| BACK GROUND: MicroRNA, which proteins to nucleic non-coding RNA and is 21~25 nt, can suppress the expression of targeted genes by reducing the stability of their mRNAs or inhibiting the translation of their protein products. Since microRNA was initially found in nematode in 1993, thousands of microRNAs have been indentified in animals. Numerous researches showed that microRNA play an important role in development, maintenance of physiological functions and genesis of disease in organisms.MiR-449a is a latest discovered miRNA,which belonged to miR-34 family.There are many literatures have reported that miR-449a have a low express level in prostate cancer, leading to histone deacetylases(HDACs) which is a oncogene over-express.The result suggest that miR-449a is a anti-oncogene,which abnormal express may relate with tumorigenesis.PURPOSES& METHODS: In the experiment, we use to carry miR-449a expression cassette of adenovirus infection of hepatoma cell line HepG2, MHCC97H, MHCC97L, HCCLM3 to examine whether the miR-449a could inhibit liver cancer cell growth, metastasis, in order to develop in order to miR-449a as a treatment for cancer gene therapy and provide a theoretical basis for new strategies.First, miR-449a of the target site was cloned into the plasmid of carrying the enhanced green fluorescent protein (enhancer Green fluorescence protein, eGFP) -pEGFP-C1 , the constructed plasmid called pEGFP-C1-miR-449aT.Then, the constructed intermediate vector pcDNA3.0-miR-449a and adenovirus shuttle plasmid pShuttle-cmv-miR-449a. The pShuttle-cmv-miR-449a and pEGFP-C1-miR-449aT co-transfected Hela cell lines by quantitative PCR detection of miR-449a expression and fluorescence quenching observed by fluorescence microscopy confirmed that exogenous expression of miRNA activity. In the verification of the pShuttle-cmv-miR-449a expression can be a normal biological function of miR-449a, adopted pShuttle-CMV-miR-449a with the adenovirus backbone plasmid AdEasy-1 recombinant, a recombinant plasmid pAdEasy-miR-449a , in the HEK293 human embryonic kidney cells carrying the successful packaging of the miR-449a expression cassette of the recombinant adenovirus Ad-miR-449a. Carrying the miR-449a adenovirus infected liver cells may be a substantial increase in the expression of miR-449a, and thus were used qRT-PCR and Western Blot immunoblot (Western blotting, WB) detection of miR-449a on cell cycle protein, Cyclin D1 (CCND1) and cancer metastasis-associated protein, c-Met at the mRNA and protein level of impact. Finally, through the Transwell used to detect miR-449a effect on HepG2 cell migration.RESULTS:①pShuttle-cmv-miR-449a and pEGFP-C1-miR-449aT co-transfected HeLa cells, qRT-PCR results showed that pShuttle-cmv-miR-449a can be highly efficient expression of miR-449a, green fluorescence is quenching through watched fluorescence microscopy, confirmed by the expression of miR-449a has a biological activity.②Adenovirus with miR-449a was successfully constructed and confirmed by qRT-PCR detection of Ad-miR-449a can be highly expressed in hepatocellular carcinoma cell miR-449a.③qRT-PCR results showed that, miR-449a expression can be reduced CCND1 and c-Met mRNA expression.④WB tests showed, Ad-miR-449a-infected cells after the CCND1 and c-Met protein expression also decreased significantly.⑤Transwell experiments showed that, miR-449a can affect the transfer of HepG2 cells.CONCLUSION: MiR-449a could targete CCND1 and c-Met; miR-449a by inhibiting the expression of c-Met inhibition of HepG2 cell migration. This miR-449a as a new tumor suppressor gene play a role in cancer therapy to provide a certain theoretical basis. |
