TCA) cycle, glucose metabolism, etc.

Figure 4

The key signalling pathways involved in the 2 groups. A – The key signalling pathways involved in the hepatocellular carcinoma tissues/distal noncancerous tissues group. B – The key signalling pathways involved in the adjacent noncancerous tissues/distal noncancerous tissues group. The top 10 enriched signalling pathways were displayed in the figures

https://www.archivesofmedicalscience.com/f/fulltexts/118871/AMS-21-4-118871-g004_min.jpg

The String analysis of the differentially expressed proteins

As shown in Figure 5 A, in the HCC/DN group, the proteins could be classified into 3 major clusters: proteins in the red region were related to protein translation and post-translation processing, proteins in the blue region were related to protein glycosylation modification, and proteins in the green region were related to biological metabolic functions dominated by glucose metabolism. While in the AN/DN group, the proteins could also be classified into 3 clusters: the red region represented proteins related to immune and metabolic functions, the green region represented proteins related to apoptosis functions, and the blue region represented proteins related to protein binding functions (Figure 5 B).

Figure 5

The interaction networks of differently abundant proteins in the 2 groups. A – The interaction networks of differently abundant proteins involved in the hepatocellular carcinoma tissues/distal noncancerous tissues group. B – The interaction networks of differently abundant proteins involved in the adjacent noncancerous tissues/distal noncancerous tissues group

https://www.archivesofmedicalscience.com/f/fulltexts/118871/AMS-21-4-118871-g005_min.jpg

Discussion

Hepatocellular carcinoma has become the third-most-common cause of cancer-related death worldwide. Most cases of HCC were developed in patients who had already had liver cirrhosis [15]. Therefore, surveillance for the early onset of HCC was recommended. The biomarkers with high sensitivity and specificity were essential for optimising the management of HCC [16]. Zhang et al. used the iTRAQ pipeline to distinguish the proteomic profiles of malignant ascites in HCC patients from those with non-malignant liver cirrhosis and found that Enolase-1 and fibrinogen are potential ascitic fluid-based biomarkers for diagnosis and prognosis of HCC [17]. Guo et al. reported that assaying CD14 levels may complement AFP measurement for the early detection of HCC [18]. Wang et al. suggested that different molecular alterations and specific signalling pathways were indeed involved in different HCC subtypes [19]. Our study aimed to investigate the molecular signatures of the HCC by quantitative proteomics using iTRAQ with LC-MS/MS.

In our study, the number of differentially expressed proteins identified in the HCC/DN group was much higher than in the AN/DN group. These findings indicate that the features between the adjacent noncancerous tissues and distant noncancerous tissues were more similar than those between the HCC tissues and the distant noncancerous tissues, which were accorded with objective existence.

The gene ontology annotation analysis showed that the cell components of the differentially expressed proteins that either overlapped in 2 groups or uniquely in 1 group were mostly located in the extracellular exosome, which indicated that the proteins extracted in this experiment were mainly secreted proteins. For the biological process analysis, the GO annotation analysis showed that the proteins overlapped in both the groups and were the major participants in the protein folding, lipid metabolic process, gluconeogenesis, nucleobase-containing compound metabolic process, and canonical glycolysis. Most of these processes focused on metabolic changes, which suggested that the changes in the material metabolism were universal, regardless of the transformation from distant cancer to adjacent cancer or the approach of adjacent cancer to HCC. The molecular function of these proteins also focuses on energy metabolism, which also supported the hypothesis [15, 2023].

There were 155 dysregulated proteins in the HCC group compared to the DN group, but these proteins were not dysregulated in the AN group compared to the DN group. We further analysed that these proteins involved the biological process by GO analysis; the results showed that these dysregulated proteins were mainly involved in signal transduction, cell proliferation, protein stabilisation, and the negative regulation of the apoptotic process. These processes might be involved in the formation or development of HCC, and it has been reported that these processes are involved in the disturbing of the signal transduction and protein degradation [2427], apoptotic process [27, 28], and cell proliferation [28, 29] in tumours. The molecular function of these proteins, such as the cadherin binding involved in cell-cell adhesion, protein homodimerisation activity, ubiquitin-protein ligase binding, calcium ion binding, GTP binding, etc., also supported this conclusion.

