| PartⅠ Production of RNA interference lentivirus targeted at hypoxia inducible factor-lalph and evaluation of interference targetsPurpose:To try whether the lentivirus vectors targeting to hypoxia inducible factor-la (HIF-la) could be transfected to McA RH7777cells, and to evaluate the degrees of HIF-1α knocked down for choosing the best interference target to carry on the following studies.Materials and methods:To design four HIF-la RNA interference targets and one negative control (NC) targets, and to attach the interference targets to pGCSIL-GFP lentivirus vectors; To transform the attaching production to the competent cells of E.coli, to choose the positive clones to carry on PCR amplification and gene sequencing for identify whether the RNA interference targets were attached to the lentivirus vectors; To amplify the lentivirus vectors with RNA interference targets and measure the titer; To transfect the lentivirus vectors with RNA interference targets to McA RH7777cells, the transfected cells were carried on cell crawling piece and DAPI staining to determine the transfection efficient, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried on to determine the mRNA degree of HIF-1α knocked down.Results:RNA interference targets were attached to lentivirus vectors and transformed to the competent cells. The amplification length of the vectors were following:NC,343bp; Target1,347bp; Target2,347bp; Target3,347bp; Target4,346bp. The gene sequencing certificated that the RNA interfence targets were attached to the vectors successfully, The amplification of lentivirus vectors and the titer measurement were carried on successfully, the titer of the vectors were following:HIF-1α-LV-NC,5E+9TU/ml, HlF-1α-LV-Targetl,4E+8TU/ml; HIF-1α-LV-Target2,5E+8TU/ml; HIF-1α-LV-Target3,7E+8TU/ml; HIF-1α-LV-Target4,7E+8TU/ml. According to cell crawling piece and DAPI staining, all the transfection rates were more than95%; After the vectors were transfected with McA RH7777cells, the relative HIF-lamRNA levels were following according to the RT-PCR analysis:Wild type,1.01±0.09; NC,1.02±0.08; Targetl,0.50±0.13; Target2,0.23±0.13; Target3,0.71±0.10; Target4,0.82±0.11. So, Target2vectors will be used for following studies.Conclusion:The HIF-la RNA interference targets were attached to the lentivirus vector pGCSIL-GFP successfully; the lentivirus vector pGCSIL-GFP transfected with McA RH7777cells successfully, and the transfection rates were higher; in McA RH7777cells, the HIF-la mRNA levels were knocked down by RNA interference. Part Ⅱ RNA interference of hypoxia inducible factor-1alph decreased the proliferation, migration, and invasion of hypoxic hepatocellular carcinoma cells in vitroPurpose:Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits HIF-1α and further VEGF (vascular endothelial growth factor) expression, cell proliferation, invasion, and migration in a hepatocellular carcinoma (HCC) cell line under hypoxia.Materials and methods:RNA interference of HIF-1α was constructed by lentivirus-mediated RNA interference tools, and a rat HCC cell line, McA RH7777, infected with short interference of HIF-1α coding virus were cultured under a hypoxia condition or a norm conditions for a series of times. The expression levels of HIF-1α and VEGF were examined using RT-PCR and Western blot. Cell proliferation, migration and invasion were measured by cell viability assay, transwell migration and invasion assay, respectively.Results:The results of RT-PCR analysis:HIF-la RNA interference-treated cells under normoxic conditions showed reduced HIF-la levels of76.94%, while VEGF levels were reduced by53.72%. Vector control cells under hypoxic conditions showed a relative increase in the mRNA expression of HIF-la and VEGF were3.83fold and2.90fold, respectively, over the levels of HIF-la RNA interference-treated cells when the mRNA expression of HIF-1α and VEGF were the most high in a series of hypoxic times. According to pearson correlation analysis for HIF-1α and VEGF mRNA in the two groups, pearson coefficient was0.84and0.83in HIF-1α RNA interference and control cells respectively, the mRNA expressions of HIF-1α and VEGF were correlated (p<0.05). HIF-1α RNA interference inhibited the hypoxia induced increment of HIF-1α and VEGF mRNA expressions.The results of Western blot analysis:HIF-la in the HIF-1α RNA interference-treated cells was0.10-fold and0.18-fold of that in the control cells under norm conditions and6h-hypoxic conditions, respectively. VEGF in the HIF-1α RNA interference-treated cells was0.71-fold and0.47-fold of that in the control cells under norm conditions and6h-hypoxic conditions, respectively. HIF-la RNA interference inhibited the hypoxia induced increment of HIF-1α and VEGF protein expressions.The results of transwell migration assay:under norm conditions, the migrated cells (35.17±8,64) in the HIF-la RNA interference-treated cells were less the migrated cells (46.83±9.50) in the vector control cells significantly (p<0.05); under