| Malignant tumor(also known as "cancer")has become the world's second leading cause of death.With the increasing number of deaths of cancer patients,the study of tumor markers has attracted much attention in the early diagnosis of cancer.It is significant to detect tumor markers for early diagnosis of cancer.For example,the overexpression of hTERT may indicate the occurrence of tumor,and the increase or decrease of microRNA content may play a role in inhibition or promotion for tumor.In recent years,scientists have been increasing the research on nanomaterials and nanotechnology in cancer treatment.Due to their inherent optical and physicochemical properties,nanomaterials can effectively improve the sensitivity,specificity,and targeting ability.DNA self-assembled nanomaterials composed of nano gold,graphene oxide and nucleic acid aptamers have potential applications in biosensing,imaging and medical diagnostics.In order to realize real-time monitoring and regulation of endogenous tumor markers in vivo,we combined nanomaterials with DNA to design and synthesize three different nanoprobes for achieving in situ detection and regulation of hTERT mRNA and microRNA-21.Firstly,we designed a multifunctional AuNP-probe that can not only achieve in situ detection and fluorescence imaging of intracellular hTERT mRNA but also down-regulate hTERT mRNA levels and inhibit telomerase expression.As hTERT mRNA/telomerase activation is critical for tumor cell development and proliferation,this nanoprobe could also induce cancer cell apoptosis.AuNP-probe consists of an oligonucleotide hybridization chain and AuNPs via Au-S bond formation.DNA duplexes are formed by the short cyanine(Cy5)dye-tagged reporter sequence(Flare-DNA)and the long thiol-terminated recognition sequence(HS-DNA).Once the AuNP-probes enter a cancer cell and bind with hTERT mRNA,Flare-DNA is released,and the fluorescence of Cy5,which was initially quenched by AuNP,is recovered.In addition,the hTERT mRNA level is down-regulated by the antisense oligonucleotide,resulting in the reduction of expression of hTERT and induction of apoptosis of telomerase-positive cancer cells.Secondly,a sticky flare probe was designed to detect,localize and silence the intracellular microRNA-21,achieving controlled release of the loaded drug doxorubicin,increasing sensitivity to cancer cells,and achieving anticancer effects of combination of gene regulation and chemotherapy in cancer cells.The AuNP-probe consists of a 13-nm AuNP core functionalized with a densely packed shell of thiol-modified oligonucleotides(HS-DNA).A fluorophore-conjugated reporter strand designed to be antisense to miRNA-21,termed the sticky-flare sequence(SF-DNA),is partly hybridized to HS-DNA.Amount of doxorubicin(Dox)molecules have been also designed to insert into the GC base pairs of the HS-DNA/SF-DNA duplexes and the fluorescence of Dox can be quenched accordingly.In the presence of miRNA-21,along with the dissociating of SF-DNA,probe-loaded Dox will be released and realize the precise drug delivery in tumor cells,achieving the intracellular miRNA-21 in situ imaging and inducing cancer cell apoptosis effectively.Finally,a specific probe of graphene oxide loaded with a large amount of peptide nucleic acid and folic acid was synthesized.The hTERT mRNA was imaged and regulated in situ,down-regulated hTERT mRNA level,inhibited telomerase expression and induced apoptosis.The probe is loaded with folic acid on the surface of graphene oxide,and then assembled with FAM-modified peptide nucleic acid(PNA)by ?-? stacking,and the fluorescent signal of FAM is quenched by graphene oxide.Then,By targeting the intracellular hTERT mRNA by the folate receptor on the surface of the cancer cell,once contacted with the target,the FAM-modified PNA will be detached from the graphene oxide and hybridized with the target hTERT mRNA to form a hybrid strand,which silences the hTERT mRNA and affects the hTERT.The expression eventually leads to apoptosis. |
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