Layered Double Hydroxide Loaded MiR-30a For The Treatment Of Breast Cancer

Purpose: Breast cancer is the most prevalent cancer among female patients.However,it is not the primary tumor that causes death in breast cancer patients,but the metastasis of the tumor.Micro RNA(miRNA)is a non-coding small RNA,which can carry out multitargeted gene regulation and plays an important regulatory role in the occurrence and development of cancer.miR-30 a has been reported to inhibit the growth and metastasis of a variety of tumors,including breast cancer.Therefore,the delivery of exogenous tumor suppressor miR-30 a is an effective means to realize breast cancer gene therapy.miRNA itself is a negatively charged polar small molecule,which is not easy to enter the cell membrane,and the exposed miRNA molecule is easily degraded by nucleases in the physiological environment in vivo,and is easily removed quickly by the liver and kidney,and its therapeutic effect in vivo is greatly limited.In order to solve the shortcomings of miRNA that is easy to degrade and not easy to enter cells,this project intends to construct a safe,efficient,and stable layered dihydroxide gene drug delivery system,in order to improve the stability of miR-30 a and prolong its role in inhibiting the growth and metastasis of breast cancer.Methods:(1)Analysis of the difference in miR-30 a expression between normal tissues and breast cancer in TCGA(The Cancer Genome Atlas)database by UALCAN,and the effect of miR-30 a on overall survival rates(OS)of patients by KM(KaplanMeier).(2)q RT-PCR(Quantitative Real-time PCR)to compare the expression differences of miR-30 a in normal breast epithelial cells MCF 10 A and breast cancer SKBR3 cells;(3)Preparation of LDH and LDH@miR-30 a by hydrothermal and electrostatic adsorption methods,respectively;(4)Detection of LDH and LDH@miR-30 a by Malvern particle size potential analyzer particle size and potential;(5)Transmission electron microscopy(TEM)to examine the morphological features of LDH and LDH@miR-30a;X-ray diffractometer(XRD)to determine the crystal structures of LDH and LDH@miR-30a;(6)The mass ratio of miRNA bound to LDH was determined by agarose gel electrophoresis;(7)The uptake of LDH@miR-30 a for SKBR3 cells at 4 h and 24 h was observed using laser confocal microscopy;(8)Lysosomal escape assay and lysosomal membrane permeability assay to investigate the mechanism of LDH@miR-30 a escape lysosomal capture and delivery;(9)CCK-8 assay and flow cytometry to determine the effect of LDH@miR-30 a on SKBR3 value-added and cell cycle arrest;(10)Wound healing assay and Transwell assay to determine the effect of LDH@miR-30 a on SKBR3 migration invasion;(11)Targetscan analyze target genes of miR-30a;(12)q RTPCR and Western Blot to detect the effect of LDH@miR-30 a on SNAI expression in SKBR3 cells;(13)Western Blot exploration to detect the effect of LDH@miR-30 a on EMT-related protein expression in SKBR3 cells;(14)Construction of a nude mouse xenograft tumor model to evaluate the effect of LDH@miR-30 a on breast cancer treatment in vivo by tumor weight,volume and Tunel staining;evaluation of LDH@miR-30 a biosafety by body weight,and HE staining.Results:(1)miR-30 a expression was decreased in breast cancer patients,and the expression level was positively correlated with the survival rates of breast cancer patients;(2)miR-30 a expression was significantly higher in MCF 10 A cells than in SKBR3 cells;(3)LDH was successfully prepared using a co-precipitation method and LDH@miR-30 a was prepared by co-incubation with miR-30 a.;(4)LDH and LDH@miR-30 a had mean particle sizes of 141.7 nm and 282.2 nm,respectively;and Zeta-potentials of 37.7 m V and 30.5 m V,respectively;(5)The XRD image of LDH@miR-30 a has the same characteristic peaks as LDH,and the morphology of LDH and LDH@miR-30 a shows a regular hexagonal layer structure;(6)LDH could stably conjugate with miR-30 a at a mass ratio of 10:1 or more;(7)LDH@miR-30 a could be successfully taken up by SKBR3 cells and was still detectable intracellularly at 24 h;(8)LDH@miR-30 a has lysosomal escape ability;(9)LDH@miR-30 a was able to inhibit SKBR3 cell proliferation and block their cell cycle in G0/G1 phase;(10)LDH@miR-30 a was able to inhibit SKBR3 cell migration and invasion;(11)LDH@miR-30 a was able to inhibit SNAI1 expression;(12)Reduced expression of N-cadherin and Vimentin and increased expression of E-cadherin in the LDH@miR-30 a group compared to the miR-30 a alone group;(13)Compared with the single miR-30 a group,the tumor volume and weight of LDH@miR-30 a group were significantly smaller,and TUNEL staining showed that the proportion of apoptotic cells in the tumor tissue of LDH@miR-30 a administration group increased more;and there was no significant difference in body weight change compared with other treatment control groups;(14)HE staining showed no significant damage of LDH@miR-30 a group on the major organs of nude mice.Conclusion: In this study,a p H-sensitive gene drug carrier LDH@miR-30 a was successfully synthesized,which can escape through the lysosomal pathway,promote the release of miR-30 a into the cytoplasm,and exert certain anti-breast cancer effects in vitro and in vivo.LDH@miR-30 a provides a new strategy for gene therapy of breast cancer,and also provides a new safe and effective transfection agent for the study of miRNArelated functions.