| The development of chemoresistance is a major hurdle for the treatment of colorectal cancer(CRC),which contributes remarkably to the poor clinical prognosis.Nanodrug delivery systems show great potential in overcoming chemoresistance,but limited by the lack of identification of chemoresistance targets from cancer patients.In the present study,we enrolled chemotherapy-resistant or sensitive CRC patients and used the next-generation RNA sequencing to reveal that Asporin(ASPN)is highly expressed in tumor tissues from oxaliplatin(OXA)-resistant patients and closely correlated with a poor prognosis of CRC.Downregulation of ASPN reversed OXA resistance and promoted cell apoptosis both in vitro and in vivo.To overcome ASPN-mediated OXA resistance,we constructed a nanoparticle-based co-delivery system(denoted as PPO-siASPN)for simultaneous de-livery of OXA and siRNA targeting ASPN(siASPN).PPO-siASPN not only facilitated the intracellular delivery of OXA through the enhanced cellular uptake,but effectively suppressed ASPN expression for synergistic antitumor activity in vitro and in vivo.In the more clinically relevant patient-derived xenograft(PDX)mouse model,systemic administration of PPO-siASPN achieved a remarkable therapeutic effect.This study uncovered the critical role of ASPN in causing OXA resistance in CRC patients and suggests a promising nanoformulation that may be more effective than current standard-of-care medications.Research background and objectivesThe occurrence of chemotherapy resistance is the main obstacle to the treatment of colorectal cancer,and is an important reason for the poor efficacy and clinical prognosis of colorectal cancer.Tumor drug.resistance involves a variety of molecular biological mechanisms,and it is currently considered to be a complex process involving multiple factors,multiple levels,and multiple genes.The exact mechanism of chemotherapy resistance in colorectal cancer patients is complex and is not fully understood.Nanodrug delivery systems show great potential in overcoming chemoresistance,but are limited by the lack of identification of chemo-resistant targets from cancer patients.Therefore,it is of great theoretical and practical significance to reveal the potential mechanism of CRC chemotherapy resistance,clarify the molecular mechanism of chemotherapy resistance,and find new treatment strategies to overcome CRC chemotherapy resistance.Methods and materials1.Firstly,the potential genes related to chemotherapy were explored by RNA sequencing and bioinformatics analysis,and the expression of ASPN in CRC was analyzed by immunohistochemistry(IHC)analysis of 96 pairs of CRC patients’ tissue chips,combined with the bioinformatics of TCGA database and GEO dataset,and its relationship with clinical prognosis was predicted.2.Furthermore,in vitro and in vivo experiments were conducted to explore the relationship between ASPN in CRC and OXA chemotherapy resistance,and we verified that ASPN knockdown combined with OXA chemotherapy inhibited tumor occurrence and progression.A nude mouse subcutaneous tumor model was constructed to verify the role of downregulating ASPN in vivo to reverse chemotherapy resistance.The potential mechanism of ASPN in CRC tumorigenesis and chemotherapy resistance was explored by bioinformatics analysis,and WB was used to detect pathway protein levels in vitro.3.The nanocarrier PPO-siASPN was constructed,and the effective binding mass ratio was measured by screening and characterization.The intracellular distribution of nanoparticles was assesseed by fluorescence confocal and the biodistribution of nanoparticles in vivo was assessed by animal imaging.WB and qPCR were conducted to detect the knockout efficiency of PPO-siRNA nanoparticles on ASPN-expression in CRC cells.The CCK-8 method was used to measure the cytotoxicity of nanocarriers.4.A nude mouse.CRC subcutaneous tumor model and a patient-derived xenograft(PDX)mouse model were constructed to explore whether systematic administration of PPO-siASPN could achieve significant therapeutic effects in vivo.Representative organ tissues such as tumor tissue,heart,liver,-spleen,lung,kidney,brain were taken for histological analysis,and mouse blood was collected for in vivo toxicity analysis.Result1.Through RNA sequencing and bioinformatics analysis,we discovered a novel key regulator of Asporin(ASPN)associated with tumorigenesis and chemotherapy resistance.Combined with clinical data analysis,we found that the expression of ASPN in tumor tissues was significantly higher than that in paracancerous tissues(P<0.001),and the expression of ASPN in patients with advanced CRC was higher than that in early stage,and the expression of ASPN in patients with OXA-resistant tumors was higher than that in patients with chemotherapy-sensitive(P<0.01).2.We further explored the relationship between ASPN and OXA chemotherapy resistance in vitro and in vivo,and found that downregulation of ASPN may reverse OXA resistance by significantly reducing the IC50 of OXA in CRC common cell lines and OXA-resistant cell lines.The silencing of ASPN combined with OXA therapy can significantly inhibit cell proliferation,migration invasion,and promote increased apoptosis.The results of in vivo experiments were consistent with in vitro cell validation:downregulation of ASPN combined with OXA chemotherapy significantly inhibited tumor genesis and progression.3.We further explore the potential mechanism of ASPN in CRC tumorigenesis and chemotherapy resistance,and find that ASPN expression level is significantly positively correlated with key regulatory molecules of Wnt/β-catenin pathway through bioinformatics.The key regulators of the Wnt/β-catenin pathway were significantly downregulated in ASPN-knocked tumor cells by WB.In addition,the analysis of the TCGA dataset showed that ASPN expression was significantly positively correlated with key regulators of the ABC transporter.4.On this basis,a nano-co-delivery system PPO-siASPN was designed and developed to realize the simultaneous delivery of small interfering RNA(siRNA)and OXA that can target ASPN for in-vivo administration to overcome the chemotherapy resistance of OXA in colorectal cancer.Systematic administration of PPO-siASPN also had significant therapeutic effects in mouse models of patient-derived xenograft(PDX)with stronger clinical relevance.In addition,for the body weight curve,blood index and organ index,and histology of mice,PPO-siASPN nanoparticles did not find obvious systemic toxic effects.Conclusion1.ASPN is overexpressed in CRC tumor tissues and suggests poor prognosis2.Down-regulation of ASPN combined with OXA chemotherapy significantly inhibited the occurrence and development of CRC tumors in vitro and reversed OXA chemotherapy resistance.3.ASPN may promote chemotherapy resistance by activating multiple targets:Wnt/β-catenin pathway and key regulators of the ABC transporter.4.The nano-co-delivery system PPO-siASPN can simultaneously deliver siRNA and OXA that can target ASPN,and overcome the chemotherapy resistance of OXA in colorectal cancer in vivo without obvious systemic toxicity. |
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