Down-regulation Of TSPYL5 Gene By Small Interfering RNA Induce Apoptosis Of A549 Cell

Objective: Lung cancer is the highest incidence of cancer in recent years, In industrialized countries, 29% of all patients die of cancer.In our country, the incidence of lung cancer is also found in a rising trend and is in the first place of various types of cancer. In recent years, the treatment of lung cancer is mainly by chemotherapy and radiotherapy, but those drugs and measures bring great pain to the patients. Therefore, we should study from the perspective of lung cancer genes, and to find effective mechanisms for the development of therapeutic targets. The development of modern molecular biology techniques and their applications for cancer gene therapy has opened a new way. In recent years, the role of class Y chromosome genes encoding testis-specific protein (TSPY-like) in cancer development and is paid much attention. TSPYL is named for the encoding of a group proteins which are highly similar with the Y chromosome testis-specific protein (Testis-specific protein Y encoded, TSPY) protein, the gene locates on chromosome 8q22.1, from of 437 amino acids, the molecular weight being of about 45kDa, containing 6 exons,which is expressed in the human lung, stomach and brain tissue and the site is closely related to tumor development. In addition, the gene play a more important role in maintaining cell morphology, viability and stability of the nervous system, embryonic development is also more closely related. Recent studies have found that members of his family TSPYL5 in lung adenocarcinoma A549 cells can affect the level of protein expression p21WAF1/Cip1, p21WAF1/Cip1 protein is an important regulator of cell cycle proteins, so TSPYL5 may play an important role in the regulation of cell cycle proteins which may has an improtant relationship with the development and promote of lung cancer, but the specific molecular mechanism is not very clear. This study aims to tranfect TSPYL5 siRNA to human adenocarcinoma A549 cells with liposomes,to observe the morphological changes of A549 cells apoptosis by flow cytometry, TSPYL5 gene mRNA by RT-PCR, TSPYL5 protein level by immunofluorescence.Methods:TSPYL5 siRNA gene fragments were synthetized and transfected into A549 cell by liposome.Cell morphological change was observed with microscope, cell proliferation and apoptosis index was detected by flow cytometry, TSPYL5mRNA expression determined by RT-PCR and protein expression of TSPYL5 was revealed by immunofluorescence analysis.Results:Cell morphological change was observed with microscope. Before transfection,the growth of cell was adherent, being in good condition, most of the cells were spindle-shaped and medium size, with the nucleolus being clear. After 48 hours of transfection,the cell became irregular shape, shrinking, poor wall adherent. Apoptosis was detected by flow cytometry: the apoptotic rate was in the siRNA transfection group(27.71±0.46)%, in the blank control (0.37±0.40)%, and in the negative control (0.47±0.37)%. There was no obvious difference between the blank control and the negative control(P>0.05),but there is obvious difference between the siRNA group and the two control groups(P<0.001).This showed that TSPYL5 siRNA could induce the apoptosis of the lung cancer A549 cells. RT-PCR analysis showed that there are two straps of 219 bp (TSPYL5) and 404 bp(β-actin) respectively.The straps ofβ-actin are similar and clear.The strap of the TSPYL5 siRNA group was weaker than the other groups.The Labworks revealed that mRNA levels of A549 were notably lower in TSPYL5 silencing group than in the blank and negative controls.The IOD ratio of TSPYL5t/β-actin of the blank control was 1.589±0.09,the negative control was 1.583±0.074,There was no significant difference between the blank control and the negative contro(lP>0.05). The siRNA group was 1.085±0.006,and there was significant difference between the siRNA group and the two controls group(P=0.000). This showed that TSPYL5 siRNA can induce the degradation of TSPYL5mRNA.Conclusion:Using RNA interference technology to silence TSPYL5 gene can significantly promote the apoptosis of A549 cell .This provides foundation for further research using RNA interference technology for the biological treatment of lung cancer foundation.