Effects Of F10 Gene On The Apoptosis, Chemosensitivity And Tumorigenicity Of A549 Cells

Background and objectiveIn our previous research of "973 project", we found a new function-unknown gene F10 (GenBank accession number:AB196290) from the difference library of cDNA between hydatidiform mole tissues and normal villi of early pregnancy by using suppressed subtractive hybridization techque. Because this gene was kept in the F10 location of 96-well plate, we tentatively named the gene as F10. Previous studies showed that the expression of F10 gene was positive and its intensity increased gradually in hydatidiform mole, invasive mole and choriocarcinoma. Moreover, the expression of F10 gene was positive in ovarian cancer and endometrial cancer. But F10 gene was negative in some normal tissues, such as endometrium and cervical epithelium. From the above results, we can conclude that F10 gene may be a new gene related to the malignant transformation of hydatidiform mole and the occurrence of gynecological cancer. At present, gynecological cancer is still a major threat to women's health. F10 gene may be a new tumor-associated gene, so it is significant to study its function for exploring pathogenesis of gynecological malignancy and guiding clinical treatments. However, as far as F10 gene function is concerned, it is just in the stage of initial exploratory, so further studies on F10 gene function is necessary.Although the function of F10 gene is not clear entirely, it has been strongly implicated that F10 gene may participate in the occurrence and development of tumors by promoting cell proliferation ability which is subject to raise the expression of proliferating cell nuclear antigen and cyclinDl. The expression of F10 gene is low in human lung cancer A549 cell. Most of scholars believe that tumorigenicity can be promoted when cell apoptosis is inhibited. Then whether F10 gene is involved in the process of apoptosis regulation of tumor cells or not? This problem offers us a new research direction for F10 gene function.Apoptosis is a complex process regulated by a variety of genes and enzymes, includes a series of signal transduction systems. Some apoptosis-related indicators can not only reflect effects of F10 gene in apoptosis of tumor cells, but also help us clarify the mechanism of F10 gene in the process of tumor formation. Cysteinyl aspartate-specific protease (CASPASE) family, one of the important components in apoptosis regulation, participates in a number of apoptosis-related physiological and pathological processes. CASPASE-3 is an important member in this family. Up-regulation of CASPASE-3 expression shows the number of apoptosis cells increase and down-regulation of CASPASE-3 expression shows the number of apoptosis cells reduce. B-cell lymphoma/leukemia-2 (BCL-2) family, one of the most important gene families associated with apoptosis, is divided into two types of anti-apoptosis and pro-apoptosis. In this family, BCL-2 and BAX are two function-opposite members and the levels of their expression have a major impact on cell survival.Gene transduction technique is a mature method and used widely in gene function research. A proper gene transduction system plays a key role in transduction. Enhanced green fluorescent protein (EGFP) is an optimized GFP. It can form a distinct green fluorescence in 488nm excitation wave. In tissues and cells, EGFP can tolerate various treatments while maintaining light-emitting property. pEGFP-N1, a eukaryotic expression vector, can carry a gene and recognize a target gene efficiently. Moreover, it is easy to operate. So it is an ideal vector and used widely. These theories provide us a reliable foundation to study F10 gene function.In this study, we decided to explore the following contents:(1)We used A549 cell as the cell model and pEGFP-N1 as the eukaryotic expression vector to construct a stable over-expression system of F10 gene in A549 cell line by gene transduction technology. (2) On this basis, we observed effects of F10 over-expression on the apoptosis of A549 cells and explored its mechanism. (3) We selected TAXOL as the chemotherapy drug to study effects of F10 over-expression on the chemosensitivity of A549 cells.