The Effects Of ASAP3 On Invasive And Metastasis Ability Of Hepatoma Carcinoma Cell Lines

Objective:Hepatoma carcinoma(HCC) is a highly malignant cancer in our country, and the metastasis and recurrence(?) the main obstacle for the living of the patients Moreover, it is the main causation which result in the death of the patients with HCC. Presently, the mechanism of the invasive and metastasis is still blurry. It has become the hotspot to open out the mechanism of the invasive and metastasis and to search the new molecular targets in order to inhibit the invasive and metastasis of tumour. ASAP3also referred to as UPLCl、DDEFL1 and ACAP4, is a new member of the ARF GAPs family. It contains the BAR PH, GAP domains and the ANK repeats. Previous studies indicated that ASAP3 might play pivotal roles in cell migration,vesicle transport, cell skelecton rearrangement and cell proliferation. It has been reported that ASAP3 was highly up-regulated in several human hepatocellular carcinoma. We cloned the cDNA of ASAP3, constructed the ASAP3-siRNA expression vector, on these basis, the roles of ASAP3 on the invasive and metastasis ability were studied. This research will provide the theoretical foundation and experimental basis for the consequent clinical application.Methods and results:1. Constructing and identifying of the ASAP3 specific siRNA expression vector: Design on web (http://www.ambion.com/techlib/misc/siRNA_finder.html) with Ambio technology and constructing the the ASAP3 specific siRNA expression vector in vitro., and bata actin as negative control. Be named pASAP3-siRNA and pGAPDH-siRNA respectively.2. The effects of pASAP3-siRNA on the invasive and metastasis ability of HCC cell:①The effect of ASAP3-siRNA on the ASAP3 mRNA expression of HCC cells: Transiently transfected MHCC97H with pASAP3-siRNA and pGAPDH-siRNA plasmids using Lipofectin, pSilencerTM3.1 H1 empty vector as control. Collect the cells at 24h after transfection, Detected ASAP3 mRNA expression of hepatocellular carcinoma cell line MHCC97H cell with RT-PCR and western blot respectively. ASAP3 mRNA bands optical density analysis showed that ASAP3 mRNA expression levels were significantly higher in the pASAP3-siRNA transfection of MHCC97H cell than the other control groups at 24h after transfection, the difference was significant (P <0.01). pASAP3-siRNA took on the efficiency on ASAP3 mRNA expression gene silencing, the silencing rate was about 67%. Furthermore, ASAP3 protein band optical density analysis showed that ASAP3 protein in MHCC97H cell transfected pASAP3-siRNA decreased significantly than other control groups, the difference was significant (P<0.01).②he effect of ASAP3-siRNA on the proliferation and apoptosis of HCC cells:Transiently transfected MHCC97H with pASAP3-siRNA and pGAPDH-siRNA plasmids using Lipofectin, pSilencerTM3.1 H1 empty vector as control. Collect the cells at 24h after transfection, Detected cell proliferation and apoptosis by MTT assayand flow cytometry respectively. Results showed that the cell proliferation of the MHCC97H cells transfected pASAP3-siRNA was significantly inhibited at 48h after transfection than the other control groups, (P<0.01), the proliferation inhibition rate was 42.03±4.36%. However the empty vector and pGAPDH- siRNA transfection can not inhibit cell proliferation on MHCC97H. Flow cytometry showed that, compare with the other control groups, the MHCC97H cells transfected pASAP3-siRNA in apoptotic peak in cell cycle patterns formed clearly, the apoptosis rate was 13.88±1.74%. There was no obvious apoptosis peak in other control group.