CDNA Cloning And Expression Of Human Insulin-like Growth Factor Binding Protein-3 And Its Inhibition Effect On Hep3B Cell

Background and Objective: More and more attention was paid toinsulin-like growth factor binding protein-3 (IGFBP-3), which acts as agrowth-inhibitory and apoptosis-inducing molecule via IGF-dependent andIGF-independent mechanisms. It has been shown to take protective effect oncommon cancers such as lung cancer, prostate cancer, breast cancer, et al. Inorder to explore its role in hepatocarcinogenesis and find new ways for thediagnosis and treatment of hepatocellular carcinoma (HCC) in the end, weinvestigate the serum IGFBP-3 levels in patients with different kinds of hepatitisand HCC. We also studied the expression of IGFBP-3 in liver tissues in patientswith chronic hepatitis,HCC and healthy controls, retrospectively. Meanwhile,we aim to construct the eukaryotic expression vector with hIGFBP-3 cDNA andstudy its inhibitory effect on Hep3B cells.Materials and Methods: 1. We collected the serum of 54 patients withdifferent kinds of hepatitis and 25 patients with HCC and 10 healthy controls.The concentration of IGFBP-3 was measured by ELISA using ACTIVE?IGFBP-3 ELISA kits (DSL-10-6600). The expression of IGFBP-3 in livertissues was examined by SABC immunohistochemistry using anti-IGFBP-3monoclonal antiboby (Sigma,I 4527). 2. Total RNA was extracted fromhealthy liver tissues by TRIzol? Reagent. The IGFBP-3 coding region, deletedall of the uncoding fragments, was amplified by RT-PCR and cloned to vectorpcDNA3.1(+), designated pcDNA3.1-BP3. The recombinant eukaryoticexpressing vector was confirmed by PCR amplification, restrictive enzymaticdigestion and sequence analysis. 3. The construct was transfected into Hep3Bcell by LipofectamineTM 2000 reagent. The stable transfectants, designatedHep3B-pcDNA3.1 and Hep3B-pcDNA3.1-BP3, were selected by the additionof G418 to the growth medium, followed by a series of confirmation, such asRT-PCR, Western Blot and immunohistochemistry. 4. The effects ofconstitutively expressed IGFBP-3 on Hep3B cells were measured by growthkinetics, TUNEL and flow cytometry.Results:1. The level of serum IGFBP-3 in healthy controls, in patients with acutehepatitis, chronic hepatitis(medium degree), chronic hepatitis(severe degree),hepatitis gravis, liver cirrhosis and HCC was 4891.63±1482.91 ,4243.88±1086.02 , 3361.38±584.11 , 3021.63±946.48 , 1951.13±791.96 ,1146.93±443.15 and 1760.61±958.88 ( ng/ml ) , respectively. There wassignificant difference between the IGFBP-3 level in healthy controls and inpatients with all kinds of hepatitis except acute hepatitis. Its value became loweras the damage degree of liver aggravated. The level of IGFBP-3 in HCC wasmuch lower compared to that in healthy controls, in patients with acute hepatitis,chronic hepatitis, but did not differ significantly from that in patients withhepatitis gravis and liver cirrhosis. Its level correlated with that of serumalbumin, prealbumin significantly, and inversely correlated with that of totalbilirubin, direct bilirubin and prothrombin time.2. The positive immunostaining rate of IGFBP-3 in liver tissues of healthycontrols, in patients with chronic hepatitis, liver cirrhosis and HCC was 100%(11/11),100%(12/12),87.5%(14/16)and 42.86%(9/21), respectively。By Ridit test, the R ± SR value of the immunostaining intensity of IGFBP-3was 0.7462 ± 0.0288,0.6958± 0.0413,0.5344 ± 0.0610,0.2330 0.0373,respectively;and accordingly the value of±R ± 1.96S R was 0.6898~0.8026,0.6149~0.7767,0.4148~0.6540,0.1599~0.3061。The intensity of staining inHCC tissues is significantly lower than in the other three groups (p<0.05). Adecreased expression of IGFBP-3 in liver cirrhosis was found compared tonormal controls (p<0.05). But there was not statistically significant differencebetween liver cirrhosis and chronic hepatitis(p>0.05)and between chronichepatitis and normal controls(p>0.05).3. The recombinant eukaryotic expressing vector pcDNA3.1-BP3 wasconstructed successfully and verified by the means mentioned above.4. Stable transfectants were obtained and confirmed by RT-PCR, Westernblot and immunohistochemistry. By western blot, a strong signal representing a40~44 kDa form of IGFBP-3 was detectable in the growth medium ofHep3B-pcDNA3.1-BP3, while in Hep3B-pcDNA3.1 and Hep3B only weaksignals were observed. Also, the IGFBP-3 in the cell of Hep3B-pcDNA3.1-BP3is more extensive than that in the latter. The IGFBP-3 cDNA is expressed fullyin the transfectants.5. The proliferation rate of Hep3B-pcDNA3.1-BP3 was considerablyslower than that of Hep3B-pcDNA3.1 and Hep3B. However, their growtharrested at a similar density. The doubling time of Hep3B-pcDNA3.1-BP3,Hep3B-pcDNA3.1 and Hep3B is 19.34, 13.82 and 14.37 hours. Much moreapoptotic cells were found in Hep3B-pcDNA3.1-BP3 cell line by TUNEL thanthe other two. By flow cytometry, the percent of apoptotic cells ofHep3B-pcDNA3.1-BP3, Hep3B-pcDNA3.1 and Hep3B was 4.15%, 0.8% and0.6%.Conclusion:1. The serum concentration of IGFBP-3 is a good marker of the liverfunction. The greatly decreased serum concentration of IGFBP-3 maycontribute to the diagnosis of HCC.2. The expression intensity of IGFBP-3 in liver tissues with cirrhosis andchronic hepatitis was decreased and can reflect the damage degree of liverfunction. The inhibition of apoptosis and enhanced proliferation of hepatocytesdue to significantly reduced expression of IGFBP-3 in HCC may be involved inhepatocarcinogenesis.3. The recombinant eukaryotic expressing plasmid pcDNA3.1-BP3 wasconstructed successfully.4. Stable transfectants Hep3B-pcDNA3.1-BP3 and Hep3B-pcDNA3.1wereobtained and the IGFBP-3 cDNA is expressed successfully inHep3B-pcDNA3.1-BP3.5. The growth-inhibitory and apoptosis-inducing effect of endogenousIGFBP-3 was shown in Hep3B cells.