(4) We inoculated A549 cells with F10 over-expression into nude mice to observe effects of F10 gene on the tumorigenicity of A549 cells. Meanwhile, we detected three apoptosis-related factors of CASPASE-3, BAX and BCL-2 to explore its mechanism.This study is an original and innovative research. By studying effects of F10 gene on the apoptosis, chemosensitivity and tumorigenicity of A549 cells, we hope get some new thoughts on the F10 gene function and provide some theoretical supports in developing new therapeutic ways for gynecological tumors.Chapter 1:Construction and identification of stable A549 cell line with F10 over-expressionF10 gene is a new gene and its function is unknown. In lung cancer A549 cells, the expression of F10 gene is low. pEGFP-N1 vector contains EGFP, which can form a distinct green fluorescence in 488nm excitation wave and indicate gene expression. Moreover, it has an efficient promoter, a strong copy power, multiple cloning sites and neo marker. These special structures make it be a eukaryotic expression vector which can carry a gene and recognize a target gene efficiently.Based on these theories, we transfected the exogenous F10 gene into A549 cells by gene transduction technology with pEGFP-N1 vector. We hope construct a stable A549 cell line with F10 over-expression to provide an experimental platform for the F10 gene function research.MethodsThe recombinant plasmids of pEGFP-Nl-F10 and pEGFP-N1-Mock were designed and constructed. The two plasmids were identified by double-digestion of EcoR I and Kpn I and sequencing DNA. Then they were transfected into A549 cell lines, which positive monoclonal cells were selected by G418 and identified by fluorescence quantitative PCR.Results1. Identification of pEGFP-N1-F10 plasmid:A clear band was obtained on the 888bp site after pEGFP-N1-F10 plasmid was double-digested. The result of sequencing DNA of F10 gene was consistent with the corresponding sequence published in GenBank. Furthermore, the Blast comparative analysis showed this sequence was identical with the F10 genome sequence.2. Observation of A549 cells with stable over-expression of F10 gene under fluorescence microscopy:The recombinant plasmids of pEGFP-N1-F10 and pEGFP-N1-Mock were transfected into A549 cell line. After G418 selection for 4 weeks, survival cells were isolated and cultured continuously for 1 week. We observed that green fluorescence existed in the two groups of A549 cells and the transfection efficiencies were nearly 100%.3. Detection the mRNA levels of F10 gene in positive clone cells by fluorescence quantitative PCR:The expression of F10 mRNA (16192.710±4061.873) in the F10+A549 group was higher than that in Mock-A549 (1.270±0.210), which the difference was statistically significant (t=-6.904, P=0.002). These results showed that F10 gene had been integrated into the A549 cells and expressed efficiently.Summary1. The recombinant plasmid of pEGFP-Nl-F10 was designed and constructed. By enzyme digestion and sequencing, F10 gene was confirmed to be inserted into pEGFP-N1 vector successfully.2. We transfected the pEGFP-N1-F10 recombinant plasmid into A549 cells with low-expression of F10 gene. The positive monoclonal cells were selected by G418 and identified by fluorescence quantitative PCR. The mRNA levels of F10 gene in the over-expression group were significantly higher than those in the negative control group. These results suggested that a stable A549 cell line with F10 over-expression was constructed successfully. This provided us an experimental platform for further studies on F10 gene function.Chapter 2:Effects of F10 over-expression on the apoptosis of A549 cellsApoptosis is a complex process regulated by various genes and enzymes. CASPASE-3 and BAX are two important factors. In recent years, researchers have found that the incidence and development of tumors are closely related to suppression of apoptosis. Based on these theories, we used the stable A549 cell line with F10 over-expression to observe effects of F10 over-expression on the apoptosis of A549 cells and explored its mechanism by detecting the expression of CASPASE-3 and BAX.MethodsA549 cells were divided into three groups:F10+A549 cells as experimental group, Mock-A549 cells as negative control group and A549-WT cells as blank control group. Cell proliferation ability was examined by MTT. Cell apoptosis was tested by TUNEL-FITC/Hoechst33258. The mRNA and protein levels of BAX and CASPASE-3 were detected by fluorescence quantitative PCR