Study On The Effect Of High Mobility Group A2 Protein In Non-small Cell Lung Cancer

PartⅠThe Expression of HMGA2 and Its Relationship with CellProliferation in Non-small Cell Lung CancerObjectiveTo evaluate the effects of the High mobility group A 2 protein(HMGA2)expressionin Non-small cell lung cancer(NSCLC)on tumor growth and metastasis,and therelationship between HMGA2 and clinicopathologic factors and cell proliferation index.MethodsImmunohistochemistry was applied to detect the expression of HMGA2 and Ki-67protein in 38 cases of NSCLC(23 cases of lung squamous cell cancer,15 cases of lungadenocarcinoma)and tumor-surrounding normal lung tissues.ResultsIn the tumor-surrounding normal lung tissues,the HMGA2 and Ki-67 was negativelyexpressed in all specimens,while HMGA2 and Ki-67 was separately positively expressedin 39.47%,44.74% NSCLC specimens,there were distinctly difference(P＜0.05)。The HMGA2 expression was positively correlated to lymph node metastasis,but there wasno correlation between the HMGA2 expression and other clinicopathologic factors such ashistological grades,TNM,tumor size and cell proliferation index Ki-67.ConclusionsThe present study demonstrated that HMGA2 is ectopically expressed in the NSCLCand has a significant impact on tumor growth and progression. PartⅡThe Construction of the Recombinant Plasmid pGCsi3.0U6/HMGA2 shRNA and its Expression in Human lung SquamousCell Carcinoma Cell line SK-MES-1 CellsObjectiveTo construct an expression vector with short hairpin RNA of HMGA2 and detect theexpression of the recombinant plasmid in SK-MES-1 cells.MethodsDNA fragment with the structure of HMGA2 shRNA was designed and synthesized.After annealed,the strands were cloned into pGCsi3.0U6/Neo/GFP/RNAi vector toconstruct the recombinant plasmid.The recombinant plasmid was verified by sequencing.The SK-MES-1 cells were cultured in vitro and divided into the blank control group,thespecific interference group and the negative control group,the transfection was made usinglipofectamine 2000.After transfection,the cells were observed under the fluorescencemicroscope.The expression of HMGA2 in SK-MES-1 cells in three groups was detected byRT-PCR and Western blot.ResultsThe sequencing result showed the shRNA coding sequence in recombinant plasmidwas consistent with the designed target HMGA2's cDNA.Cells transfected with theplasmid showed green luminescence,suggesting the correct transfection of the plasmids.Compared with the blank control group,the mRNA expression levels of HMGA2 inspecific interference group and the negative control group was(48.13±0.15)%,(97.33±0.08)% respectively.Compared with the blank control group,the proteinexpression levels of HMGA2 in the specific interference group and the negative controlgroup was(42.31±0.05)%,(96.07±0.09)% respectively.ConclusionsThe recombinant plasmid expressing short hairpin RNA of HMGA2 was successfullyconstructed,and transfection the plasmid can significantly decrease the expression ofHMGA2 in SK-MES-1 cells. PartⅢStudy on the Effect of Transient Silencing HMGA2 GeneExpression on Cell Proliferation,Apoptosis,Migration and Invasionof Human Lung Squamous Cancer Cell Line SK-MES-1ObjectiveTo explore the effects of a plasmid with short hairpin RNA(shRNA)on cellproliferation,apoptosis,migration and invasion of human lung squamous cancer cell lineSK-MES-1.MethodsThe SK-MES-1 cells were cultured in vitro and divided into the blank control group,the specific interference group and the negative control group.Cell proliferation wasanalyzed by MTT,the cell cycle and cell apoptosis were analyzed by flow cytometry.Themigration and invasion of SK-MES-1 cells were investigated by Transwell assay.ResultsCompared to the the blank control group and he negative control group,the transientsilencing of HMGA2 gene in the specific interference group resulted in reduction of tumorcell proliferation activity.The cells were stopped at G1 phase,and no apoptosis wasdetected.The migrant cells in interference group(62.3±6.8)were less than those in blankcontrol group(151.3±6.5)and negative control group(139.7±9.5)(P＜0.01).The invasivecells in