hypoxic conditions, the migrated cells (48.17±10.76)in the HIF-la RNA interference-treated cells were also less the migrated cells (75.83±8.13) in the vector control cells significantly (p<0.05). HIF-1α RNA interference inhibited the hypoxia induced increment of cell migration.The results of transwell invasion assay:under norm conditions, the invasive cells (25.33±6.92) in the HIF-1α RNA interference-treated cells were less the invasive cells (36,83±7.47) in the vector control cells significantly (p<0.05); under hypoxic conditions, the invasive cells (32.67±7.94) in the HIF-la RNA interference-treated cells were also less the invasive cells (47.33±6.95) in the vector control cells significantly (p<0.05). HIF-1α RNA interference inhibited the hypoxia induced increment of cell migration.The results of cell viability assay:hypoxia significantly reduced the viability of HIF-1α RNA interference cells, compared with that of control cells. All the coefficient of drug interaction (CDI) values for the different degree of hypoxia were less than1, indicating a synergistic effect between HIF-la RNA interference and hypoxia.Conclusion:These data suggest that short interference RNA of HIF-la inhibits the HIF-laand VEGF mRNA and protein expression, the cell migration and invasion which have been enhanced under hypoxic conditions, and synergizes with hypoxia to inhibit the cell proliferation in HCC cells. Part III RNA interference of hypoxia inducible factor-1alpha improves the effects of transcatheter arterial embolization in rat liver tumorsPurpose:In this study, we investigated whether RNA interference of HIF-1α could improve the efficacy of transcatheter arterial embolization (TAE) to treat HCC.Materials and methods:McA RH7777transfectants expressing either the empty vector or the HIF-1α RNA interference sequence were subcutaneously implanted into the rats’right thighs. Two kinds of pieces of tumor tissue were implanted into32rats’livers to induce HCC, respectively. Tumor-bearing rats were randomly assigned to one of the four treatment groups (n=8):siHIF-la+TAE, siHIF-la, TAE, and Control. Rats were treated by TAE by retrograde placement of a microcatheter into the gastroduodenal artery. In the siHIF-1α+TAE and TAE groups, rats received an intra-arterial injection of iodized oil. In the siHIF-1α and Control groups, rats received an equivalent amount of saline via intra-arterial injection. Animals were sacrificed four weeks after TAE and their livers and lungs were harvested, to study the difference of tumor volumes, to study the lung metastasis by H&E staining, to study the tissue expression difference of HIF-la, VEGF, and CD31by immunohistochemistry analysis, to study the protein expression difference of HIF-la and VEGF by Western blot analysis.Results:The results of immunohistochemistry analysis:the tumor tissue expression of HIF-1α, VEGF, and CD31in the siHIF-1αa group were less significantly than in the Control group(42.69±12.13vs59.24±15.35;51.21±12.23vs68.75±13.81;39.70±15.25vs58.90±15.12)(p<0.05); the tumor tissue expression of HIF-la, VEGF, and CD31in the siHIF-1α+TAE group were less significantly than in the TAE group (48.32±13.76vs74.26±14.78;57.39±13.41vs83.46±14.3545.20±16.44vs69.15±16.88)(p<0.05). The knock-down of HIF-1α inhibited the increased expression of HIF-1α, VEGF, and CD31caused by TAE in addition to that caused by tumor hypoxia in tumor tissues.The results of Western blot analysis:the tumor protein expression of HIF-laand VEGF in the siHIF-la group were less significantly than in the Control group (0.26±0.06vs0.40±0.06;0.61±0.07vs0.78±0.07)(p<0.05); the tumor protein expression of HIF-laand VEGF in the siHIF-la+TAE group were less significantly than in the TAE group (0.29±0.07vs0.52±0.07;0.66±0.07vs0.90±0.08)(p<0.05). HIF-1α knock-down inhibited the increased protein expression of HIF-1α and VEGF caused by TAE.The results of H&E staining analysis:The lung metastatic rate declined visibly, from87.5%in the Control group to50%in the siHIF-la group (p>0.05), and from100%in the TAE group to50%in the siHIF-la+TAE group (p<0.05). The mean number of lung nodules in each rat in the siHIF-la group were reduced significantly compared to the rats in the Control group (7.38±8.57vs32.75±16.35, p<0.05); the mean number of lung nodules in the rats in the siHIF-la+TAE group were reduced significantly compared to the rats in TAE group (10.38±11.67vs42.88±13.77, p<0.05). RNAi of HIF-la effectively inhibited lung metastasis of McA RH7777tumors enhanced by TAE.The results of tumor volume analysis:Tumors treated with siHIF-1α+TAE only reached volumes of8162±4269mm3, and were significantly smaller than Control group tumors (21489±4603mm3)(p<0.05) and the tumors treated with the siHIF-la or TAE (14501±4355and15573±4191mm3)(p<0.05). The CDI of siHIF-1α and TAE was0.78, indicating that RNAi of HIF-1α synergized with TAE to inhibit the growth of McA RH7777tumors.Conclusion:RNAi of HIF-1α reduced the expression of HIF-1α, VEGF and CD31in