Study On The Preparation Of BRD4 SiRNA Nanoparticles And Their Anti-gastric Cancer In Vitro

Objective: Gastric cancer(GC)is a widespread type of cancerous tumor.Despite the utilization of various combined treatment methods,such as surgery,chemotherapy,radiotherapy,and immunotherapy,to improve the survival rate of patients with GC,the majority of them still face a poor prognosis,high recurrence rate,and are prone to metastasis.However,with the continuous development of gene technology in recent years,the detection,monitoring,and treatment of GC has greatly benefited from the clinical significance of tumor markers.With the development of new biomarkers for the diagnosis of gastric cancer,RNA interference has become a promising gene targeting therapy,and small or short interference ribonucleic acid(Si RNA)has the advantages of high efficiency silencing of specific genes and small toxicity and side effects.However,siRNA is unstable and easily degraded,and the current delivery vector of siRNA is single.Therefore,the discovery of new biomarkers and the development of effective delivery vectors of siRNA are the focus of this research.The BRD4 protein is a member of the bromodomain and superterminal family of proteins(BET)and can play an important role in the occurrence and development of various tumours,as well as the invasion and metastasis of tumour cells.The purpose of this study was to determine the expression of BRD4 in patients with gastric cancer,construct BRD4 siRNA nanoparticles,optimize the preparation process with characterization.The delivery in gastric cancer cells and their anti-gastric cancer activity was also explored,providing reference for the discovery of new biomarkers and the development of new targeted preparations for gastric cancer.Methods: This study is mainly divided into three parts.The first part is a clinical study.Fresh tissues from 9 patients with gastric cancer were collected.The study aimed to investigate the expression level of BRD4 protein in gastric cancer tissues,para-cancerous tissues,and normal gastric tissues using Western blotting.The research collected data from 94 patients with gastric cancer and their tissue wax blocks.In addition to the detection of BRD4 protein expression in gastric cancer tissue using the immunohistochemical SP method,the relationship between BRD4 protein expression and clinicopathologic features and prognosis was also analyzed to reveal BRD4’s clinical significance in gastric cancer diagnosis and prognosis.Furthermore,Lipo 2000 was used to transfect BRD4 siRNA into human gastric cancer cells(HGC-27,SGC-7901),and its silencing effect was detected by Western blotting.The study also evaluated the effect of BRD4 on cell viability using MTT.The study evaluated the impact on cell migration and invasion ability by performing scratch assay and Transwell invasion assay,respectively.The second part is preparation.The redox responsive material Polyethylene glycol disulfide bond-linked polyethylenimin(PEG-SS-PEI)modified by Maleimide(Mal-)and cell transmembrane peptide low molecular weight protamine(LMWP)undergo amide reaction to obtain positively charged LMWP-PEG-SS-PEI,which is then mixed with negative BRD4 siRNA in a certain proportion to obtain nanoparticles by self-assembly.The study utilized dynamic light scattering(DLS)to investigate the particle size,Zeta potential,and polydispersity index(PDI)of L-NPs and NPs.Additionally,transmission electron microscopy(TEM)was used to observe the appearance and distribution of nanoparticles,while also determining the encapsulation rate of nanoparticles.The third part of the study involved in vitro testing of the preparation,where the MTT method was used to determine the optimal concentration required to inhibit the viability of gastric cancer cells.The in vitro release characteristics of the siRNA-loaded complex are investigated in the glutathione environment,and the cellular uptake,intracellular localization and lysosome escape in HGC-27 and SGC-7901 cells are studied.Transwell assay and scratch assay were used to investigate the effect of siRNA-loaded complex on cell invasion and migration.Western blotting was used to detect the expression of BRD4 and related pathway proteins in HGC-27 and SGC-7901 cells,so as to evaluate the effect of BRD4 siRNA nanoparticles on biological behavior of gastric cancer cells.Results: Statistical analysis of clinical samples showed that the expression of BRD4 in tumor and paracancer tissues was significantly higher than that in normal tissues(P<0.01).The expression level of BRD4 in gastric cancer tissues was strongly correlated with the degree of tumor differentiation(P=0.033),regional lymph node metastasis(P=0.038),clinical stage(P=0.002)and survival(P=0.000).There was no correlation with gender(P=0.564),age(P=0.926),depth of infiltration(P=0.619).Kaplan-Meier analysis showed that high BRD4 expression level was associated with poorer overall survival in gastric cancer patients(P=0.003).BRD4 siRNA transfection significantly decreased the expression of BRD4 protein in human gastric cancer cells HGC-27 and SGC-7901,and inhibited the viability,migration and invasion of HGC-27 and SGC-7901 cells.The BRD4 siRNA-loaded nanoparticles were successfully prepared by self-assembly.The optimal N/P value of the nanoparticles was 30,and the prepared BRD4 siRNA loaded nanoparticles were uniformly distributed,spherical in appearance,with a particle size of 95.21±6.23 n M and PDI of 0.229±0.016.The Zeta potential was 37.80 ± 3.45 m V,and the encapsulation rate was 91.10 ± 1.62%.The release of BRD4 siRNA-loaded nanoparticles in glutathione-containing PBS(p H7.4)buffer medium was accelerated from 12 to 24 h,and the release rate reached 92.33±3.50% at 24 h.Cellular uptake test results showed that the uptake rate of BRD4 siRNA-loaded nanoparticles was high,and they were mainly concentrated in lysosomes after uptake by cells.After 6 h of co-incubation,BRD4 siRNA escaped from lysosomes and diffused into cytoplasm.BRD4 siRNA-loaded nanoparticles significantly inhibited the viability,migration and invasion of HGC-27 and SGC-7901 cells.Meanwhile,the expression levels of PI3K/Akt and c-Myc signaling pathway proteins decreased.Conclusions: Overexpression of BRD4 in gastric cancer tissues may be a new biomarker for early diagnosis and prognosis of gastric cancer.The preparation of BRD4 siRNA-loaded nanoparticles by PEG-SS-PEI modified with LMWP can achieve efficient cell uptake and lysosome escape,and has anti-gastric cancer effect in vitro,which may be related to the inhibition of PI3K/Akt and c-Myc signaling pathways caused by BRD4 silencing.The results provide a new strategy for the diagnosis,prognosis and treatment of gastric cancer.