| Hepatocellular carcinoma(HCC)is one of the malignant tumors worldwide There is a relatively high incidence and mortality rates in China,the number of new cases and deaths cases of HCC in 2015 was 466,100 and 422,100,respectively.Clinically,most HCC patients are already in late-stage of HCC when they are diagnosis due to the lack of specific biomarkers and poor clinical symptoms,and more than 80%of the HCC patients are poor prognosis.In addition,owing to the recurrence and metastasis of HCC,the current clinical treatment methods have certain limitations in HCC treatment,and the efficacy is not to be satisfactory.Therefore,it is urgent to study the molecular mechanisms involved in the pathogenesis and development of HCC in order to discover new therapeutic targets,and to develop new and effective treatmentsExosomes are bilayer membrane particles which secreted by a various type of cells with a particle size between 30 to 150 nm.It contains a variety of protein and nucleic acid substances(including DNA and RNA)that are supplied by the donor cells.Exosomes involve in a variety of biological processes such as cell proliferation,migration and apoptosis acting as a mediator of intercellular communication.The contents of exosomes are closely related to the pathological and physiological state of human body.Exosomes play an important role in the disease,especially in the origin and development of cancer.Therefore,it is helpful to discover new therapeutic targets and develop biomarkers of diseases by analyzing the contents in exosomes and revealing the molecular mechanism of HCC.In recent years,the long non-coding RNA(IncRNA)in circulating exosomes was received increasing attention.IncRNA is an RNA that dose not have protein-coding function.It can participate in the regulation of cellular migration,proliferation and apoptosis.IncRNA plays a key role in human diseases,especially in the process of cancer occurrence and development.In the past ten years,IncRNA was widely concerned and gradually developed into a highly active research area because of the research constantly reveal the role of IncRNA in the occurrence,development and matastasis of HCC.However,the function and features of most lncRNAs are still not identified and annotatedIn addition,due to the specific affinity between the exosomes and their donor cells,and their natural biocompatibility,exosomes become an ideal carrier for therapeutic drugs delivery.At present,exosomes are successfully used as carrier of cancer chemotherapy drugs for cancer targeted therapy Copper-cysteamine(Cu-Cy NPs)is a new type of photosensitizer that can produce red light under the excitation of ultraviolet light,X-ray and microwave,and which is one of the main factors of photodynamic therapy(PDT).PDT is an ideal alternative method to traditional surgery and chemotherapy owing to its low toxicity and invasiveness.It achieves therapeutic goal by reacting light-activated photosensitizer with oxygen molecules to produce reactive oxygen species which in turn triggers apoptosis of target cells.The PDT application of Cu-Cy NPs is still in the early stages of research,and its biological toxicity,photodynamic reactivity and biological delivery way have not been fully evaluatedThis study consists of two parts:In the first part,we mainly described the expression characteristics of IncRNA in HCC circulating exosomes,explored its molecular mechanism involved in the occurrence and development of HCC,then evaluates the potential value of IncRNA as a serum biomarker of HCC.In the second part,we explored the possibility of application of Cu-Cy NPs in photodynamic therapy of HCC in vivo and in vitro,and explored the feasibility of exosomes as transport carriers for Cu-Cy NPsPart ?:Discovery of lnc85 Marker and its Related Mechanisms in HCC by Using HCC Plasma ExosomesPurpose:To deascribe the expression characteristics of IncRNA in circulating exosomes and explore the molecular mechanism of IncRNA involved in the development of HCC,and evaluate its potential value as a biomarker of HCCMethods:1.We used the exosomal extraction kit to extracts exosomes from the plasma of HCC patients and healthy controls,and detected differentially expressed lncRNA and mRNA in HCC and healthy controls by RNA-sequencing;2.The relative expression feature of IncRNA and mRNA in plasma exosomes of HCC patients was comprehensive descriped by compared the RNA-sequencing result with an open database which