title: Silencing of high mobility group A1 enhances gemcitabine chemosensitivity of lung adenocarcinoma cells.
abstract: BACKGROUND
  The high mobility group A1 (HMGA1) proteins are architectural transcription factors found to be overexpressed in lung adenocarcinoma. Lentivirus-mediated RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in human cancer cells. Our preliminary study shows that gemcitabine inhibits growth of the human lung cancer cell line SPCA-1 and induces apoptosis, and this effect might link with down-regulation of HMGA1 expression. This study aimed to investigate the chemosensitivity change of the lung adenocarcinoma cells SPCA-1 after HMGA1 inhibition by lentivirus-mediated RNAi.
  
  
  METHODS
  We studied a highly malignant lung adenocarcinoma cell line (SPCA-1 cells). Lentiviral short-hairpin RNA (shHMGA1) expression vectors targeting HMGA1 were used for generation of lentiviral particles. After being transfected into the lung adenocarcinoma cell line SPCA-1, the expression of HMGA1 was determined by retrotranscriptase polymerase chain reaction (RT-PCR) and Western blotting. The effect of gemcitabine on proliferation of positive and negative cells was observed by methyl thiazolyl tetrazolium (MTT) assay and clonogenic survival assay. Apoptosis was observed by flow cytometery. Chemosensitivity to gemcitabine was determined by IC50 analysis. Caspase activity was quantitated by a caspase colorimetric protease assay kit.
  
  
  RESULTS
  HMGA1-siRNA silenced its target mRNA specifically and effectively in SPCA-1 cells. The apoptotic rates of the scramble control group were (7.43 ± 0.21)%, (11.00 ± 0.20)%, and (14.93 ± 0.31)%, and the apoptotic rates in the silenced group were (9.53 ± 0.42)%, (16.67 ± 0.45)%, and (25.40 ± 0.79)% under exposure to 0.05, 0.5 and 5.0 µg/ml of gemcitabine (P < 0.05). The IC(50) of the silenced group was (0.309 ± 0.003) µg/ml which was significantly lower than in the scramble control group, (0.653 ± 0.003) µg/ml (P < 0.05). It reduced cancer cell proliferation and increased apoptotic cell death after being treated with gemcitabine compared with the scramble control group. HMGA1 silencing resulted in reduction in the phosphorylation of Akt, and promoted the activation of caspases 3, 8 and 9 upon exposure to gemcitabine.
  
  
  CONCLUSIONS
  Lentivirus-mediated RNA interference of HMGA1 enhanced chemosensitivity to gemcitabine in lung adenocarcinoma cells. The mechanism may be associated with the PI-3K/Akt signal pathway. HMGA1 may represent a novel therapeutic target in lung cancer.
authors.affiliation: Department of Oncology, Zhong Da Hospital Affiliated to Southeast University, Nanjing, Jiangsu 210009, China.
authors.forename: Yuan-Dong
authors.initials: YD
authors.lastname: Cao
authors.forename: Pei-Lin
authors.initials: PL
authors.lastname: Huang
authors.forename: Xin-Chen
authors.initials: XC
authors.lastname: Sun
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.forename: Zhi-Liang
authors.initials: ZL
authors.lastname: Jin
authors.forename: Hong-Yan
authors.initials: HY
authors.lastname: Cheng
authors.forename: Rui-Zhi
authors.initials: RZ
authors.lastname: Xu
authors.forename: Fan
authors.initials: F
authors.lastname: Li
authors.forename: Shu-Kui
authors.initials: SK
authors.lastname: Qin
authors.forename: Yu-Xia
authors.initials: YX
authors.lastname: Deng
authors.forename: Xiao-Lin
authors.initials: XL
authors.lastname: Ge
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D024003:HMGA Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D024003:HMGA Proteins; D003841:Deoxycytidine; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D000093542:Gemcitabine