and Western blot. Subsequently, we compared the change of parameters among the three groups by factorial designed ANOVA and one-way ANOVA.Results1. Detecting cellular proliferation ability by MTT:In the three groups of A549-WT, Mock-A549 and F10+A549, the cellular proliferation capacity showed a significant difference (F=48.039, P=0.000). The main effect of time was significantly different (F=323.264, P=0.000). The interaction effect between time and group had a significant difference (F= 11.442, P=0.000). With the time growing, the difference of cellular proliferative capacity in the three groups was significant. From the 12nd hour, F10+A549 cells, compared with Mock-A549 cells and A549-WT cells, showed faster growth, while there was no significant difference between A549-WT cells and Mock-A549 cells.2. Detecting cellular apoptosis by TUNEL-FITC/Hoechst33258:In A549-WT cells (5.800±1.643), Mock-A549 cells (5.600±1.140) and F10+A549 cells (1.400±1.140), the number of positive cells by TUNEL-FITC/Hoechst 33258 had a significant difference (F=17.472, P=0.000). In F10+A549 cells, the number of positive cells was less than that in A549-WT cells and Mock-A549 cells, while there was no significant difference between A549-WT cells and Mock-A549 cells. These results showed that apoptosis cells in A549-WT cells and Mock-A549 cells were significantly more than those in F10+A549 cells.3. Detecting the mRNA levels of BAX and CASPASE-3 by RT-PCR:In A549-WT cells (0.754±0.018; 6.603±0.172), Mock-A549 cells (0.702±0.049; 6.920±0.188) and F10+A549 cells (0.159±0.035; 1.332±0.187), the mRNA levels of BAX and CASPASE-3 had statistically significant differences (F=245.201, P= 0.000; F=889.634, P=0.000).The mRNA levels of BAX and CASPASE-3 in F10+A549 cells were lower than those in A549-WT cells and Mock-A549 cells, while there were no significant differences between A549-WT cells and Mock-A549 cells.4. Detecting the protein levels of BAX and CASPASE-3 by Western blot:In A549-WT cells (0.370±0.001; 0.367±0.020), Mock-A549 cells (0.360±0.022; 0.385±0.009) and F10+A549 cells (0.172±0.032; 0.165±0.005), the protein levels of BAX and CASPASE-3 had statistically significant differences (Welch=45.966, P=0.008; F=268.790, P=0.000).The protein levels of BAX and CASPASE-3 in F10+A549 cells were lower than those in A549-WT cells and Mock-A549 cells, while there were no significant differences between the A549-WT cells and Mock-A549 cells.Summary1. Compared with negative control group and blank control group, the growth speed of A549 cells in experimental group increased rapidly. This suggested that F10 gene may promote A549 cell proliferation. At the same time, apoptosis cells in negative control group and blank control group were significantly more than those in experimental group. This suggested that the over-expression of F10 gene may inhibit A549 cell apoptosis.2. The mRNA and protein levels of BAX and CASPASE-3 in experimental group were lower than those in negative control group and blank control group, indicating that F10 gene may down-regulate the expression of CASPASE-3 and BAX in A549 cells. These results suggested that the over-expression of F10 gene may inhibit A549 cell apoptosis, in which CASPASE-3 and BAX may participate in the regulation of apoptosis.Chapter 3:Effects of F10 over-expression on TAXOL chemosensitivity of A549 cellsChemotherapy is an important method to treat cancers. The traditional concept of cancer treatment is that chemotherapy drugs kill tumor cells by a direct effect on nucleic acids, tubulin protein and other targets. Now, people recognize that chemotherapy drugs can also induce apoptosis to kill cancer cells. TAXOL is a kind of chemotherapy drugs playing an important role in anti-tumor effect by apoptosis. In recent years, it has been discovered and widely used in lung cancer. Based on the above theories, we used the stable A549 cells with F10 over-expression and selected TAXOL as the chemotherapy drug to observe effects of F10 over-expression on the chemotherapeutic sensitivity. Meanwhile, we detected the expression of CASPASE-3 and BAX to explore its mechanism.MethodsA549 cells were divided into three groups:F10+ A549 cells as experimental group, Mock-A549 cells as negative control group and A549-WT cells as blank control group. All cells were treated with TAXOL. Cell proliferation ability was