③he effect of ASAP3-siRNA on the biological behavior of HCC cells:Transiently transfected MHCC97H with pASAP3-siRNA and pGAPDH-siRNA plasmids using Lipofectin, pSilencerTM3.1 H1 empty vector as control. Collect the cells at 24h after transfection, Detected cell adhesion rate, cell migration and cell invasion with MTT assay, cell scratch assay and Transwell small rooms assay respectively. The results of MTT colorimetric assay showed that the adhesion of MHCC97H cells transfected pASAP3-siRNA was significantly lower at 60min after cell inoculation, significant difference with the other control groups (p<0.05). The results of scratch experiments at 24h after 24h after transfection showed that the migration of MHCC97H cells transfected pASAP3-siRNA was significantly inhibited comparing with the other control groups. The invasion of MHCC97H cells transfected pASAP3-siRNA was significantly lower than the first two groups (p<0.05).3. Construction and identification of ASAP3 eukaryotic expression vector:Desighed primers referring to the ASAP3 sequence of Genbank, expended the CDS sequence of ASAP3,2753bp totally. Cloned the ASAP3 gene through the DNA reorganization technology and PCR method from person liver cancer cell line MHCC97H, inserted it in eukaryon the expression vector pcDNA3.1/HisC, constructed the eukaryon expression vector including the ASAP3, transfered the DH5acoli., filtrated the positive clone, and identified the accuracy of the reorganizated plasmid through PCR and enzyme cuts and sequence detection. Be named pASAP3.4. The effects of ASAP3 on the invasive and metastasis ability of HCC cell:①The effect of ASAP3 on the ASAP3 expression of HCC cells:Transiently transfected Hep3B with pASAP3 and pcDNA3.1/HisC empty plasmids using Lipofectin. Collect the cells at 24h after transfection. Detected ASAP3 mRNA expression of Hep3B cells with RT-PCR and western blot respectively. ASAP3 mRNA bands optical density analysis showed that ASAP3 mRNA expression levels were significantly higher in the Hep3B cells transfected pASAP3 than the other control groups at 24h after transfection, the difference was significant (P<0.01). pASAP3 can increase ASAP3 expression significantly in Hep3B cells.②The effect of ASAP3 on the proliferation and apoptosis of HCC cells:Transiently transfected Hep3B with pASAP3-siRNA and pcDNA3.1/HisC empty plasmids using Lipofectin. Collect the cells at 24h after transfection, Detected cell proliferation and apoptosis by MTT assayand flow cytometry respectively. Results showed that the Hep3B cells transfected pASAP3 multiplicated exuberantly (P<0.01). Flow cytometry showed that, compare with the other control group, the Hep3B cells transfected pASAP3-siRNA had not the apoptotic peak to appear obviously.③The effect of ASAP3 on the biological behavior of HCC cells:Transiently transfected Hep3B with pASAP3 and pcDNA3.1/HisC empty plasmids using Lipofectin. Collect the cells at 24h after transfection, Detected cell adhesion rate, cell migration and cell invasion with MTT assay, cell scratch assay and Transwell small rooms assay respectively. The results of MTT colorimetric assay showed that the adhesion to FN of Hep3B cells transfected pASAP3 was significantly higher at 60min after cell inoculation, significant difference with the other control groups (p<0.05). The results of scratch experiments at 24h after transfection showed that the metastasis of Hep3B cells transfected pASAP3 was significantly increaseded comparing with the other control groups. The invasion of Hep3B cells transfected pASAP3 was significantly higher than the control groups (p<0.05).Conclusions:In summary:①ASAP3-siRNA can markedly reduce the ASAP3 gene expression in the human liver cancer cell line MHCC97H with high shift potential;②ASAP3-siRNA may suppress the multiplication of person liver cancer cell line MHCC97H obviously, induce apoptosis, and inhibit the invasiveness obviously comparing with the control groups;③Transfecting the ASAP3 gene can clearly enhance the ASAP3 gene expression and the invasive and metastasis ability in person liver cancer cell line Hep3B which does not transfer;④Transfecting the ASAP3 gene can clearly enhance the invasive and metastasis ability in person liver cancer cell line Hep3B which does not metastasize. Indicated that ASAP3 gene expression can obviously affect the multiplication, apoptosis and the invasive and metastasis ability of the liver cancer cells, and it will be possible to take it as a target spot to carry on the HCC prevention.