The Detection And Study Of MiR-155 And Protein Oxidative Modification Regulated By Cadmium Toxicity Effects

Cadmium(Cd)is an important rare metal element,mainly existing in sulfur cadmium ore or accompanying zinc ore in nature.Cd can also be combined with sulfur group elements of the group VIA to synthesize sulfur-based cadmium(CdO,CdS,CdSe and Cd Te)semiconductor nanomaterials.These materials with unique properties are widely used in various photovoltaic appliances,photodevice,nanosesing,and biomedical imaging.Data shows that China has the richest cadmium reserves and the largest cadmium consumption,so the environmental pollution is becoming increasingly prominent.Cd pollutions in the environment mainly exist in the air,water and soil.Some enter the body directly through respiration and drinking water,while others are absorbed by crops or enter animals through the food chain,and ultimately accumulate inside the human body through diet or smoking.In addition,there is also a problem of Cd exceeding standards in some low-quality metal accessories,as well as some whitening and sunscreen products,posing a serious threat to the health of women.Cadmium has a significant bioaccumulation in the human body,with a half-life of up to 10 to 30 years,and belongs to Class I carcinogens.The clinical symptoms of Cd poisoning are closely related to the contact routes,exposure levels and contact rates of the body.Acute Cd poisoning mainly stimulates the respiratory mucosa and gastrointestinal tract,and even death may occur in severe cases.Chronic Cd poisoning has a wide range of effects,causing varying degrees of damage to various organs of the body.As an important metabolic organ,the liver has a higher cadmium retention rate compared to other organs,making it one of the most severely affected organs by cadmium.Although the breast is not the main metabolic organ,and the residual amount of cadmium is less,recent studies have shown that environmental cadmium exposure is related to the increased risk of human breast cancer.Although a lot of work has been carried out on the hepatotoxicity and breast cancer promoting effects of cadmium,the pathogenesis of corresponding organs under different exposure levels needs further research.After entering the body,Cd always causes the formation of reactive oxygen species(ROS),leading to oxidative stress that plays different roles in various mechanisms and is considered a general mechanism for Cd toxicity and disease development.The free radicals generated by oxidative stress can directly or indirectly oxidize or damage DNA,proteins and lipids,leading to genomic instability,protein oxidative damage and lipid peroxidation,thereby triggering physiological and pathological reactions in cells and tissues.However,currents studies mostly focus on the changes in inflammatory molecule levels,cell apoptosis and necrosis,or changes in metabolism levels after oxidative stress to evaluate the toxic effects of Cd,and the elaborate molecular mechanisms are unclear.Gene regulation and protein oxidative modification serve as the source of cellular response to oxidative stress,and their role in the mechanism of Cd toxicity remains to be further explored.Therefore,basing on the oxidative stress by Cd,studying the regulatory interactions between molecules such as ROS,genes,and proteins is of great significance for a deeper understanding of the potential toxic effects of Cd.In addition,the development of precise detection probes targeting abnormal molecular markers in the toxic mechanism of Cd can not only provide assistance for the study of Cd toxicity mechanisms,but also provide early detection and prognostic guidance for related disease.Based on this,we study the potential toxicity mechanism of Cd and its nanomaterials at the molecular level,analyze the interaction of ROS,gene and oxidative modification of some key proteins in promoting the development of breast cancer and hepatotoxicity of Cd,and investigate the macro expression of toxic effects of Cd at the animal level,so as to provide a new perspective for the oxidative toxicity mechanism of Cd,and build a visual detection probe to provide a new platform for the early detection of Cd toxicity and breast cancer.Specifically include:1.Environmental Cd exposure is related to the occurrence and development of breast cancer,but the related mechanism of still unclear.Based on the oxidative stress by Cd,we study the interaction between ROS,antioxidant defense system Keap1-Nrf2,and breast cancer related gene miR-155,etc.,so as to clarify the potential mechanism of Cd in promoting the evolution of breast cancer.MCF-10 A cells were exposed to lower concentrations of Cd2+(1 μg/m L),and to evaluate its toxic effect in promoting breast cancer.On the one hand,under oxidative stress,H2O2 shows a positive correlation with miR-155,increasing the proliferation ability of cells.On the other hand,mass spectrometry results showed that excessive H2O2 caused sulfination(-SO2H)and sulfonation(-SO3H)modification of active