"Aged" Zinc Oxide Nanoparticles Promote Aggressiveness Of Human Hepatocarcinoma Cell Line HepG2 And Its Mechanism

BackgroundFor a long time,the incidence and mortality of tumors have remained high in various countries,and the infiltration and metastasis of tumor cells is also one of the important reasons why tumor patients are difficult to cure.Among the various types of cancer,hepatocellular carcinoma is the 5th most common malignant tumor in China,with the second highest mortality rate.The two major factors that promote the malignant progression of tumors are genetic factors and environmental factors.While many people focus on the genetics of cancer,environmental factors play an equally important role in the development and progression of cancer.Studies have shown that nanomaterials can promote tumor metastasisZinc oxide nanoparticles(Zn O NPs)have aroused the curiosity of researchers around the world due to their wide range of biological activities.It has been reported that zinc oxide nanoparticles can accumulate in cells,cause cell death through oxidative stress and inflammatory response,and can also cause toxicity to animals and humans,including neurotoxicity,cardiotoxicity,liver toxicity and nephrotoxicity.More notably,studies have found that Zn O NPs tend to dissolve in the environment and undergo structural transformations,eventually leading to changes in their storage status and bioavailability,a process we call aging.And the “aged” nano-zinc oxide has stronger genotoxicity and lower cytotoxicity.Compared with cytotoxicity,the genotoxicity of “aged” Zn O NPs adversely affects cells and even the entire organism,causing genetic mutations.While mutagenicity is thought to be one of the earliest cellular responses caused by physical and chemical carcinogens,it may play an important role in the initiation and progression of cancer.Previous experimental results of our group showed that long-term treatment of nano-zinc oxide in a low-dose aged state can promote the malignant transformation of embryonic fibroblasts in MEF mice and tumorigenesis in nude mice.Therefore,we wonder whether aged nano-zinc oxide can promote its malignant progression in tumor cells and hepatocarcinoma cells.Based on the hypothesis,based on the fact that nanomaterials are released at lower concentrations in the environment,in vitro cytology experiments used low-concentration(1.5μg/ml)nano-zinc oxide to induce human Hep G2 carcinoma cells for a long time with low-concentration(1.5μg/ml)nano-zinc oxide(4 months),observe its effect on Hep G2 cells,and explore possible mechanisms.Research methods1.Formulate nanomaterials and undergo 60 days to simulate the natural aging process.2.Long-term chronic induction of human hepatoma cell Hep G2(4 months)using low-concentration(1.5 μg/ml)of primary and aging nano-zinc oxide.3.Cytobehavioral experiments using chronically treated Hep G2 cells of nanomaterials,including: plate cloning experiments,soft agar colony formation experiments,transwell migration experiments,transwell invasion experiments.4.Malignantly enhanced Hep G2 cells were used to construct a mouse xenograft in situ model to detect the effect of aged nano-zinc oxide on tumor growth and metastasis in vivo.5.Analyze changes in transcriptome levels of Hep G2 cells chronically treated with nanomaterials by RNA sequencing.6.Verify changes in relevant genes and proteins by q PCR and Western Bloting.7.By transfecting the virus,knock down the expression level of relevant genes and reverse verify the role of related genes in cells.Experimental results1.Successfully prepared aged nano-zinc oxide,and successfully established a chronic-induced cell model.2.The experimental results of transwell and plate cloning showed that the migration invasion ability and proliferation ability of Hep G2 cells were promoted after material induction,that is,malignant transformation occurred.3.Key genes that promote the malignant transformation of Hep G2 cells were found:cldn2,krt16.ConclusionLong-term treatment of low-dose aging Zn O NPs can promote malignant transformation of Hep G2 cells and tumorigenesis in nude mice.This promotion may be the result of high expression of two genes,cldn2 and krt16.