The Influence Of Small Interfere RNA Targeting STAT3 Expression Vector To The Expression Of Im-2 In Liver Cancer Cell

Objective: This study choice signal transduction and activator of transcription3 (STAT3) as a target gene to construct short hairpin RNA ( shRNA) expression vector that interference STAT3 gene in liver cells, explore the expression of pim-2 gene after STAT3 changes.Method:1 STAT3-siRNA expression vector preparation. According to certain principles of design and synthesis of siRNA against STAT3 gene expression cassette (stat3-SECs).STAT3-SECs will be connected with the psiLentGeneTM vector,take appropriate product of transformation to Ecoil DH5αcompetent cells,select positive recombinant clones,recombinant plasmid was digested by endonuclease,plasmid required for experimental confirmation.2 Cell culture Select human liver cancer cells SMMC-7721 cultured for conducting experiments.3 Transfected cells According to the Transfection reagent instructions,transfect STAT3-siRNA plasmid into SMMC-7721cells.At the same time,set SMMC-7721control,SMMC-772 1 transfection reagent and SMMC-7721 mismatch-SECs expression vector groups,SMMC-7721empty plasmid group respectively.4 Observe the morphologic change of the cells After 24-hour transfection,study the modality change under invert light microscope.5 RT-PCR After 24-hours transfection, the total RNA, abstracted respectively from the cells with Trizol reagent was reverse-transcribed into cDNA. Use this cDNA as template, amplify pim-2 andβ-actin gene fragment. Then contrast the difference of expression level in pim-2 gene by gel imaging analysis sofeware.6 Western Blot Solubilized protein samples from the SMMC-7721 cell line by using SDS-PAGE,and then electrophoretically transferred to a nitrocellulose membrance. After blocking with a protein blocking agent,the membrance is probed with the primary antibody.The antibody-antigen complexes are then identified by the secondary anti-IgG antibody, which is conjugated to horseradish peroxidase (HRP) enzymes.Finally,chromogenic substrates are used to visualize the activity.In the whole procedure,theβ-actin gene was used as the inner reference to compare expression of the PIM-2.7 Statistical analysis Biostatistic analyses were done by SPSS 11.0 software package.Date from the experiment was analyzed by ANOVA,α= 0.05 was the remarkable Standard. Results:1 Cloning1.1 The DNA sequence of siRNA expression cassettes shows that the component of siRNA expression cassettes is matched with the anticipation.1.2 Recombinant plasmid was identified by digestion as requirement of experimental expression vector.2 Cell morphologyInverted microscope, The liver cancer cells in mismatch chain expression vector group, empty plasmid group,transfection reagent and control groups were multiplied and well adherenced. Cells appeared as fusiform shape, spreaded out of the field of vision with clear nucleoli and caryocinesis phase. The liver cancer cells of SMMC-7721 STAT3 expression vector group were decreased and bad adherenced.With more floating ones, cells were deranged and inequality of size,shrinkage and vacuoluse,appearing karyoplasmic ratio inversion and diminution of karyokinesis phase.There occured effervescencing anddescented-deuterium,while cell membrane were integrity.We saw many round micro-bubbles and buddings in cells and losing normal contact inhibition infracells.Moreover, the cell debris increased and background got muddy.With the concertration of SMMC-7721 STAT3 expression vector grandualy increased, the change-degree of cells increased correspondingly. 3 RT-PCR resultsTheβ-actin expression remained unchanged in SMMC-7721 control group,SMMC-7721 reagent idling group,SMMC-7721 mismatch chain expression vector group,SMMC-7721 siRNA-stat3 expression vector group,while the pim-2 expression lower than the other three groups in the SMMC-7721 siRNA-stat3 expression vector group.4 Western Blot ResultsTheβ-actin protein expression remained unchanged in SMMC-7721 control group, MMC-7721 reagent empty plasmid group,SMMC-7721reagent idling group,SMMC-7721 mismatch chain expression vector group,SMMC-7721 stat3-siRNA expression vector group,while the PIM -2 protein expression lower than the other three groups in the SMMC-7721 siRNA-stat3 expression vector group.Conclusion:1 The STAT3-siRNA expression vector synthesize successful in vitro.2 STAT3-siRNA expression vector can effectively inhibit the expression of pim-2,and inhibit human liver cancer cell proliferation and promote apoptosis.3 The treatment strategy of RNA interference silences stat3 may provide the new mentality for liver cancer treatment.