The Composite Fluorescent Nanoprobes For Visualizing MiRNA Regulation Of Apoptosis In Cancer Cells

Cancer is a serious disease threatened human health,and its incidence is increasing year by year.The diagnosis and treatment of cancer is a challenge that researchers have been working to overcome.Mi RNA plays an important role in cancer development,and the abnormal expression of miRNA is closely related to the development of various cancers.Mi RNAs regulated development of cancer as oncogenes or tumor suppressoe genes.Apoptosis is autonomous cellular programmed death.Regulation of miRNA expression can induce apoptosis,which can achieve good therapeutic effects.Real-time monitoring of apoptosis is critical in drug efficacy and prognosis evaluation.Mi RNA-targeted therapy is gaining popularity among researchers and has become a current research hotspot in the field of precision therapy.Therefore,developing a real-time,in situ detection method for tracking intracellular miRNA and apoptosis,and visualizing the effect of changes in miRNA expression on apoptosis is important.This is particularly important when evaluating the feasibility of miRNA-targeted therapy and realizing precise and personalized treatment of cancer patients.Current methods for detecting intracellular miRNAs and aopotosis proteins are mainly based on molecular biology techniques.These methods detect the targets from cell homogenate and dead cells—they cannot accurately reflect the real-time changes in miRNA expression and apoptosis in living cells.Therefore,developing effective methods that can detect miRNA and apopyosis in living cells can help to understand and observe the effect of miRNA expression changes on apoptosis.It has practical value for the miRNA-targeted cancer precision therapy and drug evaluation.For the past few years,as the nanotechnology develops,fluorescent nanoprobes have been widespread applied to the domain of analytical chemistry,biology and medicine.Among them,gold nanoparticles,polydopamine and graphere oxide have received much attention in biomolecule detection,cell imaging and cancer detetction with their unique optical properties and excellent bioaffinity.However,as far as we know,existing fluorescence imaging methods mostly realize single detection of miRNA or apoptosis in living cells—they cannot visually monitor the effects of miRNA expression changes during apoptosis.In order to better understand the machanisms of miRNAs in cancer development and the regulation of apoptosis by miRNAs,we need to develop a fluorescent nanoscale imaging technology to simultaneously detect miRNAs and apoptosis proteins in living cells,and monitor the effect of miRNA expression changes on apoptosis in living cells in situ in real time.The technonly can achieve miRNA-targeted precision therapy effectively and evaluated the efficacy of anticancer frugs accurately.Based on miRNAs related to cancer development and apoptotic proteins related to apoptosis,we synthesized two novel composite fluorescent nanoprobes for detecting miRNAs and apoptotic proteins in living cells simultaneously using gold nanoparticles(Au NPs),polydopamine(PDA),graphene oxide(GO),oligonucleotide chains and peptide chains.These strategies successfully achieved real-time monitoring of the regulation of miRNAs on apoptosis.Firstly,we designed a composite nanoprobe(Au@PDA)with gold nanoparticles as the core and polydopamine as the shell.Based on electrostatic interaction and π-πadsorption,single strand DNA(ss DNA)and oligopeptides labeled with different fluorescent dyes were modified stepwise on the surface of the PDA.The ss DNA could specifically respond to miRNA-21,and the oligopeptide could be specifically lysed by the apoptotic marker caspase-3.Since Au NPs and PDA have high fluorescence quenching effect,dyes on DNA and peptide chains can be effectively quenched.The ss DNA will become double-stranded with the target chain and the oligopeptide will be cleaved by caspase-3 when the nanomaterial encounters the corresponding targets miRNA-21 and caspase-3 after the probe entered into cells,leading to the release of fluorophores from the PDA surface to restore the fluorescence.The caomposite nanoprobe is able to simultaneously detect miRNA and apoptotic proteins in living cells,providing a simple,non-invasive and visual method to in suit and real-time monitor the regulation of miRNA on apoptosis,which provides a reference value for further research on miRNA-targeted therapy.We further known that molecular beacons(MBs)have higher specificity and selectivity in recognizing individual mismatched bases compared to DNA single strands.To improve the specificity of nanoprobe targeting miRNA,we selected molecular beacons as the detection unit.In addition,GO is a novel two-dimensional carbon-based material.It has a larger specific surface area and can be loaded with more detection units.Therefore,we alternatively designed a novel fluorescent composite nanoprobe with GO loaded with MB and peptide chains.GO can effectively quench the fluorescence on MB and peptide chains.When encountering miRNA-221 and apoptotic protein caspase-3,MB specifically forms a double-strand with miRNA-221 and caspase-3 specifically cleaves the peptide chain.Then the fluorescence is restored.This probe we designed not only realized the simultaneous detection of miRNA and apoptotic protein in living cells,but also improved the specificity of the detection,making the detection results more accurate and valid,and providing stronger support for miRNA targeting research and evaluation of drug efficacy.