Study Of A Novel Tumor Targeting Peptide Gene Vector And Its Therapeutic Effects On Liver Cancer Cells

BackgroundLiver cancer is a malignant tumor derived from liver and its morbidity and mortality rates in the top five in China.The main therapeutic method for liver cancer is treated with small-molecule drugs,which susceptible to drug resistance,therefore,the therapeutic effect of small-molecule drugs is limited.Anti-tumor peptides have attracted extensive attention as it characterized as hypotoxicity,variety,low immunogenicity and non-resistance.Anti-tumor peptides original abroad,the natural ones are abundant and which were applied extensively to basic research and clinical trials.However,some anti-tumor peptides source from nature presented as instability and unmodifiability,which limited its application.Artificial peptides presented great application prospect as its flexible modification.Plenty of studies revealed that anti-tumor peptides worked by multiple mechanisms such as disrupting cell membrane,inducing cell apoptosis,inhibiting cell transition and invasion,and regulating angiogenesis.Most of peptides affect with variety ways.Although the advantages of anti-tumor peptides are apparently,the ability of target to cancer cells is deficient.Therefore,the way to combine anti-tumor peptides with gene-target therapy will be a potential research orientation.The activity of human telomerase reverse transcriptase?hTERT?promoter was close to Cytomegalovirus?CMV?promoter in cancer cells,which was almost inactive in normal cells,these discoveries provide theoretical basis for applicating hTERT promoter in tumor-target research.PurposeIn this study,multiple peptides mimic designed by computer software and the physicochemical properties were assessed by bioinformatics tools,the optimized peptide named KK-64.The biological activity of KK-64 in liver cancer cells were determined.Then,kk-64 DNA sequence was inserted in pcDNA3.1 vector which contained hTERT promoter and further explored the activity of recombinant plasmid in liver cancer cells.MethodsMimic the properties of anti-tumor peptides and designed KK-64 peptide by computer,then predict the properties such as molecular weight,hydrophilicity or hydrophobicity,secondary structure and stability.Synthesis the KK-64 peptide by chemical method,after that,detected the effect of KK-64 to human liver cell line HepG2 and human normal liver cell line HL-7702cell viability.Secretion of lactic dehydrogenase?LDH?was determined to assessed the membrane-breaking ability of KK-64 peptide.Reverse-translated the KK-64 peptide to DNA sequence by human preference codon,then inserted kk-64 sequence to pcTERT vector which containing hTERT promoter?The vector was constructed successfully former in this laboratory?,next,identified the recombinant plasmid pcTERT-kk-64 by restriction enzyme digestion and gene sequencing.Transfected the recombinant plasmid pcTERT-kk-64 to HepG2 and HL-7702cells by lipidosome,the ability of proliferation was detected by Cell viability detection kit.The cell apoptosis rate was determined by Annexin V/PI staining and tested by flow cytometry.Expression and activation of proteins related to endogenous mitochondrial apoptosis pathway and exogenous death receptor pathway were analyzed by western blot.HepG2 cell migration was detected used transwell cell and cell invasion was determined used transwell cell which paved matrigel after transfected with pcTERT-kk-64.Furthermore,the expression of genes MMP-2 and MMP-9 which related to cell migration and invasion were analyzed by Real-time PCR.We observed the morphologic change of HepG2 and HL-7702 cell membrane by confocal laser scanning microscopy after transfected with recombinant plasmids and stained Dioctadecyloxacarbocyanine perchlorate membrane dye.ResultsIn the bioinformatics results,the property of KK-64 peptide was stable and presented bipolarity,and characterized as a cationic peptide with?-helical structure.In biological test results,KK-64 peptide can obviously inhibit the proliferation of HepG2 and HL-7702 cells,the IC₅₀ value were 3.256?M and 3.603?M respectively.KK-64 peptide can break the cell membrane in a short time.After enzyme digestion and sequencing we identified that kk-64 sequence was inserted to pcTERT vector successful.In the results,the viability of HepG2 cells were remarkably inhibited after transfected with recombinant plasmid pcTERT-kk-64,while no change was detected in HL-7702 cells after transfection.pcTERT-kk-64 also induced obviously cell apoptosis in HepG2 cells and activated mitochondrial apoptosis pathway and death receptor pathway.However,pcTERT-kk-64 cannot triggered HL-7702 cell apoptosis.Furthermore,the abilities of migration and invasion of HepG2 cells were suppressed after transfected with pcTERT-kk-64.The membrane of HepG2 was broken after transfected with pcTERT-kk-64 plasmid while no change was observed in cell membrane of HL-7702 cells.Conclusion and significanceTo sum up,KK-64 peptides displayed a well biological activity in liver cancer cells.Recombinant plasmid pcTERT-kk-64 can inhibit the proliferation,induce cell apoptosis and suppress cell migration and invasion,even break the cell membrane of liver cancer cells.Furthermore,pcTERT-kk-64 presented tumor-target property.The completion of this study provides a novel idea for targeting study of anti-tumor peptides and affords a new insights and research basis for the development of new therapeutic drugs for liver cancer.