examined by MTT. Cell apoptosis was tested by TUNEL-FITC/Hoechst33258. The mRNA and protein levels of BAX and CASPASE-3 were detected by fluorescence quantitative PCR and Western blot. Subsequently, we compared the change of parameters among the three groups by factorial designed ANOVA and one-way ANOVA.Results1. Detecting cellular proliferation ability by MTT:In the three groups of A549-WT +TAXOL,Mock-A549+TAXOL and F10+A549+TAXOL,the cellular proliferative capacity showed a significant difference (F=75.926, P=0.000).The main effect of time was significantly different(F=270.007, P=0.000). The interaction effect between time and group had a significant difference (F=8.815, P=0.000). With the time growing, the difference of cellular proliferation capacity in the three groups was significant. From the 12nd hour, A549 cells in the two groups of A549-WT+TAXOL and Mock-A549+TAXOL, compared with the group of F10+A549+TAXOL, showed slower growth, while there was no significant difference between the A549-WT+TAXOL group and the Mock-A549+TAXOL group.2. Detecting cellular apoptosis by TUNEL-FITC/Hoechst33258:In the three groups of A549-WT+TAXOL (40.200±3.114), Mock-A549+TAXOL (38.200±2.775) and F10+A549+TAXOL (9.800±2.049), the number of positive cells by TUNEL-FITC/Hoechst33258 had a significant difference (F=200.778, P=0.000). In the group of F10+A549+TAXOL, the number of positive cells was less than that in the two groups of A549-WT+TAXOL and Mock-A549+TAXOL, while there was no significant difference between the A549-WT+TAXOL group and the Mock-A549+TAXOL group. These results showed that after A549 cells were treated with TAXOL, apoptosis cells in the two groups of A549-WT and Mock-A549 were significantly more than those in the F10+A549 group.3. Detecting the mRNA levels of BAX and CASPASE-3 by RT-PCR:In the three groups of A549-WT+TAXOL (0.695±0.162; 6.779±0.736), Mock-A549 +TAXOL (0.873±0.093; 7.751±0.436) and F10+A549+TAXOL (0.253±0.008; 0.528±0.009), the mRNA levels of BAX and CASPASE-3 had statistically significant differences (Welch=61.749, P=0.006; F=188.849, P=0.000). The mRNA levels of BAX and CASPASE-3 in the F10+A549+TAXOL group were lower than those in the A549-WT+TAXOL group and the Mock-A549+TAXOL group, while there were no significant differences between A549-WT+TAXOL and Mock-A549+TAXOL4. Detecting the protein levels of BAX and CASPASE-3 by Western blot:In the three groups of A549-WT+TAXOL (0.976±0.005; 0.553±0.108), Mock-A549+ TAXOL (0.989±0.007; 0.629±0.063) and F10+A549+TAXOL (0.743±0.020; 0.067±0.024), the protein levels of BAX and CASPASE-3 had statistically significant differences (Welch=166.892, P=0.000; F=51.231, P=0.000).The protein levels of BAX and CASPASE-3 in the F10+A549+TAXOL group were lower than those in the A549-WT+TAXOL group and the Mock-A549+TAXOL group, while there were no significant differences between A549-WT+TAXOL and Mock-A549+TAXOL.Summary1. A549 cells in each group were treated with TAXOL. Cellular proliferation ability was detected by MTT. Apoptosis was tested by TUNEL-FITC/Hoechst33258. Compared with negative control group and blank control group, the growth speed of A549 cells in experimental group increased rapidly and apoptosis cells reduced. This suggested that F10 over-expression may inhibit TAXOL chemosensitivity of A549 cells.2. The mRNA and protein levels of BAX and CASPASE-3 in experimental group were lower than those in negative control group and blank control group, indicating that F10 gene may down-regulate the expression of CASPASE-3 and BAX in A549 cells treated with TAXOL. These results suggested that the over-expression of F10 gene down-regulate the expression of CASPASE-3 and BAX, which may further inhibit TAXOL chemosensitivity of A549 cells.Chapter 4:Effects of F10 over-expression on the tumorigenicity of A549 cellsSince we found F10 gene, our previous researches have focused on the cellular level. However, the nude mice experiment can imitate an environment in human body, which we can observe effects of F10 gene on the tumorigenicity of tumor cells in live animals. Therefore, we inoculated A549 cells with over-expression F10 into nude mice to observe effects of F10 gene on the tumorigenicity of A549 cells and explore its mechanism.MethodsA549 cells with over-expression F10 were used to produce animal models of lung cancer in nude mice. Tumorigenic