The Effects Of Cholesterol-conjugated Let-7a MicroRNAs Mimics On The Growth Of Hepatocellular Carcinoma In Vitro And In Vivo

Hepatocellular carcinoma (HCC)has thefifth highest incidence and the third most common cause of cancer-related deathsin the world.HCC is associated with poor prognosis, mainly due to metastasis. There remains no effective therapeutic strategy because most patients are diagnosed during the latter stages of the disease.Recently, many studies indicate that miRNA expression signaturescan serve as diagnosis and prognosis predictorsfor HCC and provide a new insight into HCC treatment. MicroRNAs are a class of19to22nucleotides non-coding RNA which are widely involved in the process of cell life such as cell development, differentiation, metabolism and associated with development of many types of cancer including HCC. It indicatesthat microRNAs may have potential value for HCC clinical treatment. let-7is one of firstly discoveredmicroRNA families.let-7now has a widely known role in progression of malignancies. It demonstrates let-7inhibits growth of cancer by targeting Ras. let-7is lowly expressed in HCC and associated with early recurrence of HCC.Therefore, targeting let-7andRas may be a promising approach for cancer therapy.However,there remains obstacles for clinical application with microRNAs because there are not appropriate vectors which can deliver the drugs into targeted cancer cellseffectively. Recently, microRNA mimics are reported to be used for restoration of microRNAs which are downregulated in cancer.We selected let-7a as the study object and intended to investigate the role of let-7a in the growth of HCC in vitro and in vivo. Compared with let-7a mimic, cholesterol conjugated let-7a mimic(chol-let-7a mimic) is found to deliver into HCC cells more efficiently and level of let-7a elevated more severely.Simultaneously, we discovered chol-let-7a mimic had high affinity of liver after systemically administration. As a result, we chose chol-let-7a mimic to overexpress let-7a.On this basis, our research can be divided into two aspects.First, we studied the role of let-7a in proliferation,apoptosis, migration, invasion and effects on ultra microstructure of HepG2and SMMC-7721cell lines. It showed that proliferation, migration, invasion weresuppressed, apoptosis was prompted and the ultrastructure of HCC cells wasdamaged by chol-let-7a mimic. Second, we discussed the effect of chol-let-7a mimic on growth and metastasis of HCC in vivo. We constructed subcutaneous and orthotopic liver cancer xenografts model in nude mice.At the same time, cholesterol conjugated chol-let-7a mimic or NC was injected intratumorallyor systemically. The ultrasound machine vevo2100was applied for the primary and metastatic liver cancer detection. At the end of the animal experiment the fresh orthotopic liver cancer tissues were collected for the ultra microstructure of cancer observation with electron microscopy and histological analysis. Simultaneously, we studied the inhibition of let-7a in Ras after systemically administration by using RT-PCR and Western blot. We proved chol-let-7a mimicmore directly targeted tumors than NC.We demonstrated exogenous chol-let-7a mimic could enter into solid tumor, level of let-7a was increased in the tumor and the proliferation, metastasis of HCC was inhibited in vivo.We also demonstrated the level of three Ras genes was higher in the transplanted HCC tumor tissue than in the normal liver tissue of mice. Moreover, we found upregulation of let-7a inhibited level of protein and mRNA in three Ras genes and demonstrated let-7a directly regulatedKras and Nras through the3’UTR binding sites.In this study,we systematicallydemonstrated over expression of let-7a could significantly inhibit growth and metastasis of HCC in vitro and in vivo. Above all HCC cell lines were transfected with cholesterol conjugated let-7a mimic and then proliferation, migration and invasion of HCC cells were suppressed. In addition, cholesterol-conjugated let-7a mimic was applied for local injection and systemicaladministration in subcutaneous and orthotopic liver cancer xenografts. We demonstrated the growth and metastasis of transplanted liver cancer was inhibited. Meanwhile the protein and mRNA level of three Ras genes were restrained. It indicatesthe stability of let-7a mimic in peripheral blood was strengthened with chemical modification of let-7a mimic. At the same time cholesterol-conjugated Iet-7a mimic could enhance the affinity of liver which could directly targeted the solid tumor and level of let-7a maintained high and expression of three Ras genes were decreased for a long time. We show that let-7a might become a future development of an alternative strategy in targeted cancer therapy.