Application Research Of Formiminotransferase Cyclodeaminase Conjugated Hollow Mesoporous Organosilica Nanospheres For The Treatment Of Hepatocellular Carcinoma

BackgroundLiver cancer is the Sixth-most common malignancy and the fourth leading cause of cancer-related mortality in the world.Liver cancer can be divided into three categories,including hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma and mixed hepatocarcinoma,according to the origin of the cancer.To date,the radical resection accompanied with radiotherapy and/or chemotherapy are still the main treatment for HCC.Recently,the molecular targeted therapy as well as immunotherapy have made a big through for the treatment of HCC,but the 5-year survival rate of HCC patients was still not satisfactory.Actually,the main reason for the lower survival rate is the accumulation of genetic mutations and epigenetic changes,thus it is imperative to identify the novel liver cancer marker and therapeutic target.Formiminotransferase cyclodeaminase(FTCD)is a bifunctional enzyme contained formic acid aminotransferase and cyclic deaminase domains,which plays an important role in histidine catabolism metabolism and folic acid metabolism respectively.Interestingly,previous studies discovered that FTCD is generally expressed in all mammalian cells,especially in hepatocytes.Moreover,the expression level of FTCD was closely related to the normal functional of the intracellular golgi complex.In addition,the expression level of FTCD can affect the sensitivity of tumor cells to chemotherapy.But the efficiency of traditional plasmid transfection techniques for the construction of FTCD-overexpressed HCC cell lines was too limited to achieve clinical transformation.Recently,due to the efficiently delivery capacity of Nano-carriers,the Nano drugs delivery systems were the suitable selection for drug modification and intelligent drug delivery.Therefore,to construct a FTCD plasmid carried nano delivery system will be the key method to achieve efficient and targeted treatment for HCC.Among them,hollow mesoporous organosilica nanospheres(HMON)is a specific pore structure,accompanied with hollow,small density and large specific surface area.Due to its unique permeability,sieving molecular ability,optical properties and adsorption,HMON nano carrier could be widely used in biomedical field.In this study,we successfully constructed the manganese(Mn)ions and FTCD plasmid loaded mesoporous hollow silica nanocarriers(HMON@Mn-PEI@FTCD),which could exert enhanced tumor suppression and nuclear magnetic imaging,thus HMON@Mn-PEI@FTCD would be a promising therapeutic method for the treatment of HCC.Part Ⅰ.The expression levels of FTCD and its clinical significance in hepatocellular carcinomaObjective:To evaluate the expression of FTCD in HCC tissue samples,and clarify its relationship with clinicopathological features and prognosis of patients.Method:1.The expression levels of FTCD gene in cancer tissues and adjacent tissues and the survival data of patients in the LIHC-TCGA database were obtained,expression levels were compared and total survival was analyzed.2.Sixty paired of HCC tissue samples and adjacent normal tissues were obtained for immunohistochemical techniques to detect the expression levels of FTCD.Result:1.FTCD expression showed a lower expression in HCC tissues,when compared with adjacent normal tissues,and low expression of FTCD indicated shorter overall survival of HCC patients(P<0.0001).2.Immunohistochemical analysis showed that FTCD protein was mainly located in the cytoplasm,and its expression was low in HCC tissues.3.Low expression of FTCD was closely association with the high AFP levels(P=0.009),larger tumor size(P=0.013),presence of vascular invasion(P=0.001),advanced BCLC stage(P=0.024),and advanced pTNM stage(P=0.000).Conclusion:In HCC tissues,the expression of FTCD was significantly lower than that in adjacent normal tissues.Down-regulation of FTCD was closely associated with poor prognosis in HCC patients.FTCD would be a promising prognostic marker for HCC.Part Ⅱ.The biological role of FTCD in hepatocellular carcinoma and its molecular mechanismObjective:To study the biological effects of FTCD on HCC cell lines and its underlining molecular mechanism.Method:1.The expression levels of FTCD in HCC cell lines(Huh7,BEL-7402,SNU449 and SK-Hep1 cells)and normal liver cell line(HL-7702 cells)were detected.2.FTCD over-expressed BEL-7402 and SNU449 cell lines were constructed by plasmid transfection technology.3.Cell counting kit-8 and BrdU assays were used to evaluate cell proliferation ability of BEL-7402 and SNU449 cell lines with FTCD overexpression.4.Cell apoptosis and DNA damage were detected by flow cytometry in BEL-7402 and SNU449 cell lines with FTCD overexpression.5.The expression levels of PI3K/AKT signaling pathway related proteins in BEL-7402 and SNU449 cell lines with FTCD overexpression were detected by Western blot.Result:1.The expression levels of FTCD was lower in HCC cells than that in normal cells,especially lower in BEL-7402 and SNU449 cell lines.2.CCK-8 and BrdU experiments showed that proliferation abilities were significantly inhibited in BEL-7402 and SNU449 cell lines with FTCD overexpression(P<0.001).3.Apoptosis rate and intracellular DNA damage markers(γ-H2AX)were significantly increased in BEL-7402 and SNU449 cell lines with FTCD overexpression(P<0.001).4.The expression of PI3K/AKT signaling pathway related proteins were inhibited in BEL-7402 and SNU449 cell lines with FTCD overexpression(P<0.001).Conclusion:Overexpression of FTCD significantly suppressed PI3K/AKT signaling pathway related proteins,which then inhibit cell proliferation and induce cell apoptosis in HCC cells.Our findings consistently suggest that FTCD would be a promising regulatory target for the treatment of HCC patients.Part Ⅲ.The synthetic and exploration of hollow mesoporous organosilica nanotheranostics(HMON)drug delivery systemsObjective:To construct the manganese ions(Mn)and FTCD plasmid loaded hollow mesoporous organosilica nanotheranostics drug delivery systems(HMON@Mn-PEI@FTCD),and explore its nuclear magnetic imaging characteristics and anti-tumor effects.Methods:Based on the cationic adsorption effect of PEI,manganese ions(Mn)and FTCD plasmid were loaded on hollow silica nanocarriers(HMON).The physicochemical characters of nanodrug delivery system were detected by Ma Erwen Particle size Analyzer,transmission electron microscopy.The safety of nano carriers for normal liver cells was evaluated by CCK-8 assays.The Immunofluorescence assay and RT-qPCR assay were used to verify its tumor targeting ability.Moreover,the cytotoxicity,flow analysis,western blotting,and tumor-forming nude mice were used to detect the in vivo and in vitro anti-tumor toxicity and nuclear magnetic imaging characteristics.Results:1.The HMON@Mn-PEI@FTCD nanoparticles have high biocompatibility.2.The HMON@Mn-PEI@FTCD nanoparticles could efficiently and selectively enter HCC cells.3.The HMON@Mn-PEI@FTCD nanoparticles can inhibit the proliferation ability,promote more apoptosis rate via reducing the expression level of tetrahydrofolic(THF)and inducing oxidative stress damage.4.The HMON@Mn-PEI@FTCD nanoparticles can achieve MRI imaging.Conclusion:The HMON@Mn-PEI@FTCD nanoparticles could selectively enter into HCC cells,and exert enhanced anti-tumor effect,which has wide application value in clinic.