Polyethyleneimine-coated Fe₃O₄ Nanoparticles For Efficient MiR-100 Delivery To Reduce Malignant Biological Behavior Of Hepatocellular Carcinoma

Backgrounds:Hepatocellular carcinoma?HCC?is the third leading cause of cancer-related deaths in the world,and seriously endangering human health.Similar to other diseases,the occurrence and development of liver cancer involves a variety of complex pathogenesis,and mi RNAs is also involved in it,plays a role in the oncogene or anti-oncogene of cancer.MiRNAs,also called microRNA,is a kind of non-encoding single stranded RNA molecules encoding by the endogenous gene length of about 18-24 nucleotides,has a post transcriptional regulation function.It can regulate growth,differentiation,proliferation,apoptosis and other life activities of cell.At present,abnormal expression of certain miRNA has been observed in a variety of tumors,including liver cancer.In addition,several types of mi RNAs have been reported to regulate the growth,migration and invasion of hepatocytes,including miR-143,-203 and-221.MiRNA plays the role of oncogene or tumor suppressor gene in cancer.For the anti-cancer effect of miR-100,people are trying to deliver them to the tumor so as to achieve the aim of treatment.Many articles have been reported,microRNA-100?miR-100?was investigated as it is known to be down-expressed in a variety of cancer cells,for example,miR-100 inhibits growth,migration and invasion of hepatocellular carcinoma cells.The current difficulty lies in the successful delivery of the carrier system,such as safety and targeting.At present,more and more research shows that nanocrystalline particles can be used as a good medium for the transfer of miRNA.Magnetic ferriferrous oxide nanoparticles are widely used in the clinical diagnosis and treatment of cancer and in gene transfer.They have many uses such as detection of different diseases,including degenerative diseases and inflammatory,and early detection of tumors.The outer coating of magnetic nanoparticles can be combined with various ligands such as siRNA and mi RNA to form a targeted carrier system.In the present study,the delivery efficiency and therapeutic effect of this particular magnetic nanoparticle-based miR-100 carrier in HepG2 and BEL-7404 cells were assessed in vivo experiment.Our results demonstrated that Fe₃O₄ nanoparticles conjugated with miR-100 can effectively deliver miR-100-3p to hepatocellular cells and reduce the malignant biological behavior.Research contents:1.Measured the particle size and potential of Fe₃O₄.2.Detected the expression of miR-100-3p in human normal hepatocytes?HL-02?and several HCC cell line?HepG2,SMMC-7721,QGY-7701,BEL-7404 and Hep3B?were by qRT-PCR.Then selected two kinds of HCC cells with relatively low miR-100-3p expression?HepG2 and BEL-7404?.3.Detected the toxicity of carrier material by PI and DAPI staining.4.Detected the ability of hepatocellular carcinoma cells to uptake carrier materials by Prussian staining.5.Tested the target ability of carrier material by magnetic targeting experiment.6.Determined the mass ratio of miR-100-3p and Fe₃O₄,and then measured the particle size and potential of each mass ratio.7.Fe₃O₄ and miR-100-3p were co-incubated and transported into two kinds of hepatoma cells respectively,and the biological function of the successfully transported hepatoma cells was detected.Followed by:?1?Transwell/Matrigel assay observed HepG2 and BEL-7404 cells migration and invasion abilities.?2?Scratch test observed HepG2 and BEL-7404 cells migration abilities and wound healing ability.?3?Cell proliferation test detected HepG2 and BEL-7404 cells proliferation abilities.?4?Adhesion assay detected HepG2 and BEL-7404 cells adhesion change.Research results:1.The TEM results showed that the Fe₃O₄ particles were well water-soluble,and the particle size and potential showed that the particle size of Fe₃O₄ was at the nanometer level,which was in the nanometer level.The electric potential is positive charge.2.Compared with HL-02 cells of human normal hepatocytes,miR-100-3p is generally low expressed in several hepatoma cell lines.Especially in HepG2 and BEL-7404 cell lines.3.PI staining:The results showed that the red fluorescence of the cells incubated with different concentrations of the material was very little,which proved that the toxicity of the material to the two cells was very small,and the biological affinity was good.4.Prussian blue staining:The results show that the cells incubated with different concentrations of materials were restained red.Blue particles were found in the cytoplasm,indicating that the nanoparticles could be adsorbed into the cytoplasm.5.Magnetic targeting experiment:The results show that the material aggregation occurred on the side of the external magnetic field.PI staining also showed that there were few dead cells on the side of material aggregation.There is little toxicity to cells.6.Then miR-100-3p and Fe₃O₄ were incubated at different mass ratios at room temperature,and then combined to detect the expression of miR-100 with PCR.it is decided to take the mass ratio of 1:8 as the ratio of the two to carry out follow-up experiments.7.Effects of Fe₃O₄/miR-100-3p on the biological behavior of HepG2 and BEL-7404hepatoma cell lines:?1?The effect on the migration and invasion ability of hepatoma cells was detected by the cell test of Transwell.After Fe₃O₄ transports miR-100-3p to HepG2 and BEL-7404cells,the migration and invasion ability of the cells decreased significantly.?2?Effect on migration and invasion of hepatoma cells:Scratch results showed that miR-100-3p was transported into HepG2 and BEL-7404 cells by Fe₃O₄.The ability of cell migration and wound healing was significantly decreased.?3?The effect on the proliferation ability of hepatoma cells:the results of cell proliferation test showed that after Fe₃O₄ transports miR-100-3p into HepG2 and BEL-7404 cell lines,the cell proliferation ability decreased slightly.?4?The effect of Fe₃O₄ on the adhesion ability of hepatoma cells:the results of adhesion test showed that the adhesion ability of hepatoma cells decreased slightly after Fe₃O₄transports miR-100-3p into HepG2 and BEL-7404 cell lines.Conclusion:1.Fe₃O₄/PEI magnetic nanocarriers were successfully constructed by high temperature hydrothermal method.2.The Fe₃O₄ magnetic nanocarrier has the advantages of low toxicity,good affinity and targeting to hepatoma cells.3.MiR-100-3p was successfully transfected into hepatoma cells by Fe₃O₄ magnetic nanoparticles,which inhibited the migration,invasion,proliferation and adhesion of HepG2 and BEL-7404 cells.It plays the role of tumor suppressor gene in the progression of liver cancer.