| Background: In recent years,the morbidity and mortality of Lung cancer run thefirst place in all tumors and impact seriously on human’s life and death.In industrializedcontries29%of all patients die of lung cancer.In our country,the incidence of lungcancer is also found in a rising trend and is the first place of various types of cancer. Butbecause of the development of cancer is a complex process that many factors and genesparticipate in it and the metastasis and multidrug,results that survival rate of non-smalllung cancer cell(NSCLC) and small lung cancer cell(SCLC) is very low. Though manytheraoeutic measures have been taken,such as operation,radiotherapy,and chemothera-py.But still have no obvious curative effect.So we have to clarty the machanism ofcancer and find the newest and effective target of gene therapy.RASSF10is the latest member of the RASSF family with Ras effector. RASSF10have been frequently inactivated by aberrant promoter hyper-methylation in severalhuman cancers，such as thyroid cancer, malignant melanoma of the skin and glioma.Butits research of biological function in lung cancer is still unclear and need to be furtherresearch.So the purpose of our study is to detect the expression condition of RASSF10in lung cancer SPCA-1cells,and use gene interference methods to knockdown theRASSF10expression status in SPCA-1,thereby to further discuss the role of RASSF10in lung tumorigenesis and cell apoptosis.Objective:To investigate the morphology changes of lung tumor cell and detectthe expression status of RASSF10after gene interference in SPCA-1cells,so as to givea comprehensive analysis of role of RASSF10in lung cancer occurrence,and offer anew theoretical basis for clinical treatment of lung cancer.Methods:Liposomes(LipofectamineTM2000) was used to transfect human shRNA-RASSF10plasmid into human lung cancer SPCA-1cells,in order to knockdown theexpression of RASSF10gene.Fluorescence microscopy was used to detect transfection efficiency and investigate the morphology changes of lung tumor cells.RT-PCR wasused to detect the level of mRNA expression.Immunofluorescence and Western blottechniques was used to detect the level of RASSF10protein expression of transfectedSPCA-1cells. CCK8and Flow cytometry techniques was used to detect the prolife-ration and apoptosis of transfected SPCA-1cells respectively.Results:Cell morphological changes were observed with microscope.Beforetransfection, the spindle cells were moderate-sized with clear nucleoli. At48h aftertransfection, the number of cells and nuclear division were increased.RT-PCR analysisshowed that there were two straps of318bp(RASSF10) and984bp(GAPDH)respectively.The straps of GAPDH were similar and clear.The strap of the RASSF10sh-RNA group was weaker than the other groups.The labworks revealed that mRNAlevels of RASSF10were notably lower in RASSF10silencing transfectants than in theirblack and negative controls.There was obvious difference between the sh-RNA groupand the other two group controls(P＜0.05).But there was no significant differencebetween the black control and the negative control(P＞0.05).Western blot analysisshowed there were two straps of57KD(RASSF10)and43KD(β-actin) respectively.Thestraps of β-actin were similar and clear. The strap of the RASSF10sh-RNA group wasweaker than the other two groups. The labworks revealed that the protein levels ofRASSF10were notably lower in RASSF10silencing groups than in their black andnegative controls.The difference between the sh-RNA group and the other two groupcontrols was obviously(P＜0.05). But there was no obvious difference between theblack control and negative control(P＞0.05). CCK8experimental was used to detectcell proliferation and the results showed that in0h,48h,72h,96h four time points aftertransfected, RNA interference group absorbance were0.31±0.003,0.62±0.010,1.05±0.024,1.44±0.018.Compared with cells in normal control group absorbance were0.30±0.021、0.49±0.012、0.75±0.031、1.17±0.014. Compared by t test analysis of thetwo groups was statistically significant(P<0.05).Apoptosis was detected by flowcytometry:the apoptotic rate in the sh-RNA transfection group was (5.12±0.36)%,whilein the black control was(7.23±0.54)%,and in the negative control was (7.40±0.42)%.there was no obvious difference between the black control and the negative control(P＞0.05),but there was significant difference between sh-RNA group and the two controlgroups(P＜0.05).Conclusion:Using RNA interference technology to silence RASSF10gene cansignificantly promote the proliferation and arrest the apoptosis of SPCA-1cell lines.This provides foundation for further research using RNAi for the biological treatment of lungcancer. |
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