World J Gastroenterol  19:1239 (2013)

Effect of silencing of high mobility group A2 gene on gastric cancer MKN-45 cells.

Wei CH, Wei LX, Lai MY, Chen JZ, Mo XJ.


摘要:

AIM:
To investigate the effect of high mobility group A2 () gene silencing on gastric cancer MKN-45 cells .

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 , 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 ( < 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 0.069 ± 0.003, 0.067 ± 0.05), Akt (0.248 ± 0.004 0.489 ± 0.006, 0.496 ± 0.104) and Bcl-2 (0.295 ± 0.084 0.592 ± 0.072, 0.594 ± 0.109) were significantly reduced. The protein expression levels of P27 (0.151 ± 0.010 0.068 ± 0.014, 0.060 ± 0.013) and caspase-9 (0.136 ± 0.042 0.075 ± 0.010, 0.073 ± 0.072) were significantly upregulated.

CONCLUSION:
shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.

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