mice were randomly divided into the three groups of A549-WT, Mock-A549 and F10+A549. Through the nude mice experiments, we observed effects of F10 gene on the tumorigenicity of A549 cells in nude mice. Meanwhile, we detected the protein expression of three apoptosis-related factors of CASPASE-3, BAX and BCL-2 by immunohistochemistry. Subsequently, we compared the change of parameters among the three groups by repeated measures ANOVA and one-way ANOVA.Results1. The tumor formation rate and the tumor formation time:The tumor formation rates in three groups were all 100%. In the groups of A549-WT, Mock-A549 and F10+A549, the tumor formation time in nude mice had a significant difference (F=13.523, P=0.000). The tumor formation time in the two groups of A549-WT and Mock-A549 was longer than that in the F10+A549 group, while there was no significant difference between A549-WT and Mock-A549.2. General observation of subcutaneous tumor tissues:The tumor volume in the F10+A549 group was increased significantly, compared with that in the two groups of A549-WT and Mock-A549.3. The growth rate analysis of subcutaneous tumor:The main effect in the different groups had a significant difference (F=23.140, P=0.000). When F10+A549 cells were compared with A549-WT cells and Mock-A549 cells, the tumor volume at the different times had a significant difference (F=350.516, P=0.000). The interaction effect between time and group had a significant difference (F=216.835, P=0.000).The growth rate in the F10+A549 group was increased significantly, compared with that in the two groups of A549-WT and Mock-A549, while there was no significant difference between A549-WT and Mock-A549.4. HE staining and slicing:In the two groups of A549-WT and Mock-A549, tumor tissues appeared more necrotic cells, which HE staining showed homogeneous red staining and amorphous material structure. But this phenomenon was not observed in the group of F10+A549 cells.5. Analyzing the protein expression of BCL-2, BAX and CASPASE-3 in subcutaneous tumor tissues by immunohistochemistry:In the two groups of A549-WT and Mock-A549, the expression of BCL-2 was almost none. However, in the group of F10+A549 cells, the expression of BCL-2 was more and positive cells showed diffuse distribution. In the two groups of A549-WT and Mock-A549, the expression of BAX and CASPASE-3 was strong, especially in the group of A549-WT cells. But the expression of BAX and CASPASE-3 was weak in the group of F10+A549 cells.SummaryWe inoculated A549 cells with over-expression F10 into nude mice and found that in the group of F10+A549 cells, the tumor formation time reduced, the tumor volume increased, the tumor growth accelerated. Immunohistochemistry showed a relatively low level of CASPASE-3 and BAX and a high level of BCL-2. This suggested that F10 gene may down-regulate the expression of CASPASE-3 and BAX and up-regulate the expression of BCL-2, which further enhance the tumorigenicity of lung cancer cell line A549 in nude mice.Conclusions1. A stable A549 cell line with F10 over-expression was successfully constructed by pEGFP-N1 vector. This provided us an experimental platform to study the function of F10 gene.2. By MTT, TUNEL-FITC/Hoechst33258, RT-PCR and Western blot, we found that F10 gene may promote A549 cell proliferation, inhibit A549 cell apoptosis and down-regulate the expression of CASPASE-3 and BAX in A549 cells. These results suggested that the over-expression of F10 gene may inhibit A549 cell apoptosis.CASPASE-3 and BAX may participate in the regulation of apoptosis.3. F10 gene down-regulated the mRNA and protein expression of CASPASE-3 and BAX in A549 cells treated with TAXOL. These results suggested that the over-expression of F10 gene may inhibit TAXOL chemosensitivity of A549 cells by down-regulating the expression of CASPASE-3 and BAX.4. We inoculated the A549 cells with A549 cells with over-expression F10 into nude mice and found that in the group of F10+A549 cells, the tumor formation time reduced, the tumor volume increased and the tumor growth accelerated. Immunohistochemistry showed a relatively low level of CASPASE-3 and BAX and a high level of BCL-2. This suggested that F10 gene may down-regulate the expression of CASPASE-3 and BAX and up-regulate the expression of BCL-2, which further enhance the tumorigenicity of lung cancer cell line A549 in nude mice.