Functional Localization Of Metastasis Suppressor Gene For Liver Cancer On Human Chromosome 8 And Relation Studies

PART IFunctional location of metastasis suppressor gene for HCCon human chromosome 8Objective: We previously showed that introduction of a normal,neomycin-tagged human chromosome 8 reduces the metastatic capcity of C5F rat liver cancer cell line,which had high metastatic, without affecting tumorigenicity suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 8. To define further the region harboring the metastasis suppressor gene,STS markes were used.We determined the random loss of human chromosome 8 by PCR amplification of STS markers.Methods: The relative genetic distances of the STS markers were according to the National Center for Biotechnology Information Database(NCBI).C5F genomic DNA and A9/neo8 genomic DNA were used as negative and positive controls for chromosome 8 amplification,respectively. Genomic DNA (A9/C5F-1 and A9/C5F-2 microcell hybrid clones used as metastasis-unsuppressed groups, A9/C5F-4, A9/C5F-8 and A9/C5F-10 microcell hybrid clones used as metastasis suppressed groups) was isolated and quantified from cultured hybrid clones.STS-PCR products were separated by electrophoresis through 2% agarose gel.Results: Metastasis-suppressed microcell hybrid clones (A9/C5F-4, A9/C5F-8 and A9/C5F-10) conserved STS markes between D8S542→D8S1973(8p21.1-23.1). In contrast, metastasis -unsuppressed clones (A9/C5F-1 and A9/C5F-2) lacked several markes in this region. To refine futher the region retaind in microcell suppressed clones, more densely spaced STS markes in the human chromosome 8p21.1-23.1 were used. The metastasis-suppressed clones retained ~ 18cM region between D8S542 and D8S1973 (8P21.1 -23.1), where the metastasis-unsuppressed clones lacked the region.Conclusion: These resuots suggest that that the metasasis suppressor gene is located within the interval between D8S542 and D8S1973 on human chromosome 8p21.1-23.1.PART IIRelationship between DLC-1 expressions and metastasis in hepatocellular carcinoma —real-time quantitative PCR studyObjective: Chromosome 8p deletion has been found with association to human hepatocellular carcinoma(HCC) metastasis. This paper aimed to study the relationship between the expression level of DLC-1 mRNA(located in 8p) and invasion/metastasis of HCC.Methods: Fifty-one surgical specimens of human HCC were divided into high-invasiveness and low invasiveness groups according to the clinicopathological features. DLC-1 mRNA expression was studied in the 51 HCC specimens as well as 5 different metastasis potential cell lines using real-time quantitative PCR (RQ-PCR).Results: The expression level of DLC-1 mRNA in HCC specimens with high invasiveness was significantly lower than that with low invasiveness. (p<0.05); The expression levels of DLC-1 mRNA were significantly different between non-metastasis ( Hep3B and HepG2) and metastasis(MHCC97-H,MHCC97-L and HCCLM3) cell lines(p<0.05).From MHCC97-L to HCCLM3, with the increase of invasiveness and metastatic potentials, the expression levels of DLC-1 decreased correspondingly, its expression level in HCCLM3 was significantly lower than thatConclusion: the expression of DLC-1 mRNA may play an important role in inhibiting the invasiveness and metastasis of HCC.PART IIIIn vitro studies of DLC-1 gene exprressionObjective: Chromosome 8p21.1 -23.1 has been found with assoxiation to human hepatocellular carcinoma metastasis. DLC-1 gene is just located within the region. We constructed the recombiant expression plasmid of pcDNA3.1-DLC-1 and observedthe changes of biological properties after HCC cells transfected to futher study the effect of DLC-1 on HCC cell invasiveness and metastasis in vitro.Methods: To further explore the effect of DLC-1 overexpression on HCC cell invasiveness and metastasis in vitro, recombinant expression plasmid vector of pcDNA3.1(-) —DLC-1 was constructed and then transfected into high metastasis MHCC97-H> HCCLM3 cell lines ,by LipofectamineTM 2000 transfection.Then a series of cell biology function assays related to invasiveness and metastasis (cell proliferation,invasion and metastasis , extracellular matrix metalloproteinase, apoptosis etc. )were applied in our project.Results: We constructed recombinant plasmid vector of pcDNA3.1(-)—DLC-1. After the transfection of recombinant plasmid , HCCLM3 cells showed a significant high expression of DLC-1 by Real-Time PCR and Western Blot assays.Results of cell proliferation assays showed that expression of DLC-1 inhibited the proliferation of HCCLM3 cells .In day 3> 4, absorbance at 450nm , which standed for the number of vital cells, was significantly lower in the group of recombinant transfection than in the control(in day 3: 0.734±0.073 vs. 1.038±0.081 vs. 1.149±0.099;inday4: 0.982±0.029 vs. 1.858±0.054 vs. 1.926±0.086; P<0.05).The high expression of DLC-1 can also inhibited the proliferation of MHCC97-H cells.Transfected cell apoptosis ration was 21.55% (HCCLM3/pcDNA3.1(-) AS DLC-1) x 14.26% (HCCLM3/pcDNA3.1(-) respectively by FCM analysis. The significant changes in apoptosis of MHCC97-H cells transfected with DLC-1 (MHCC97-H/pcDNA3.1(-) AS DLC-1, 12.57% vs. MHCC97-H/pcDNA3.1(-), 7.45% vs. MHCC97-H, 4.80%) implied that DLC-1 could improve the apoptosis ration. But no significant changes in cell cycles can be observed in transfected cells.Cell invasion assay in vitro showed the average invading cell numbers per field were 22.833±1.137 (MHCC97-H), 21.000±1.211(MHCC97-H/pcDNA3.1(-)), 12.833±0.601 (MHCC97-H/pcDNA3.1(-)-DLC-l)* 45.000±2.608 (HCCLM3) , 44.833±1.941 (HCCLM3/pcDNA3.1(-)) , 19.333±4.131(HCCLM3/pcDNA3.1(-)-DLC-l) respectively. This result demonstrated the significant difference of cell invasion potential between recombinant plasmid of pcDNA3.1(-) —DLC-1 and controls. In the group with recombinant vectors transfection, the motile ability was significantly lower than that of empty...