Specific Knock-out Of MiR-100 In Liver Promotes The Development Of Hepatocellular Carcinoma In Mice Induced By DEN

[Background]Hepatocellular carcinoma（HCC）,a common malignant tumor of the digestive system with a high mortality rate,is the fifth most common cancer in the world and the third leading cause of cancer-related death in the world.The incidence of liver cancer is increasing year by year.HCC is characterized by solid tumors with a high degree of vascularization.It is well known that angiogenesis plays an important role in tumor growth and development as well as treatment prognosis.Therefore,many chemotherapeutic drug trials conducted in patients with advanced disease have had disappointing results and poor tolerability.There are various risk factors for HCC,among which genetic factors account for a large proportion.A large number of studies have shown that miRNAs play an important role in the occurrence and development of tumors.Significant expression of miRNAs was found in serum,tissues and even exosomes of cells,providing a basis for early detection,diagnosis and treatment of HCC.Oncogenes are divided into proto-oncogenes and tumor suppressor genes,and this widely recognized classification has been extended to a class of small non-coding RNAs that are highly conserved in evolution,namely micro RNAs（miRNAs）.The 3’untranslated region（3-UTR）of the target m RNA is the target for miRNA pairing,which is complete or incomplete.To regulate a large number of protein-coding genes.In addition,the vast majority of human protein-coding genes are predicted to contain sites where miRNAs can bind,which enables miRNAs to have a strong ability to regulate a variety of biological processes such as cell differentiation,migration,apoptosis and proliferation.As key regulatory factors of many diseases,miRNAs are closely related to cancer.It has been reported in many literatures that miR-100 is differentially expressed in different cancers,and specific functional loss or gain experiments on miR-100 can be used to more specifically study the specific role of miR-100 on tumor genesis and development.MiR-100 presents different expression patterns in different cancers,a large number of data have shown that miR-100 is low expressed in liver cancer,which suggesting that miR-100 plays an important role in the development of liver cancer.Diethylnitrosamine（DEN）is a well-known genotoxic carcinogen that induces liver cancer.A large number of literatures have reported that DEN can be used for acute or chronic induction of liver cancer in rodents,which is a relatively classic animal model of mouse liver cancer.DEN damages DNA through alkylation and produces reactive oxygen species that promote oxidative stress.If this occurs in young mice,this damage accumulates with the proliferation of liver cells,leading to liver cancer in mice.The mammalian target of rapamycin（mTOR）,a so important molecule in PI3K/Akt and the Akt/mTOR signaling pathway,of course,plays a central role in protein translation and a variety of other cellular functions,such as survival,proliferation,metabolism and growth.Activated mTOR can phosphorylate ribosomal protein S6kinase（S6K）and 4E-BP1 to increase protein translation.β-catenin,a subunit of the cell-surface cadherin protein complex,acts as an intracellular signal transduction in the Wnt signaling pathway,and its altered activity has been associated with the development of hepatocellular carcinoma and other liver diseases.Mitogen-activated protein kinases（MAPKs）are important molecules that related to signal transduction that regulate cell differentiation,proliferation and apoptosis.MAPKs,which promote MAPK activation,play a role by phosphorylation of su/serine residues.But there is no doubt that animals environment is unpredictable,involving more tissues,organs and systems,liver specificity miR-100 knock out only on mice liver,whether to participate in the development of hepatocellular carcinoma（HCC）or,and miR-100 knock out whether activation of MAPK pathway,the mechanism how,these have not been reported,need to be researched.[Methods]1 MiR-100^Flox/Floxmice were hybridized with Alb-Cre⁺mice.After several generations of breeding and screening,liver-specific knockout mice of miR-100 gene were obtained.2 The same dose of DEN or PBS was given to knocked and unknocked mice3 Use the technology of HE and immunohistochemistry to detect the pathological and related antibody expression changes in the liver tissues which belong to miR-100 liver specific knockout and unknockout mice.4 The expression of signal molecules involved in MAPK pathway was detected with Western blot.[Results]1.After breeding,screening and identification,mice with liver specific knockout and unknockdown miR-100 gene were obtained,and the mice were in good condition and could be used for experiment and breeding.2.Under the carcinogenic effect of low dose diethylnitrosamine,the naked eye tumor nodules appeared in mice when mice at the age of 10 months.So the liver carcinoma model was established within mice.3.HE and immunohistochemistry showed that liver specific knockdown miR-100 mice had significantly disordered liver tissue morphologic structure compared with unknockout mice.4.The expression of mTOR and p-ERK1/2 were highly upregulated in the mice of liver specific knockout miR-100(miR-100^flox/floxAlb Cre⁺)when they compared with miR-100^flox/floxmice,while EGFR,p-STAT1 levels were not changed significantly.[Conclusion]1.Liver-specific knockout of miR-100 gene had no effect on the early phenotype of mice.2.Combined with the carcinogen DEN,liver specific knockdown of miR-100 is more likely to develop liver tumors.3.The low expression of miR-100 may promote the development of liver tumors in mice through mTOR,phosphorylated STAT3 and phosphorylated ERK1/2 pathways.