Study Of ShRNA Silencing Kir6.2 Gene In SK-Hep1 Cell Strains Of Human Hepatocellular Carcinoma

Ion channels are fundamental to the life.The relationship between ion channels and cancer is now gradually attracting the interest,To deeply understanf the effect of ion chanels on the tumor development and metastasis will be helpful not only to completely elucidate the cancer mechanism but also to provide new targets and biological intervention methods for the cancer prevention and ttreatment.Potassium channels have many kinds and are the more complicated ion channel,they play the key role on the signal transduction of the excitable cells and non-excitable cells,meanwhile take part in the regulation of tumour cell proliferation and differentiation.The relationship between the potassium channels and tumour cell proliferation has been the new hot spot of the tumour fundamental research.ATP-sensitive potassium channels couple membrane excitability to energy metabolism.Many studies in the cell biology and pharmacology have proved that potassium channels have effect on the tumour cell proliferation and survival. RNAi technology can not only discover the gene change bot also be a method to treat cancer.ShRNA (short hairpin RNA) can stably silence target gene.Human primary hepatocellular carcinoma (HCC) is one of the most common maligent tumour in our country,the tendency of HCC incidence is gradually increasing.Though great progress in diagnosis and treatment of HCC has been made over the past decades,the lower survival rate and poorer prognosis remains in patients with HCC.Basic research should be strengthenedObjective: To construct the plasmid vector expresses shRNA.The recombinant plasmid was transfectd to SK-Hep1 cell strains, KATP currents were detected by patch clamp. The characteristic of cell biology was investigated.Methods1.To construct the plasmid-mediated shRNA Fragment inserting the plasmid was obtained from the complementary oligonucleotide annealing was linked to the linearization empty pGenesil-3 zymased by ,The recombinant plasmid was transformed into the competence germ DH5a,The germ was cultured by using LB medium to extract the plasmid which was incisived and sequenced.2.Culture,passage,transfection,screen of the SK-Hep1 cell strains SK-Hep1 was continuously cultured in use of DMEM with 10% FBS(fetal bovine serum),passage was done when the cell density reached 80-90%.Transfection was done with the help of liposome 2000.3. I-V curves was drawed.4.Effect of shRNA silencing Kir6.2 gene on the characteristic of cell biology Cell cycle was detected with flow cytometry. probe incubated cells in each group .Fluorescent density of intracellular Ca2+ concentration was achieved. Cell proliferation was detectd with MTT assay. The cell invasive ability in vivo was obtained by using Transwell cabin.Results1.Identification by incision enzyme and sequencing Report showed the recombinant plasmid expressing shRNA was successful.2.Transfection,screening,amplification RT-PCR showed that expression of Kir6.2 gene in SK-K1 and SK-K2 groups remarkably decreased compared with that in SK and SK-HK groups,Kir6.2 protein in SK and SK-HK groups was more than that in SK-K1 and SK-K2 groups.3.Current of KATP channels in each group The KATP current in SK, SK-HK, SK-K1, SK-K2 groups (nA) was 1.80±0.673,1.87±0.541,5.16±1.011,3.89±1.287, respectively. Compared the curren in K1and K2 groups with that in SK group, there was significantly statistical. the current in HK group was much lower than that in K1 and K2 groups. I-V curves in K1 and K2 groups were close to the vertical axis.4.Change of the characteristic of cell biology Cells in G0-G1 of SK, SK-HK groups were more than that in SK-K1, SK-K2 groups.Cells in S phase of SK-K1, SK-K2 groups were more than that in SK, SK-HK groups. Calcium concentration SK-K1, SK-K2 groupswas much lower than that in SK group. Calcium concentration in HK groups was remarkably higher than that in SK-K1, SK-K2 groups Underexpression of kir6.2 reduced significantly cell proliferation and invasion..ConclusionRT-PCR and Western Blot identified that shRNA successfully silenced Kir6.2 gene.Current of KATP channels in SK-K1, SK-K2 groups enchanced more. The characteristic of cell biology in SK-K1, SK-K2 groups was affected.