containing 34 cells gene transcripts;3.Exosomes of HepG2 and Huh7 cells were extracted by ultracentrifugation and verified by Nanosight,TEM and Western blot;4.The IncRNA that significantly increased in plasma exosomes was selected for qRT-PCR verification in cells and cellular exosomes;5.The cells(HL-7702,HepG2 and Huh7)were transfected with siRNA(designed for the candidate IncRNAs respectively).The effects of candidate 1ncRNA on cell proliferation,migration,and apoptosis were detected by using CCK-8 kit,traswell assay and flow cytometry.RP11-85G21.1(1nc85)which had a significant influence on proliferation and migration of HCC cells was selected to further study;6.The Ensembl,LNCipedia version and IncLocator database were used to query the basic information such as genomic location,specific sequence information,sequence length,subcellular localization of Inc85.The RegRNA database was used to predict the targeting microRNA of Inc85;7.qRT-PCR was used to explore the expressed correlation between Inc85 and mir-324-5p in HL-7702,HepG2 and Huh7 cells.The expression of mir-324-5p downstream gene in Huh7 cells after silenced of Inc85 was detected by Western blot;8.qRT-PCR was used to detect the expression of Inc85 in serum of 112 HCC patients and 52 corresponding healthy controls,and ROC was used to evaluate the potential value of Inc85 as a biomarker for HCC serum.Results:1.Exosomal RNA-sequencing in plasma of HCC patients and healthy controls:There were 8,572 differentially expressed IncRNAs in plasma exosomes of HCC patients,of which 8,447 were up-regulated and 125 were down-regulated;There were 9,440 differentially expressed mRNAs of which 8,963 were up-regulated and 477 were down-regulated;2.Relative expression characteristic of IncRNA and mRNA in plasma exosomes of HCC:In HCC plasma exosomes,IncRNA had higher expressed level(lncRNA vs.mRNA=4.13 vs.2.21,P<0.01),higher expression specificity(lncRNA vs.mRNA=25.48%vs.18.99%),lower splicing efficiency(lncRNA vs.mRNA=0.38 vs.0.53,P<0.01),and lower individual variability(lncRNA vs.mRNA=0.4 vs.0.68,P<0.01)than mRNA;3.Verification of cellular exosomes:Nanosight results showed that more than 80%of the particles had a particle size between 30 nm and 150 nm,which was consistent with the size range of exosomes;TEM result showed a typical bilayer membrane vesicle-like structure of the exosomes;three known exosome surface biomarkers were detected by western blot(Alix,HSP70 and CD9);4.qRT-PCR verified candidate differential expression IncRNA in HCC cells and cellular exosomes:6 candidate differentially expressed lncRNAs were selected from RNA-sequencing results according to Fold Change>6 and P value<0.05(lnc544,Inc 380;lnc239,lnc959,lnc171 and lnc85);qRT-PCR results showed that among the 6 lncRNAs,excepted for Inc380,the other 5 lncRNA were significantly increased in HCC cells and their cellular exosomes(P<0.01);consistent with the RNA-sequencing results;5.Investigation of the effects of 5 candidate IncRNA on cell proliferation,apoptosis,and migration:5.1 The effect on cell proliferation activity:After silenced of lnc85,the proliferation activity of three cells(HL-7702,HepG2 and Huh7)were significantly decreased,especially in HepG2 and Huh7 cells(HepG2:P<0.01;Huh7:P<0.01);silenced of lnc171 slightly reduced the proliferation activity of HepG2 and Huh7 cells(HepG2:P=0.023;Huh7:P=O.030);While silenced of lnc544,lnc239 and lnc959 only slightly affected the proliferation of one type of cell(lnc544 Huh7:P=0.035;lnc239 HL-7702:P=0.017;lnc959 Huh7:P=0.020;5.2 The effect on cell apoptosis:Silenced of lnc85 and lnc544 promoted the apoptosis of Huh7 cells(lnc544,p<0.01;lnc85,p=0.014),whereas inhibited of lnc171 increased apoptosis of HepG2 cells(P<0.01),and silenced of lnc239 and 1nc959 had no obvious effect on cell apoptosis;5.3 The effect on cell invasion:Silenced of lnc85 obviously inhibited the invasion ability of three cells,including HL-7702(HL-7702,P=0.013;HepG2,P=0.015;Huh7,P<0.01),while the remaining four IncRNA had no significant effect on cell invasion ability;6.The basic information of lnc85 and its target microRNA:lnc85 was located in 157232231-157237136 of chromosome 1th,and there was no sequence encoding any proteins within 10 kb before and after the gene.The length of lnc85 transcript was 399 bp,and its mature body had two exons.Inc85 was mainly located in the cytoplasm(score:0.60).lnc85's target miRNA was mir-324-5p;7.Expression correlation between lnc85 