title: Effect of silencing of high mobility group A2 gene on gastric cancer MKN-45 cells.
abstract: OBJECTIVE
  To investigate the effect of high mobility group A2 (HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.
  
  
  METHODS
  HMGA2 short hairpin RNA (shRNA) expression plasmids were constructed, including a pair of random scrambled sequences. Human gastric cancer cell line MKN-45 cells were divided into three groups: blank control group (non-transfected cells), transfected group (cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group (transfected with random scrambled plasmid). Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro, and the cells transfection efficiency was assayed by fluorescence microscopy. The HMGA2 messenger RNA (mRNA) expression was detected by reverse transcription polymerase chain reaction, gastric cancer cells apoptosis was detected by flow cytometry, cell proliferation was detected by methyl thiazol tetrazolium, and the protein expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), P27, caspase-9 and B-cell leukemia/lymphoma-2 (Bcl-2) were analyzed by Western blotting.
  
  
  RESULTS
  Compared with the blank control group and the scrambled sequence group, the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced (P < 0.05). The relative HMGA2 mRNA expression levels of the blank control group, transfected group and scrambled sequence group were 0.674 ± 0.129, 0.374 ± 0.048 and 0.689 ± 0.124, respectively. The relative HMGA2 protein expression levels of the blank control group, transfected group and scrambled sequence group were 0.554 ± 0.082, 0.113 ± 0.032 and 0.484 ± 0.123, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of HMGA2. After being transfected with shRNA for 24, 48 and 72 h, the cell apoptotic rates of the transfected group were 21.65% ± 0.28%, 39.98% ± 1.82% and 24.51% ± 0.93%, respectively, which significantly higher than those of blank control group (4.72% ± 1.34%, 5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group (4.28% ± 1.33%, 7.87% ± 1.43% and 6.71% ± 0.92%). After 24, 48 and 72 h, the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%, 39.45% ± 2.07% and 37.56% ± 2.32%, respectively; the most obvious cell proliferation inhibition appeared at 48 h after transfection. Compared with the blank control group and scrambled sequence group, after transfection of shRNA for 72 h, the protein expression levels of PI3K (0.042 ± 0.005 vs 0.069 ± 0.003, 0.067 ± 0.05), Akt (0.248 ± 0.004 vs 0.489 ± 0.006, 0.496 ± 0.104) and Bcl-2 (0.295 ± 0.084 vs 0.592 ± 0.072, 0.594 ± 0.109) were significantly reduced. The protein expression levels of P27 (0.151 ± 0.010 vs 0.068 ± 0.014, 0.060 ± 0.013) and caspase-9 (0.136 ± 0.042 vs 0.075 ± 0.010, 0.073 ± 0.072) were significantly upregulated.
  
  
  CONCLUSIONS
  HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.
authors.affiliation: Department of Geriatric Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China.
authors.forename: Chun-Hui
authors.initials: CH
authors.lastname: Wei
authors.forename: Li-Xiu
authors.initials: LX
authors.lastname: Wei
authors.forename: Ming-Yu
authors.initials: MY
authors.lastname: Lai
authors.forename: Jia-Zhuang
authors.initials: JZ
authors.lastname: Chen
authors.forename: Xi-Jing
authors.initials: XJ
authors.lastname: Mo
mesh_terms: D017209:Apoptosis; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D025743:HMGA2 Protein / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014162:Transfection
chemical_list: C436291:CDKN1B protein, human; D025743:HMGA2 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D050760:Cyclin-Dependent Kinase Inhibitor p27; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; C506433:CASP9 protein, human; D053453:Caspase 9
keywords: Apoptosis; Gastric cancer cells; High mobility group A2; Proliferation; RNA interference