Effect Of Heparanase RNA Interference On Biological Behaviour Of HepG2 Liver Cancer Cells

Background and ObjectiveMalignant tumor is one of the most severe deseases that are threatening human health. Tumor invasion and metastasis are main causes leading to patients' death.Although early diagnosis and treatment would be the best way for patients obtaining long term survival, many patients are found to be metastasized and with little hope on surgery,chemotherapy and radiotherapy.Hence how to last the survival time of these patients is becoming an ever important goal.Heparanase(Hpa)is the only endogenous endo-glycosidase found by now that is able to degradate heparan sulfate proteoglycans(HSPG) contained in extracellular matrix(ECM) and base memebrance(BM).In normal cells,except lymphocyte and bone marrow cells, heparanase is not expressed in most mature nonimmune tissue(such as heart,lung,liver, skeletal muscle and pancreas).However,it exists in almost all metastatic malignant tumor cells.Studies have shown that down-regulating expression of heparanase could obviously inhibit proliferation and metastasis of tumor cells.Heparanase is gaining more and more attention as a new target for tumor gene therapy.RNA interference(RNAi) is gene silencing after the transcription of micromolecule double strands RNA(siRNA).siRNA selectively combines with it's complementary mRNA and induces it's degradation.The technology of RNAi has developed very fast recently.Specially after the report,on 2001 Science,of siRNA successfully inducing selective mammalian cell gene silencing,RNAi gradually becomes an important tool for biomedical research.Now RNAi technology has been vastly use in many hot field such as gene function test,post-transcriptional control of gene expression,replacing the conventional antisense nucleic acids method inducing specific gene silencing.It provides a new possibility of gene therapy for many kinds of desease. Based on above analysis,this study uses RNAi technology to induce Hpa silencing in liver cancer cell line HepG2,investigating the Hpa silenced HepG2 status on tumor growth, invasion and so on,providing theoretical basis for target choosing of tumor gene therapy.Method1.Heparanase mRNA sequence is available in Genebank.We selected 3 interference sequences through on line design tool in web site,and a group of independence sequence by BLAST as negative control group.We further designed dsDNA including interference sequences and independence sequence for synthesis.Then double restricted enzyme digested pGenesil-1 was ligated with synthesized dsDNA.After being verified by sequencing we had it amplified and extracted.2.HepG2 liver cancer cells were stablely transfected with each recombinant interference plasmid by DOTAP lipofection method.After 4 weeks of G418 selection,drug resistant clones were picked from each group for amplified culturing and were named as: HepG2/RNAi/N,HepG2/RNAi/1-1,HepG2/RNAi/2-2,HepG2/RNAi/3-1 and HepG2/RNAi/3-3,respectively.3.Real Time RT-PCR and Western blot were carried out to screen effective interference sequences.4.Changes of biological behaviour of HepG2 after heparanase RNA interference were investigated respectively on proliferation,cell cycle distribution,clone formation,invasion and tumorigenesis in nude mice.Result1.Three interference sequences were determined by online tools of www. genscript.com.They are:No.1(1214-1232):5' -GGCTATCTCTTCTGTTCAA-3',No.2 (167-185):5' -TCCTGTCCGTCACCATTGA-3',No.3(611-629):5' -CTCAGTTGCT CCTGGACTA-3',and negative control sequence 5' -ACTACCGTTGTATAGGTGT-3'; dsDNA that including interference and control sequences were successfully cloned into pGenesil-1 plasmid and confirmed correct by sequencing.These plasmids were named as pGenesil-1/Hpa/siRNA-1,pGenesil-1 /Hpa/siRNA-2,pGenesil-1/Hpa/siRNA-3 and pGenesil-1/Hpa/siRNA-N,respectively.2.Each group of recombinant interference plasmids was transfected into HepG2 cells by DOTAP lipofection method.After 3 weeks of G418 selection,drug resistant clones were picked and named as HepG2/RNAi/1-1,HepG2/RNAi/2-2,HepG2/RNAi/3-1, HepG2/RNAi/3-3 and HepG2/RNAi/N cells respectively.3.Real Time RT-PCR result showed that compared with HepG2 and HepG2/RNAi/N cells,mRNA level of Hpa was significantly down-regulated in the group of HepG2/RNAi/1-1,HepG2/RNAi/2-2 and HepG2/RNAi/3-3 cells except HepG2/RNAi/3-1 cells.Western blot result showed that Hpa protein level in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 was significantly decreased compared with HepG2 and HepG2/RNAi/N cells.The inhibiting rate of heparanase protein was 57%and 71%respectively.This results confirm that sequence 5'- GGCTATCTCTTCTGTTCAA-3'(1214-1232) and 5'-CTCAG TTGCTCCTGGACTA-3'(611-629) are the effective interference sequences of heparanase.4.Western blot testing various generations of interfered cells for heparanase protein level showed that before the 10th generation,No.1(1214-1232) and No.3(611-629) interference sequences could effectively inhibit expression of heparanase protein.5.MTT result showed that cell proliferation ability of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was significantly lower than that of HepG2 and HepG2/RNAi/N cells.Plate clone formation experiment demonstrated that single cell clone formation capacity of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was obviously lower than that of HepG2 and HepG2/RNAi/N cells(34±4,26±5 vs 138±7,123±22,p＜0.05).Flow cytometry demonstrated that No.1(1214-1232) and No.3(611-629) interference sequence could increase HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells arresting in G₀/G₁ and decrease proliferative index(PI) of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells.Transwell in vitro invasion experiment showed that penetrated cell number in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was much lower than in HepG2 and HepG2/RNAi/N cells(85.1±9.1, 78.1±10.4 vs 182.2±9.7,183.5±9.3,p＜0.05).Tumorigenesis in nude mice experiment demonstrated that HepG2 and HepG2/RNAi/N cells had a tumor formation ratio of 100%. Although HepG2/RNAi/1-1 cells also had a tumor formation ratio of 100%,but its subcutaneous tumor volume is much smaller than of HepG2 and HepG2/RNAi/N cells (0.099±0.030 vs 0.585±0.135,0.690±0.099,p＜0.01),while no subcutaneous tumor formed in group of HepG2/RNAi/3-3 cells.Conclusion1.Through bioinformatics technology,we choose three heparanase RNA interference sequences and a negative control sequence.We successfully construct three heparanase RNAi plasmids which are then stablely transfected into HepG2 liver cancer cells.Drug resistant clones are picked out after G418 selection for 4 weeks.By realtime time RT-PCR and Western blot,we successfully screened that 5'-GGCTATCT CTTCTGTTCAA-3' (1214-1232) and 5'-CTCAGTTGCTCCTGGACTA-3'(611-629) are the effective interference sequences of heparanase.Western blot further confirms that before the 10th generation,above interference sequences could effectively inhibit expression of heparanase protein in HepG2 cells.2.A serial of in vivo and in vitro study indicated that after effectively interfered by the above sequences,the cell growth velocity,single cell clone formation capacity,in vitro invasion capacity and tumorigenesis in nude mice are significantly decreased in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells compared with HepG2 and HepG2/RNAi/N cells.The above results suggest that the heparanase RNAi sequences we screened can effectively inhibit HepG2 liver cancer cells in proliferation,invasion,and tumorigenesis in nude mice.The above heparanase RNAi sequences may therefore represent interesting candidates for the treatment of patients with advanced liver cancer and possibly also with other malignanices.