interference group(37.7±3.2)were less than those in blank control group(84.0±3.6)and negative control group(72.3±7.7)(P＜0.01).ConclusionsThe shRNA-mediated HMGA2 gene silencing could inhibit the proliferation,migration and invasion of SK-MES-1 cell line,but has no effect on apoptosis of the cells.HMGA2 perhaps is involved in the tumorigenesis and development of non-small lung cancer andprobably was one of the ideal targets for treating lung cancer. PartⅣSilencing of HMGA2 Gene by Stable Transfection of shRNAInhibits Growth of Xenografted HumanLung Squamous Cancer in Nude MiceObjectiveTo investigate the effect of silencing HMGA2 by short hairpin RNA(shRNA)inhuman lung squamous cancer cell SK-MES-1 on the growth of xenografted human lungsquamous cancer in nude mice.MethodsThe SK-MES-1 cells were cultured in vitro and divided into the specific interferencegroup and the negative control group,which was transfected by the plasmidpGCsi3.0U6/HMGA2 shRNA,pGCsi3.0U6/Neo/GFP/NON RNAi respectively.Cells withstable transfection were aquired by screening the above transfected cells with G418 andobserved under the fluorescence microscope.The expression of HMGA2 protein inSK-MES-1 cells in the two groups was detected by Western blot.The xenografted tumormodels were established in nude mice by subcutaneous inoculation of the above two cellswith stable transfecion.The growth of the xenografted tumor was observed,and the nudemices were sacrificed in the fifth week.The xenografted tumor was resected and weighed;pathological section was examined under microscope which was stained with hematoxylinand eosin(HE).The transplanted success rate,tumor size and quality were analyzedbetween the two groups.ResultsCells with stable transfection in the two groups screening by G418 showed greenluminescence,suggesting indireatly the expression of the plasmids in cells with stabletransfection.Compared with the negative control group,the protein expression levels ofHMGA2 in the specific interference group was(41±0.20)%,which decreased statistically(P＜0.05).3 out of 5 xenografted tumors were aquired in the specific interference group,but4 out of 5 xenografted tumors in the negative control group,there was no statisticallydifference in the transplanted success rate between the two groups.The volume of thexenografted tumors was(0.147±0.058)cm~3 vs(0.324±0.069)cm~3 in the specific interference group and negative control group respectively.The quality of the xenograftedtumors was(0.109±0.027)mg vs(0.238±0.045)mgin the specific interference group andnegative control group respectively.There was statistically difference in the volume andquality of the xenografted tumors between the two groups.Some nested of cancer cellswere obverved in the pathological section with HE stains under the microscope.ConclusionsSilencing HMGA2 by short hairpin RNA(shRNA)in human lung squamous cancercell SK-MES-1 can decrease the protein expression level of HMGA2 and inhibits growth ofxenografted human lung squamous cancer in nude mice. Expression of Syk in non-small cell lung cancer and itsrelationshipwith clinicopathological parametersObjectiveThis study aims to research the expression of spleen tyrosine kinase(Syk)innon-small cell lung cancer(NSCLC)and the relationship between Syk andclinicopathologic factors and p53.MethodsImmunohistochemistry was applied to detect the expressions of Syk and p53 protein in39 cases of NSCLC(23 cases of lung squamous cell cancer,16 cases of lungadenocarcinoma)and tumor-surrounding normal lung tissues.ResultsThe positive rates of Syk were 46.15 %(18/39),100%(39/39)in NSCLC andtumor-surrounding normal lung tissues respectively.The expression of Syk in NSCLC wassignificantly lower than that in tumor-surrounding normal lung tissues(P = 0.000).The Sykexpression was positively correlated to p53 expression in NSCLC specimens(P=0.025).There was no significant association between Syk expression and lymph node metastasis,differential degree,tumor size and TNM.ConclusionsThe present study demonstrated that Syk was aberrant expressed in the NSCLC andmay have a significant impact on tumor growth and progression.