| Objective: To construct a lentivirus vector for RNA interference of HDAC2 gene, and detect the silence of the vertor in human hepatocellular liver carcinoma cell line(Hep G2).The proliferation mechanism of 1,25(OH)2D3 to Hep G2 is researched. From the m RNA and protein levels in 1,25(OH) 2D3 is thus promoting tumor suppressor gene p21 WAFI / CIP1 function realization by inhibiting the expression of HDAC2, thereby regulate the proliferation and differentiation of Hep G2 cells. Methods: 1、3 pairs of m RNA interference target sequences for the HDAC2 m RNA sequences and one pair of negative control sequence are designed and used to synthesize oligonucleotide sequences that target short-hairpin structure annealed to form double-stranded DNA and clone ligase into Eco RI digested by Age I and clone to lentiviral expression plasmid p LKO.1- TRC cloning vector, and the to build targeted inhibition of double-stranded short hairpin RNA(sh RNA) HDAC2 expression of eukaryotic expression vector p LKO.1-sh RNA-HDAC2. The recombinant plasmids were then digested with Eco RI and Xho I single agarose gel electrophoresis analysis. Interfere with digestion fragments were sequenced to ensure that the recombinant plasmid is inserted correctly, that is to show sh RNA lentivirus vector for gene HDAC2 are successfully constructed. 2、Correctly identified recombinant plasmid p LKO.1-sh RNA-HDAC2 were transfected human embryonic kidney cells 293 T to produce infectious and stably expressing human HDAC2 sh RNA lentiviral particles. 3、 Lentiviral particles infecting Hep G2 cells was recombinanted, and added the corresponding recombinant lentivirus and control lentiviruses. HDAC2 gene m RNA and protein expressing levels are checked by real-time PCR and Western blotting respectively and recombinant plasmids interfering effect of each group are indentified and analyzed. The Hep G2 cell growth condition was checked by CCK-8 method and the cell growth curves were plotted to observe the effect on the biological characteristics of Hep G cells after HDAC2 gene silencing. 4、Hep G2 cells were cultured with lentiviral infection and 1,25(OH)2D3 treatment. m RNA level of HDAC2 and p21 WAFI/CIP1 gene and protein expressing situation of RNA interference and 1,25(OH)2D3 treatment to Hep G2 cells was checked by real-time PCR and Western blotting. Results: 1.、sh RNA lentivirus annealing connected to p LKO.1- TRC cloning vector, positive clones were screened by restriction enzyme digestion and DNA sequencing revealed the same validated screening recombinant sequencing results with the expected target sequence. So p LKO.1-sh RNA recombinant plasmid was successfully constructed. 2. 、 Effective concentration of stable expression of recombinant sh RNA lentiviral particles of HDAC2 are obtained. 3、Realtime PCR and Western blotting checking results showed that compared with the control group, three interference sequences transfected Hep G2 cells could inhibit endogenous HDAC2 gene and protein expression(P<0.05), which interference effects p LKO.1-HDAC2-sh RNA1,3 the most obvious difference between the two was not statistically significant compared to(P>0.05), so it showed that interference HDAC2 gene lentiviral vectors have been successfully constructed. 4、CCK-8 checking results shows that protein expression in Hep G2 cells HDAC2 decreasing will lead to inhibition of cell proliferation. 5、 Comparing the Hep G2 cell treated by1,25(OH) 2D3 after HDAC2 gene silencing with HDAC2 gene silencing group, both the m RNA and the protein levels show that 1,25(OH)2D3 treatment will not further promote p21 WAFI / CIP1 gene high expression(P>0.05). Conclusion: 1、Interference HDAC2 gene can promote p21 WAFI / CIP1 m RNA and protein expression. 2、1,25(OH)2D3 can promote the m RNA and protein expression of p21 WAFI / CIP1 gene is related with inhibiting the expression of HDAC2. |
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