and mir-324-5p7.1 The results of qRT-PCR showed that the expression of lnc85 in two HCC cells was significantly higher than that in HL-7702 before lnc85 silenced,while the expression of mir-324-5p was significantly lower in HCC cells(P<0.01);After silenced of lnc85,the expression of lnc85 was significantly decreased,in contrast,the expression of mir-324-5p was significantly increased(P<0.01);7.2 qRT-PCR was used to detect the mRNA expression level of downstream gene of mir-324-5p after silenced of lnc85,the result showed that silenced of lnc85 significantly inhibited the mRNA expression level of cyclinin D1,cyclinin B1 and c-myc in HCC cells(P<0.01).In additionl1nc85's silenced led to a reduction mRNA levels of apoptosis protein(BCL-2)and migration protein(MMP 2 and MMP 9)(P<0.01)7.3 Western blot further confirmed the cellular cycle proteins after silenced of lnc85,the results showed that silenced of lnc85 resulted in down-regulation of cyclin D1 and cyclin B1 proteins levels;7.4 The mode of lnc85 regulation of the cellular activity in HCC:In HCC,the expression level of lnc85 was increased,and the combination between lnc85 and mir-324-5p relieved the inhibitory effect of mir-324-5p on its downstream genes.As the result,the expression of the gene that downstream of mir-324-5p was increased,which eventually enhanced the activity of cell proliferation and invasion,decreased the activity of cell apoptosis;8.Potential of lnc85 as a biomarker of HCC:The expression of lnc85 in serum of HCC patients was significantly higher than that of healthy control(P<0.01),and lnc85 also high expressed in AFP negative patients than healthy control.The results of ROC analysis showed that,when distinguishing between healthy controls and HCC patients,the area under curve(AUC)of lnc85 was 0.861,when the cut off value was 1.514,the sensitivity and specificity of lnc85 were 80.9%and 74.5%,respectively.While distinguishing between healthy controls and AFP negative patients,the AUC was 0.872,the sensitivity and specificity of lnc85 were 80.5%and 76.4%when the cut off value was 1.692,respectively.Conclusions:1.Compare with mRNA,lncRNA has higher expression level,higher expression specificity,lower splicing efficiency and lower individual variation;2.Differential expressed IncRNA of plasma exosomal in HCC patients could regulate cell proliferation,apoptosis or migration activities;3.lnc85 regulates the proliferation of HCC cells by inhibiting the expression of mir-324-5p to regulate its downstream genes;4.lnc85 has the potential to act as an HCC serum biomarker.Part ?:Anti-HCC and exosome transfer effect of New PhotosensitizerCopper-cysteamine NanoparticlesObjective:To investigate the effect of photodynamic therapy mediated by Copper-cysteamine Nanoparticles(Cu-Cy NPs)on HCC in vivo and in vitro,and the feasibility of using exosome as Cu-Cy NPs' transporter.Methods:1.Anti-HCC effect of Cu-Cy NPs1.1 Cu-Cy NPs were synthesized by chemical synthesis(solution-gel method),and the physical characteristics of Cu-Cy NPs were characterized by UV light,transmission electron microscope(TEM)and fluorescence spectrometer;1.2 RNO decolorization assay was used to detect the singlet oxygen(102)production rate;MTT assay was used to detect the cell toxicity of Cu-Cy NPs and ultraviolet light;1.3 The cellular uptake rate of Cu-Cy NPs was observed by optical microscope;and the PDT effect of Cu-Cy NPs on cells were detected by calcein-AM and EthD-1 fluorescence double staining;1.4 MTT assay and cell cloning assay were used to detect the effect of Cu-Cy NPs-PDT on the proliferation in HCC cells;1.5 Hoechst 33342 fluorescence staining and flow cytometry were used to detect the killing pathway of Cu-Cy NPs-PDT on HCC cells;1.6 The tumor inhibitory effect of Cu-Cy NPs-PDT was observed by using nude tumor-forming model;2.Effect of transfer Cu-Cy NPs by exosomes2.1 Soluble Cu-Cy NPs were co-cultured with HCC cells,the supernatants were collected,then the exosomes were separated by using ultracentrifugation;Then used Nanosight,TEM and western blot to verify the exosomes;2.2 RNO decolorization assay was used to confirm whether Cu-Cy NPs can be encased in cellular exosomesResults:1.The anti-HCC effect of Cu-Cy NPs1.1 Characterization of Cu-Cy NPs:a.TEM observed that the Cu-Cy NPs was a highly crystalline crystal with a particle size of about 100 nm;b.Cu-Cy NPs were produced red fluorescence under the excitation of ultraviolet light,while the natural