Lentiviral vector-mediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>iASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types. This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis.</p><p><b>METHODS</b>A pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control were synthesized, then ligated with pLVTHM vector and sequenced. The fragment containing the shRNA cassette was cloned to pLVCT-tTR-KRAB plasmid. The recombinant vectors were co-transfected with viral packaging mix into 293T cells, and viral supernatant was harvested to determine the titer. After treatment with or without doxycycline, HepG2 cells infected with virus were harvested and the expression of iASPP was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Its effects on tumor growth were characterized using MTS assay, soft agar colony formation, and flow cytometry analysis.</p><p><b>RESULTS</b>The lentiviral vector expressing shRNA that targets to the oncogene iASPP was constructed successfully. HepG2 infected with the lentivirus expressing shRNA against iASPP inhibited the expression of iASPP in the presence of doxycycline, which resulted in the repression of tumor cell proliferation and anchorage-independent growth potential.</p><p><b>CONCLUSIONS</b>The lentiviral vector-mediated tet-on system demonstrates efficient and inducible knockdown of iASPP in hepatocellular carcinoma cells. iASPP gene may be involved in tumorigenesis and progression of human tumors.</p>

Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.</p><p><b>METHODS</b>Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.</p>

Effects of tumor suppressing gene TIP30/CC3 on the growth of tumor cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To introduce the newly found gene TIP30/CC3 into a hepatoma cell line PLC/PRF/5 and select the stable expression clones. The growth and cell cycles were studied with the clones stably expressing TIP30/CC3 or anti-TIP30/CC3, and the effects of TIP30/CC3 gene on hepatoma cells were analyzed.</p><p><b>METHODS</b>The internal expression of TIP30/CC3 protein was detected with Western blot, then TIP30/CC3 or anti-TIP30/CC3 cDNA was subcloned into a constitutive vector pcDNA3 followed by transfection into PLC/PRF/5. Stable expression clones were selected. The cell growth curve was made and cell cycles detected using flow cytometry. To confirm the results in vitro, stable-expressing cells were implanted subcutaneously into nude mice and time of tumor formation recorded and tumor volume measured.</p><p><b>RESULTS</b>PLC-anti-TIP30 grew faster than the others. Three days after transfection, live cells of PLC-anti-TIP30 were 14.0*10(4), in comparison with the control PLC-DNA3 and PLC/PRF/5, the differences were statistically significant. Live cells of PLC-TIP30 were 4.9*10(4), significantly less than the two control groups. Six days after transfection, live cells of PLC-anti-TIP30 were 25.0*10(4), significantly more than the controls PLC-DNA3 and PLC/PRF/5. Live cells of PLC-TIP30 were 12.4*10(4), significantly less than the two control groups. Cell cycle analysis showed that PLC-anti-TIP30 proliferated faster, 22.4% cells were in G0/G1 (gap) phases and 58.6% cells in S (DNA synthesis) phase. The growth of the PLC-anti-TIP30 cell was retarded and many cells were arrested from G1 to S phases. Cells in G0/G1 and S phase were 44.2% and 33.3% respectively. Furthermore, the average time of tumor formation was shorter in anti-TIP30 group and longer in TIP30/CC3 group, and times were 6.0 d (with control groups) and 15.6 d (with control groups) respectively. Tumors in the nude mice grew faster in PLC-anti-TIP30 group and slower in PLC-TIP30 group.</p><p><b>CONCLUSION</b>Tumor suppressor gene TIP30/CC3 can inhibit the proliferation of tumor cells and interfere in its cell cycles. It can be used as a valuable tool for hepatoma biotherapy including gene therapy.</p>

Construction of adenovirus vector expressing TIP30 and its tumor suppressive effect in vitro and in vivo / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct an adenovirus vector expressing TIP30 gene (Ad-TIP30) and investigate its tumor suppressive effect in vitro and in vivo.</p><p><b>METHODS</b>Ad-Easy system was used to construct Ad-TIP30 by recombination in E. coli. The virus was packaged in 293 cells and subsequently identified valid. Human HCC (hepatocellular carcinoma) cell lines HepG(2) (p53-wt), PLC/PRL/5 (p53-mut), and osteosarcoma cell line Saos-2 (p53-null) with different p53 genotype were infected with Ad-TIP30 and control virus with Ad-GFP, respectively. The tumor suppressive effect of TIP30 in vitro was examined by trypan blue exclusion method. The expression level of p53 was determined by RT-PCR before and after Ad-TIP30 infection. The in vivo tumor suppressive effect was detected in nude mice with human HCC xenograft.</p><p><b>RESULTS</b>The expression of TIP30 significantly inhibited the in vitro proliferation of tumor cells, among which HepG(2) with wild type p53 gene was most susceptible to Ad-TIP30 induced growth inhibition. The expression of p53 was significantly up-regulated in HepG(2) after Ad-TIP30 infection as determined by RT-PCR. The growth in nude mice of HCC infected with Ad-TIP30 was significantly inhibited with an inhibition rate of 62.9%.</p><p><b>CONCLUSION</b>The expression of TIP30 could inhibit the proliferation of tumor cell lines through both p53-dependent and p53-independent pathways, and may be used as a potential tool for cancer therapy.</p>

