| Objectives Lung cancer is one of the world’s most common respiratory cancers in recent years, of which the incidence and mortality have risen. In 2008 Epidemiology Statistics found that the age of onset of lung cancer had become increasingly younger, the average of which was 5 to 10 years earlier. Over the years, many researchers and clinicians dedicated to the research of lung cancer, having made early diagnosis and comprehensive treatment of lung cancer improved significantly. However, prevention and treatment of lung cancer are still difficult and there is no efficient treatment yet. Overall treatment effects and long-term prognosis of patients with lung cancer are still poor. In recent years, more and more scholars have concerned about gene therapy because of its characteristics of efficient, specific and strong targeted. Currently in vitro, the pre-clinical trials of lung cancer gene therapy have achieved satisfactory results. However, long-term efforts are needed if we want to apply it to clinical treatment of lung cancer.PDCD4 is a kind of tumor suppressor gene that is discovered in the year of 1995. It can suppress tumor development mainly through inhibiting transcription and translation of the tumor cell, as well as through upregulating the expression of p21. Existing studies have shown that loss of expression or down-regulation of PDCD4 in lung cancer cells are widespread. Therefore, there are some issues that are very worthwhile to explore:(1)If the exogenous tumor suppressor gene PDCD4 transfected into lung cancer cell lines can be expressed successfully? (2)What is the direct impact on cell morphology and cell cycle of lung cancer after the expression of PDCD4? In this study, after PDCD4 is transfected into lung cancer cell lines, we will detect its expression and observe its effect on cell morphology, the growth rate and the cell cycle of lung cancer cell lines. And then our study could provide basis for the gene therapy of lung cancer.Methods1. PDCD4 eukaryotic expression vector and empty vector were transfected into lung cancer cell lines respectively, then use the fluorescence microscopy to observe the changes of growth and morphological of lung cancer cell lines.2. The analysis of PDCD4 expression after transfection was by FQ-PCR and Western blot.3. By cell counting method, analyze the changes of PDCD4 lung cancer cell growth rate after transfection.4. The analysis of lung cancer cell growth cycle after transfection was by flow cytometry.Results1. Exogenous PDCD4 mRNA and protein were successfully expressed in lung cancer cell lines (A549).(1) PDCD4 mRNA expression was detected by FQ-PCR. The expression of non-transfected lung cancer cell lines mRNA was regarded as normalized standard. Repeat the procedure three times and the results were as follows: the expression of A549-PDCD4 mRNA was 45.99±8.705,27.82±6.114 and 48.35±2.024, the expression of A549-vector mRNA was 0.4628±4.67E-2,0.3252±6.40E-2 and 0.4679±1.718E-2. Put the triplicate experimental data into pairwise comparisons(ais corrected to 0.05/3=0.017) and the results indicated that there was no significant difference among the three groups (P>0.017).(1) PDCD4 protein expression was detected by Western blot. The results were as follows:the expression of non-transfected lung cancer cell and A540-vector protein were relatively low, while the expression of A549-PDCD4 protein was relatively high.In summary, exogenous PDCD4 gene was stably expressed in lung cancer cell lines.2. Expression of exogenous PDCD4 can alter morphology of lung cancer cell lines.Non-Transfected and transfected with empty vector lung cancer cells have grown well and have normal morphology. But the lung cancer cells which were transfected with PDCD4 gene have some significant changes in cell numbers and morphology:The number of cells was significantly reduced compared with the control group; The volume of most lung cancer cells reduced; The cell transferred into round shapes whose protrusions disappeared.3. Expression of exogenous PDCD4 can inhibit the growth rate of lung cancer cells.The day before transfection, put the lung cancer cells into six-well plates with proper density (5×105 per well). After 48h, three kinds of lung cancer cells (non-transfected A549, A549-vector and A549-PDCD4) were counted. Repeat the procedure three times and the results were as follows:A549-PDCD4: 5.27×105±0.68×105;A549-vector:8.90×105±0.40×105;A549:9.40×105±0.79×105 Put the triplicate experimental data into pairwise comparisons (ais corrected to 0.05/3=0.017) and the results indicated that the A549-PDCD4 cell number was smaller significantly than the other two cell lines (P<0.017). However, there was no significant difference between A549-vector and non-transfected A549 (P> 0.017). Exogenous PDCD4 gene can inhibit the growth rate of lung cancer cells.4. Expression of exogenous PDCD4 can block the cell cycle of lung cancer cells.We use flow cytometry to analyze the lung cancer cell cycle after transfection. The results were as follows:The proportion of A549-PDCD4 cells in G0G1 phase (79.92% ± 2.72%) was larger than that of A549-vector cells (67.7% ± 4.56%) and non-transfected A549 cells (69.52% ± 3.76%) (P<0.017). And there was no significant difference between A549-vector cells and non-transfected cells in G0G1 phase (P> 0.017). Whereas the proportion of A549-PDCD4 cells in S+G2M phase(20.07%±1.91%)was smaller than that of A549-vector cells (32.3%±2.37%) and non-transfected A549 cells (30.48%±4.86%) (P<0.017). And there was no significant difference between A549-vector cells and non-transfected cells in G0G1 phase (P> 0.017) either. The results indicates that exogenous PDCD4 can block the cell cycle of lung cancer cells and make cell cycle arrest in G0G1 phase, which can inhibit the cell from proliferating. This result has important clinical value for reducing the degree of malignancy of lung cancer and improving patients prognosis by upregulating the expression of PDCD4.Conclusion PDCD4 can change the morphology of lung cancer, inhibit the growth rate and block the cell cycle, which may be one of important mechanisms that PDCD4 suppress lung cancer development. Our study provides an important experimental basis for the PDCD4 targeting gene therapy applying to clinical work. With the discovery of PDCD4 and other tumor suppressor genes, and gene therapy in the treatment of malignant tumors will become an important part in the prevention and treatment of tumor occurrence, metastasis and relapse. |
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