Expression characteristics and diagnostic value of annexin A2 in hepatocellular carcinoma

AIM:To investigate the characteristics and diagnostic value of annexin A2(ANXA2) expression in cancerous tissues and sera of patients with hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).METHODS:Levels of liver ANXA2 gene transcription or protein expression were analyzed in HCC-,their selfcontrolled precancerous-,and distant cancerous-tissues from 30 HCC.Serum levels of ANXA2 expression in 115 patients with HCC,25 with metastatic liver can-cer,35 with chronic hepatitis,28 with acute hepatitis,38 with cirrhosis,and 30 healthy controls were determined.Clinicopathological characteristics of circulating ANXA2 expression were analyzed,and its diagnostic efficiency and clinical values in HCC were evaluated.RESULTS:ANXA2 expression was localized in both cell membrane and cytoplasm in HCC tissue,mainly in the cytoplasm of matched adjacent cancerous tissue,and there was almost no positive staining in matched distant cancerous tissue.Abnormal expression of liver ANXA2 was present in HCC tissues compared with self-controlled adjacent-and distant-cancerous tissues at protein or mRNA level.Circulating ANXA2 in HCC patients was significantly higher than that of other liver diseases(P < 0.01) except metastatic liver cancer.If the diagnostic cutoff value of ANXA2 level was more than 18 ng/mL,the incidence of serum ANXA2 was 86.96% in the HCC group,80% in the metastatic liver cancer group,31.58% in the liver cirrhosis group,none in the chronic hepatitis or acute hepatitis or normal control group,respectively.Serum ANXA2 expression in HCC patients was correlated with HBV infection(27.38 ± 5.67 ng/mL vs 18.58 ± 7.83 ng/mL,P < 0.01),extrahepatic metastasis(26.11 ± 5.43 ng/mL vs 22.79 ± 5.64 ng/mL,P < 0.01),and portal vein thrombus(26.03 ± 5.99 ng/mL vs 23.06 ± 5.03 ng/mL,P < 0.01),and was significantly higher(P < 0.01) in the moderately-(26.19 ± 5.34 ng/mL) or the poorly-differentiated group(27.05 ± 5.13 ng/mL) than in the well differentiated group(20.43 ± 4.97 ng/mL),and in the tumor node metastasis stages ⅢⅣ(P < 0.01) than in stages ⅠⅡ.ANXA2 was not correlated with patient sex,age,size or-fetoprotein(AFP) level.Area under the receiver operating characteristic curve for the whole range of sensitivities and specificities was 0.796 for ANXA2 and 0.782 for AFP.Combining detection of serum ANXA2 and AFP substantially improved the diagnostic efficiency(96.52%) and the negative predictive value(96.61%) for HCC.CONCLUSION:The characteristics and distributionof ANXA2 expression has good diagnostic potential for HCC diagnosis.

Overexpression of insulin-like growth factor-Ⅰ receptor as a pertinent biomarker for hepatocytes malignant transformation

AIM:To investigate the dynamic features of insulinlike growth factor-Ⅰreceptor(IGF-ⅠR)expression in rat hepatocarcinogenesis,and the relationship between IGF-ⅠR and hepatocytes malignant transformation at mRNA or protein level.METHODS:Hepatoma models were made by inducing with 2-fluorenylacetamide(2-FAA)on male SpragueDawley rats.Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining,the dynamic expressions of liver and serum IGF-ⅠR were quantitatively analyzed by an enzymelinked immunosorbent assay.The distribution of hepatic IGF-ⅠR was located by immunohistochemistry.The fragments of IGF-ⅠR gene were amplified by reverse transcription-polymerase chain reaction,and confirmed by sequencing.RESULTS:Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration,precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-ⅠR expression.The incidences of liver IGF-ⅠR,IGF-ⅠR mRNA,specific IGF-ⅠR concentration(ng/mg wet liver),and serum IGF-ⅠR level(ng/mL)were 0.0%,0.0%,0.63±0.17,and 1.33±0.47 in the control;50.0%,61.1%,0.65±0.2,and 1.51±0.46 in the degeneration;88.9%,100%,0.66±0.14,and 1.92±0.29 in the precancerosis;and 100%,100%,0.96±0.09,and2.43±0.57 in the cancerous group,respectively.IGF-ⅠR expression in the cancerous group was significantly higher(P<0.01)than that in any of other groups at mRNA or protein level.The closely positive IGF-ⅠR relationship was found between livers and sera(r=0.91,t=14.222,P<0.01),respectively.CONCLUSION:IGF-ⅠR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells

AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line(LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, re-spectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA target-ing ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analy-ses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) as-say (in vitro ) and tumour-growth assay (in vivo ), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro ), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro ). RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest lev-el of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expres-sion was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cel-lular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shR-NA-mediated ANXA2 silencing in vitro , and the tumour growth inhibition rate was 38.24% in vivo . The per-centage of MHCC97-H cells in S phase dramatically de-creased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells de-creased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis. CONCLUSION: shRNA-mediated silencing of ANXA2suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.

Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.