sites(Cys151 and Cys288)of Keap1 protein,and Nrf2 was released and phosphorylated,activating the antioxidant defense system and increasing the level of peroxiredoxin II(PrxⅡ).Elevated PrxⅡ protects the active site of vascular endothelial cell growth factor receptor 2(VEGFR2)from oxidative stress,and sustained activation leads to further abnormal proliferation of MCF-10 A cells.These results indicated that Cd can increase the risk of breast cancer by regulating the expression of oncogenes and oxidative modification of some key proteins through oxidative stress,providing new ideas for the mechanism of Cd promoting breast cancer.In addition,we also assessed the risk of promoting breast cancer of three kinds of Cd-QDs(Cd S,Cd Se and Cd Te QDs),to help screen safe and reliable Cd containing nanoforms.2.As one of the important metabolic organs in human body,the liver is particularly severely affected by Cd toxicity.The hepatotoxicity of Cd-QDs has become the focus with their extensive applications in biomedicine.Previous reports have demonstrated that high oxidative stress and consequent redox imbalance play critical roles in their toxicity mechanisms.Intracellular antioxidant proteins,such as thioredoxin 1(Trx1)and peroxiredoxin 1(Prx1),could regulate redox homeostasis through thiol-disulfide exchange.Herein,we hypothesized that the excessive reactive oxygen species(ROS)induced by Cd-QD exposure affects the functions of Trx1 or Prx1,which further causes abnormal apoptosis of liver cells and hepatotoxicity.Thereby,three types of Cd-QDs,Cd S,Cd Se,and Cd Te QDs,were selected for conducting an intensive study.Under the same conditions(10 μg/m L,24 h),the H2O2 level in the Cd Te QD group was much higher than that of Cd S or Cd Se QDs,and it also corresponded to the higher hepatotoxicity.Mass spectrometry(MS)results show that excessive H2O2 leads to sulfonation modification(-SO3H)at the active sites of Trx1(Cys32 and Cys35)and Prx1(Cys52 and Cys173).The irreversible oxidative modifications broke their cross-linking with the apoptosis signal-regulating kinase 1(ASK1),resulting in the release and activation of ASK1,and activation of the downstream JNK/p38 signaling finally promoted liver cell apoptosis.These results highlight the key effect of the high oxidative stress,which caused irreversible oxidative modifications of Trx1 and Prx1 in the mechanisms involved in Cd-QD-induced hepatotoxicity.This work provides a new perspective on the hepatotoxicity mechanisms of Cd-QDs and helps design safe and reliable Cdcontaining nanoplatforms.3.Micro RNA-155(miR-155),which facilitates breast tumor growth and invasion by promoting tumor cell proliferation and inhibiting cell apoptosis,is considered an ideal early diagnostic and prognostic marker.Herein,we developed a self-assembled hybridization chain reaction(HCR)-based photoacoustic(PA)nanoprobe for highly sensitive in situ monitoring of dynamic changes in miR-155 expression during breast tumorigenesis and chemotherapy.The PA nanoprobes(Au-H1/PEG and Au-H2/PEG)were constructed by linking poly(ethylene glycol)(PEG)and two hairpin DNA strands(H1 and H2,respectively)to the surface of gold nanoparticles(AuNPs).In the presence of miR-155,the PA nanoprobes self-assembled into Au aggregates via HCR between H1,H2,and miR-155.The decreased interparticle distance in these aggregates enhanced the surface plasmon resonance(SPR)in the AuNPs.Consequently,the absorption peak of the PA nanoprobes red-shifted,and strong PA signals were generated.This strategy enabled the sensitive and quantitative detection of miR-155 with a low detection limit of0.25 n M.As a result,PA signals of miR-155 were captured on the second day after tumor inoculation when the solid tumor had not yet formed.Dynamic changes in miR-155 during tumor growth and chemotherapy were also monitored in real time to assess the therapeutic effects via PA imaging.By virtue of these advantages,the PA nanoprobes may provide a powerful platform for in situ detection of miR-155 and thus real-time monitoring of tumorigenesis and drug response in breast cancer.4.The toxic effects of Cd were studied at the animal level.We continuously exposed nude mice to low doses of Cd2+(1 mg/kg)to explore the effects of chronic Cd exposure on breast and liver function.The study founded that the weight of mice was significantly lower than that of normal mice after 12 weeks of Cd exposure.The level of liver injury markers increased,and functional and physiological disorders began to appear.The levels of miR-155 and p-VEGFR2 in breast tissue showed an increasing trend and significantly promoted the growth of breast tumors.These experimental results suggested that long-term low-dose Cd exposure affects the growth and development of mice,and interferes with the normal function of the liver by generating oxidative stress,promoting the development of breast cancer.This study provides a macro data reference for the oxidative toxic effects of chronic Cd exposure in promoting liver injury and breast cancer.