The expression of platelet-derived growth factor (PDGF) receptor-beta and its correlation with extracellular matrix in hepatic tissue in hepatic fibrosis rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression of PDGF receptor-beta and its correlation with extracellular matrix in hepatic tissue during hepatic fibrosis.</p><p><b>METHODS</b>The model of hepatic fibrosis in rats was induced by carbon tetrachloride. PDGF receptor-beta subunit, collagen I, collagen III and a-SMA in hepatic tissues of these rats were examined using immunohistochemistry. The correlation between PDGF receptor-beta subunit and collagen I, III was analyzed using SAS software after the results of immunohistochemistry were semi-quantified.</p><p><b>RESULTS</b>PDGF receptor-beta subunit and a-SMA were not detected in normal controls. Collagen I and III were distributed in the portal tracts and beneath the endothelia of the central veins and of the Disse spaces. Two weeks after CCl4 injection, the PDGF receptor-beta and a-SMA were detected, and the expression of collagen I and III increased. At the end of 4 and 6 weeks, the above four proteins were further increased. Two weeks after CCl4 injection, PDGF receptor-beta had no apparent correlation with collagen I and III. However, PDGF receptor-beta had a significant correlation with collagen I and III 2 weeks later, and the correlation coefficient was 0.74 and 0.60 respectively at 4 weeks, and 0.83 and 0.67 respectively at 6 weeks. PDGF receptor-beta had a significant correlation with a-SMA during the whole process of hepatic fibrosis and the correlation coefficient was 0.62, 0.69 and 0.81, respectively at the time of 2, 4 and 6 weeks after CCl4 injection.</p><p><b>CONCLUSION</b>The PDGF receptor-beta was overexpressed during the process of hepatic fibrosis development, and it significantly correlated with collagen I and collagen III.</p>

Effect of ribozyme against platelet-derived growth factor receptor beta subunit mRNA on the biological characters of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs.</p><p><b>METHODS</b>Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection. The expression of PDGFR- beta, alpha-smooth muscle actin (alpha-SMA), and type I and type III collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was demonstrated with flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.</p><p><b>RESULTS</b>The expression of PDGFR- beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSCs only 43% to 51% of that in control cells (t > or = 3.957, P < 0.05), and alpha-SMA expression level, type I and type III collagen synthesis ability were also reduced (t > or = 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t > or = 3.858, P < 0.05), and the proliferation response to PDGF BB was markedly inhibited. However the apoptotic rate was significantly increased in ribozyme-transfected HSCs (chi2 > or = 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.</p><p><b>CONCLUSIONS</b>The anti-PDGFR- beta ribozyme can be expressed stably in HSCs, cleave the target RNA effectively, inhibit HSCs proliferation and collagen synthesis, and induce HSC apoptosis. The results suggest that inhibiting PDGFR- beta expression in HSCs may be a new therapy for liver fibrosis.</p>

Establishment of a mouse hepatocellular carcinoma cell line producing mMIP-1α chemokines and the tomorigenicity of mMIP-1α transfected Hepa1-6 / 第二军医大学学报

Objective: To establish a mouse hepatocellular carcinoma cell line that can produce mMIP-1α and to evaluate the possibility of cancer gene therapy by mMIP-1α. Methods: mMIP-1α cDNA was cloned into retrovirus vector pBabe puro and pBabe puro-mMIP-1α was constructed, then pBabe puro-mMIP-1α was used to transfect packaging cells, anti-puromycin cells was proliferated, the supernatant was used to infect hepa1-6, the anti-puromycin clone (hepa1-6 mMIP-1α) and hepa1-6 were analysed for the expression of mMIP-1α mRNA and protein by RT-PCR and immunohistochemistry respectively. The growth curve of hepa1-6 and hepa1-6 mMIP-1α was drawn. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed on agarose gel. C57B/L mouse was inoculated with the tumor cell and the tumorigenicity was studied. Results: Recombinant retrovirus vector pBabe puro-mMIP-1α with mMIP-1α cDNA was constructed. Hepa1-6 did not produce mMIP-1α mRNA and protein, while hepa1-6 mMIP-1α could produce mMIP-1α mRNA and protein. The growth curve of hepa1-6 and hepa1-6 mMIP-1α showed no difference. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed. The tumorigenicity was reduced. Conclusion: A mouse hepatocellular carcinoma Hepa1-6 mMIP-1α is established and mMIP-1α can affect the tumorigenecity of hepa1-6.

Establishment of a mouse hepatocellular carcinoma cell line producing mMIP-1α chemokines and the tomorigenicity of mMIP-1α transfected Hepa1-6 / 第二军医大学学报

Objective: To establish a mouse hepatocellular carcinoma cell line that can produce mMIP-1α and to evaluate the possibility of cancer gene therapy by mMIP-1α. Methods: mMIP-1α cDNA was cloned into retrovirus vector pBabe puro and pBabe puro-mMIP-1α was constructed, then pBabe puro-mMIP-1α was used to transfect packaging cells, anti-puromycin cells was proliferated, the supernatant was used to infect hepa1-6, the anti-puromycin clone (hepa1-6 mMIP-1α) and hepa1-6 were analysed for the expression of mMIP-1α mRNA and protein by RT-PCR and immunohistochemistry respectively. The growth curve of hepa1-6 and hepa1-6 mMIP-1α was drawn. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed on agarose gel. C57B/L mouse was inoculated with the tumor cell and the tumorigenicity was studied. Results: Recombinant retrovirus vector pBabe puro-mMIP-1α with mMIP-1α cDNA was constructed. Hepa1-6 did not produce mMIP-1α mRNA and protein, while hepa1-6 mMIP-1α could produce mMIP-1α mRNA and protein. The growth curve of hepa1-6 and hepa1-6 mMIP-1α showed no difference. The chemotaxis of mMIP-1α produced by hepa1-6 mMIP-1α to mouse spleen cells was observed. The tumorigenicity was reduced. Conclusion: A mouse hepatocellular carcinoma Hepa1-6 mMIP-1α is established and mMIP-1α can affect the tumorigenecity of hepa1-6.