Construction Of Expression Vector For RNA Interference Of Hypoxia-inducible Factor 1alpha And Its Function In The Study Of Prostatic Carcinoma Cell Line PC-3

Objective The expression vector of RNA interfering with the hypoxia-inducible factor 1alpha (HIF-1α) was constructed and then its function was studied on Prostatic carcinoma cell line PC-3.Methods According to the sequence of hypoxia-inducible factor 1alpha in the GenBank, the oliga-nucletides strand to the gene of hypoxia-inducible factor 1alpha was synthesed chemically. After annealing and replicating, it was linked into the vector pSUPER.retro.puro, then it was transformed into the DH5αstrain. And then the plasmid was attracted, cut by the indo-nuclease and its sequence was analysed. While the recombination plasmid was used to transform the Prostatic carcinoma cell line PC-3, its efficiency was assessed. These transformed cells were collected after 48 hours, whose hypoxia-inducible factor 1alpha total RNA and expression protein were detected. Those cells were checked out by the plasmid resistance against the certain biotin, the clones of which were spectacled and were enlarged for two weeks. Then the cell strain of pSUPER-siHIF-1α/PC-3 was collected and its expression of hypoxia-inducible factor 1alpha in was rechecked.Results The sequence analysis of the double nucelease electrophoresis for the recombination plasmid illustrated that the objective fragments inserted to the progra mmed locus with a completely common line, which mean the success of the expression vector of RNA interfering with the hypoxia-inducible factor 1alpha. The efficiency was (87.15±2.36) % after 36 hours when the constructed plasmid and green fluorescent protein were co-transformed into the Prostatic carcinoma cell line PC-3. This plasmid could decrease the expression of total RNA in the Prostatic carcinoma cell line PC-3 significantly(P < 0. 05), and the suppress efficiency were 75.2%, 72.8%, respectively. The monoclone was seen in two weeks with the selective drugadded. And the hypoxia-inducible factor 1alpha protein expression of the pSUPER-siHIF-1α/PC-3 cells strain of decreased significantly(P < 0. 05), its suppress efficiency was 83.2%.Conclusions The RNA interference could be used in the construction of expression vector of constructed siRNA inhibiting the expression of hypoxia- inducible factor 1alpha with Prostatic carcinoma cell, which is the basis of researching the pathology, multiplication, and metastasis of hypoxia- inducible factor 1alpha in the Prostatic carcinoma and other cancers.