light source were colorless;c.Fluorescence spectrometer Cu-Cy NPs had the largest excitation wavelength at the wavelength of 360 nm,while the strongest emission wavelength is 600 nm;1.2 Experimental conditions of Cu-Cy NPs:a.RNO decolorization assay Cu-Cy NPs could produce 1O2 under UV light excitation,the longer ultraviolet light excitation would produc more 1O2 production;b.MTT assay:when the concentration of Cu-Cy NPs was less or equal to 25 ?g/ml,there was no significant effect on cell activity in the absence of UV light;c.MTT assay:when irradiated with ultraviolet light,the cell growth activity decreased gradually with the extension of UV time,and the cell growth activity decreased significantly when the UV time reached 6 min(P<0.01).Therefore,25 ?g/ml and 5 min UV excitation were selected to the follow-up test.1.3 Cell uptake rate and the PDT effect of Cu-Cy NPs at different uptake rate:Cu-Cy NPs were added to the culture media to co-culture with cells,and the proportion of cells uptake of Cu-Cy NPs were increased when prolong the culture time.Under the fluorescence staining,the Cu-Cy NPs-PDT effect on cells in different degrees of cell uptake were observed,however there was no significant difference in cell killing rate whether the cells uptake Cu-Cy NPs or not;1.4 Effects of Cu-Cy NPs-PDT on HCC cell proliferation:a.MTT assay:Cu-Cy NPs(25 ?g/ml)had no significant effect on cell viability when absence of UV light,while it was stimulated by UV light,Cu-Cy NPs can lead to a significant reduction in cell activity(P<0.01);b.Cell cloning assay:Cu-Cy NPs only,there was no significant difference between Cu-Cy NPs group and control group in the number of cell clones,when exposed to UV light,the number of cell clones was significantly lower in Cu-Cy NPs group than that in control group(P<0.01);1.5 Exploration of cell killing pathway of Cu-Cy NPs-PDT in HCC:a.Hoechst 33342 staining:The nuclear of HepG2 cells became smaller and marginalized in Cu-Cy NPs+UV group.The nuclear membrane was cleavage and divided into smaller pieces-apoptotic bodies.While cellular apoptosis was not or rarely seen in control group;b.Flow cytometry:Compared to the control and the 5-Fluorouracil(5-FU)group,Cu-Cy NPs+UV group could significantly induce cell apoptosis(P<0.01);c.Western blot:The expression of cleaved-APRP and cleaved-caspase3 protein were increased in Cu-Cy NPs+UV group,in addition,the expression of these apoptosis proteins were significantly higher in 36 h treatment than that in 24 h treatment;1.6 Inhibition of tumor growth im vivo by Cu-Cy NPs-PDT:HepG2 cells were injected subcutaneously into the flank and shoulder of nude mice.When the diameter of the subcutaneous mass was reach 5 mm,HE staining was applied.After the mass was stain,we could see that the typical cancer cell morphology(nuclear was deep stained and marginalized,and there were different nuclear morphology);Randomized the nude mice into five groups,then gave them different treatments and observed,the growth rates of tumors in the 5-FU and Cu-Cy NPs+UV groups were obviously slower than the control(P<0.01),while the UV,Cu-Cy NPs and the control group were no statistical difference;2.Study on the effect of transferring Cu-Cy NPs by exosomes2.1 Verification of cellular exosomes:Nanosight imaging showed that more than 80?/?of the particle size was distributed between 30?150 nm and the maximum particle size was 148 nm,which was consistent with the particle size range of the exosome;We could clearly observe the typical double-layer membrane vesicle structure of the exosomes in TEM image;Western blot also showed the marker proteins of exsomes(Alix,HSP70 and CD9);2.2 Exploration of Cu-Cy NPs transport carrier:Compared to the control(RNO),the exosomes which did not contain Cu-Cy NPs could not produce singlet oxygen no matter whether the ultraviolet light was applied;While the exosomes which contained Cu-Cy NPs could produce 1O2 under ultraviolet light irradiation(P<0.01)Conclusions:1.Cu-Cy NPs can be activated by ultraviolet light to produced 1O2;2.The combination of Cu-Cy NPs and UV light significantly decreased the HepG2 cell activity,and the 1O2 produced by Cu-Cy NPs killed cancer cells and inhibited the growth of tumors in nude mice by inducing cell apoptosis;3.Cu-Cy NPs were encased in the exosomes,and the exosome might be used as a carrier for Cu-Cy NPs to target transport in cancer cells. |
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