vivo. RNAi of HIF-1α improved the efficacy of TAE by inhibiting tumor angiogenesis, growth, and metastasis. The combination of RNAi of HIF-1α and TAE had a marked synergistic effect on tumor growth inhibition. RNA interference of HIF-1α augmented the therapeutic effects of TAE for HCC. Part IV RNA interference of hypoxia inducible factor-1alpha under the guidance of B ultrasound improves the effects of transcatheter arterial chemoembolization in rat liver tumorsPurpose:In this study, we investigated whether RNA interference of HIF-la under B ultrasound guidance could improve the efficacy of transcatheter arterial chemoembolization (TACE) to treat HCC.Materials and methods:Wild type McA RH7777cells were subcutaneously implanted into the rats’ right thighs. Pieces of tumor tissue were implanted into40rats’livers to induce HCC, respectively. Tumor-bearing rats were randomly assigned to one of the four treatment groups (n=10):siHIF-1α+TACE, siHIF-1α, TACE, and Control.RNA interference of HIF-1α under B ultrasound guidance was performed15days after tumor implantation. In the siHIF-1α+TACE and siHIF-1α groups, rats received an intra-tumor injection of RNA interference vectors. In the TACE and Control groups, rats received an equivalent amount of negative control vector via intra-tumor injection. TACE was performed in the next day.Rats were treated by TACE by retrograde placement of a microcatheter into the gastroduodenal artery. In the siHIF-1αa+TACE and TACE groups, rats received an intra-arterial injection of iodized oil and doxorubicin. In the siHIF-la and Control groups, rats received an equivalent amount of saline via intra-arterial injection.The change of animal body weight after TACE was observed, the tumor volumes were measured by B ultrasound every week after TACE, and the overall survival of each rats were recorded. Animals’livers and lungs were harvested, to measure the lung metastasis by H&E staining, to measure the tissue expression difference of HIF-la, VEGF, and CD31by immunohistochemistry, to measure the protein expression difference of HIF-la and VEGF by RT-PCR.Results:The results of rats’weight analysis:the rats’weight of each groups had not a upward or a downward trend during the first week after treatment; the rats’weight of each groups had a upward trend during the second week; during the third week, the rats’weight in siHIF-la+TACE group had still a upward trend, the rats’weight in siHIF-la and TACE groups had a slight upward trend, the rats’weight in Control group had a downward trend; during the fourth week, the rats’weight in siHIF-1αa+TACE group had still a upward trend; the rats’weight in siHIF-lα, TACE, and Control group had a downward trend, and the latter was more significant.The results of overall survival analysis:the median overall survivals of the4treatment groups:siHIF-1α+TACE,40days (95%CI:37.93-42.07days); siHIF-1α,33days (95%CI:29.90-36.10days); TACE,35days (95%CI:31.90-38.10days); Control,30days (95%CI:28.99-31.01days). The Kaplan-Meier analysis of the4treatment groups overall survival showed that p<0.05. The knock down of HIF-la improved the efficacy of TACE by prolonging the overall survival.The results of H&E staining analysis:The lung metastatic rate declined visibly, from90%in the Control group to40%in the siHIF-la group (p>0.05), and from100%in the TACE group to50%in the siHIF-1α+TACE group (p<0.05). The mean number of lung nodules in each rat from the siHIF-la group were reduced significantly compared to the rats in the Control group (7.10±10.14vs32.80±15.85, p<0.05); the mean number of lung nodules in the rats in the siHIF-1α+TACE group were reduced significantly compared to the rats in TACE group (11.90±13.58vs44.30±12.64, p<0.05). RNA interference of HIF-1α improved the efficacy of TACE by inhibiting lung metastasis of McA RH7777tumors.The results of immunohistochemistry analysis:In siHIF-la group, the knock-down of HIF-lasignificantly inhibited the increased expression of HIF-la, VEGF, and CD31caused by tumor hypoxia (p<0.05for all factors, compared to Control group); in siHIF-la+TACE, the knock-down of HIF-la also inhibited the increased expression of HIF-1α, VEGF, and CD31caused by TACE in addition to that caused by tumor hypoxia (p<0.05for all factors, compared to the TACE group).The results of RT-PCR analysis:In siHIF-la group, the knock-down of HIF-la inhibited the increased protein expression of HIF-la and VEGF caused by tumor hypoxia (p<0.05compared to the Control group); in siHIF-la group, HIF-la knock-down also inhibited the increased protein expression of HIF-la and VEGF caused by TACE plus tumor hypoxia (p<0.05for all factors, compared to the TACE group).Conclusion:RNA interference of HIF-la under B ultrasound guidance improved the efficacy of TACE for HCC by effectively inhibiting increased HIF-la, VEGF, and CD31after TACE, suppressing the tumor growth, prolonging the overall survival, and reducing the lung metastasis. |
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