Interestingly, there were 9 dysregulated proteins in the AN group compared to the DN group but no dysregulation in the HCC group compared to the DN group, and the GO results showed that these dysregulated proteins were mainly involved in desmosome organisation, positive regulation of sister chromatid cohesion, translation, rRNA processing, nuclear-transcribed mRNA catabolic process, translational initiation, and SRP-dependent co-translational protein targeting the membrane. The results also showed that the dysregulated proteins may have affected the incidence and progress of HCC, such as the change of the combination of the protein and the RNA function presenting the disorder of the transcription and translation function, which suggested that the surrounding noncancerous cells might increase the expression of the nucleic acid and enzyme by tumour microenvironment to promote the HCC proliferation and growth [30], and that the changes of telomere and telomerase in the surrounding noncancerous cells revealed the dysregulation on the chromosome stability, repair, and proliferation, which were all closely related to the incidence of HCC development [31, 32]. Similarly, the molecular function of these proteins, such as cadherin binding-involved nucleotide binding, RNA binding, calcium ion binding, chromatin binding, transcription regulatory region DNA binding, identical protein binding, etc., also supported this conclusion.

To further reveal the possible molecular mechanisms of the tumourigenesis and the development of the primary HCC, we applied the KEGG database to analyse the signalling pathways in which the differentially expressed proteins were involved. Our study also analysed the pathway of metabolism, genetic information processing, environmental information processing, and cellular. According to the results of the analysis, the dysregulated proteins in HCC are mostly involved in the JAK-STAT pathway and MAPK pathway. All the above-mentioned signalling pathways are actively associated with cancers [3336]. It has been reported that the MAPK signalling pathway played an essential role in the development and aggressive behaviour of tumours by enhancing tumour cell proliferation, differentiation, apoptosis, and cell cycle [37, 38]. Therefore, it is not surprising that the MAPK signalling pathway is involved in HCC tissues. Interestingly, the JAK-STAT pathway was only enriched in the HCC group but not in the AN group. JAK-STAT pathway has been regarded as one of the main molecular pathways in HCC progression [39].

However, the signalling pathway only enriched in the AN group comprised mainly basic metabolisms, such as biological oxidations, amino acids metabolism, TCA cycle, glucose metabolism, and so on. All of these processes belong to the material metabolism and illustrate that the primary material changes play an important role in the tumourigenesis and development of HCC. Also, the different pathways in the HCC and the AN group suggest that there might be different molecular mechanisms in the carcinogenesis and development of the primary HCC in the HCC tissue and the surrounding noncancerous tissues. The abovementioned results that were analysed demonstrate that our quantitative proteomics approach is suitable in studying the overall molecular profile changes of HCC and could give further insight into the possible molecular mechanisms.

In our study, the proteins in the HCC/DN group could be classified into 3 major clusters: proteins in the red region were related to protein translation and post-translation processing, proteins in the blue region were related to protein glycosylation modification, and proteins in the green region were related to biological metabolic functions dominated by glucose metabolism. As is already known, the malignant proliferation of tumour cells was a process of energy consumption, so the hyperactive glucose metabolism in the HCC group might provide the necessary conditions for the progression of HCC [40, 41]. Glycosylation was involved in the folding, aggregation, maturation, and transportation of protein-peptide chains and was a terminal signal on the surface of the cancer cells in carcinogenesis [42, 43]. The incidence, development, and invasion of HCC were accompanied by glycosylation changes of relevant glycoproteins, so the change of the carbohydrate structure on the surface of the HCC cells played an important role in the occurrence and development progress of HCC [44, 45].

The proteins in the AN/DN group could also be classified into 3 clusters: the red region represented proteins related to immune and metabolic functions, the green region represented proteins related to apoptosis functions, and the blue region represented proteins related to protein binding functions. This indicated that immune and metabolic changes were relatively active in the para cancer tissues, which might be related to changes in the tumour microenvironment [4649]. All these results suggest that the evolution of the tissues adjacent to HCC promoted the incidence of HCC.

In summary, this study applied the iTRAQ-based quantitative proteomic approach to analyse the secretome of the primary cultures of HCC tumour tissues. The results visibly showed that the secretome profile alternations and signalling pathways were associated with HCC occurrence and development. The dysregulated proteins in the HCC/DN group were concentrated in the MAPK signalling and JAK-STAT signalling, but the dysregulated proteins in the AN/DN group were more concentrated in the basal material metabolism. The different protein expression profiles in the primary HCC tissues, the surrounding non-cancerous tissues, and the distal noncancerous tissues might also reveal different underlying molecular mechanisms. This study provides a valuable resource of the HCC tissue secretome to investigate the molecular mechanism of HCC incidence and development.

In conclusion, the secretome profile alternations and signalling pathways were associated with HCC incidence and development. The dysregulated proteins in the HCC/DN group were concentrated in the MAPK signalling and JAK-STAT signalling, but the dysregulated proteins in the AN/DN group were more concentrated in the basal material metabolism.