Effect Of PTEN Gene On Proliferation, Apoptosis And Invasion Ability Of Multiple Myeloma Cells And Its Mechanism

Background: Multiple myeloma is a kind of incurable malignancy of end stage B-lineage cells, and characterized by an accumulation of neoplastic plasma cells in the bone marrow and invasion into other organs. Plasma leukemia develops in about 5% of MM patients as a terminal disease manifestation. Other extramedullary infiltrations are observed with increasing frequency as the overall survival of MM patients has been prolonged by current therapy. Prognosis of patients with MM remains poor despite the advanced therapeutic protocols. Invasiveness of tumor cells is not only an important indicator of the malignancy, but is also closely related with poor clinical prognosis. Migration of myeloma cells is a very harmful disease process fundamental to the invasion and dissemination of myeloma cells. However at present, little is known about the mechanisms regulating myeloma cell migration in the bone marrow and metastasizing to secondary sites. If these mechanisms are elucidated, it would be of great significance and very beneficial for looking for new treatment strategies. Extramedullary disease is almost always accompanied with complex cytogenetic abnormalities. Genetic abnormality is considered to be one of the important pathogenic factors for the genesis of MM. However, the identities of molecular alterations that allow these cancer cells to have increased metastatic potential are not clear.Phosphatase and tensin hemology deleted on chromosome ten gene (PTEN) is a recently identified tumor suppressor gene. PTEN protein has protein phosphatase and lipid phosphatase dual activity. PTEN can inhibit proliferation, induce apoptosis and regulate cell cycle progression by lipid phosphatase activity. The protein phosphatase activity of PTEN can regulate cellular bioactivities such as migration and focal adhesion, by targetting mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK) pathway and inhibiting phosphorylation of several proteins. Briefly PTEN can regulate the growth, differentiation, adhesion and cell cycle of normal cells; supress the proliferation, invasion and metastasis of tumor cells; induce apoptosis of tumor cells.Previous studies found that genetic mutation/deletion or lower expression of PTEN gene is so common in several types of human solid cancers, indicating that PTEN is one of the most frequently candidate of tumor suppressors. The function of PTEN gene in the genesis of hematopoietic malignancies such as leukemia has been focused generally. PTEN protein played an important role in the differentiation of hematopoietic stem cells and in the prevention of leukemia genesis and relapse. In MM, previous studies found hemizygous deletions of PTEN in 5% of MM patients, in 20% of plasma cell leukemias (PCL) and in 20% of human myeloma cell lines (HMCLs) as determined by inter-phase cIg-FISH methods. PTEN deletion was detected in advanced disease, such as PCL or HMCLs, suggesting that PTEN alterations occurred as a secondary change associated with disease progression in MM.However, little is known about the relationship between the genesis of MM and the activity of PTEN signal transduction pathways and whether PTEN affect the invasion ability of MM cells. To explore these questions, RPMI 8226 cells (a human MM cell line) and purified myeloma cells from MM patients were transfected with a recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP) or an adenovirus vectors only expressing green fluorescent protein (Ad-GFP). Since RPMI 8226 cells natively express the PTEN gene, we down regulated the expression of wild type PTEN by using PTEN-siRNA as a control. The effects and the mechanism of wild-type PTEN gene on the proliferation, apoptosis, cell cycle progression and invasion activity of MM cells were studied. The result of this study may provid an important theoretical basis for MM therapy with PTEN gene. This paper consists of the following three parts:Part one: Expression levels of PTEN, FAK in multiple myeloma patients and its relationship with clinical stage and extramedullary infiltrationObjective To investigate PTEN, FAK mRNA and protein levels in MM patients, analyze their relationship with clinical stage of multiple myeloma and extramedullary infiltration, and explore its role in Multiple Myeloma.Methods Bone Marrow Mononuclear Cells (BMMNCs) were isolated from 55 patients with MM and 20 Control patients. Bone marrow CD138+ plasma cells were purified from the isolated BMMNCs with an immuno-magnetic method using anti-CD138 monoclonal antibody (mAb)–coated microbeads. MM patients were grouped according to Durie-Salmon stage, with or without extramedullary infiltration. The mRNA expression levels of PTEN, FAK were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR). The expression levels of PTEN, T-FAK and p-FAK were determined by western blotting method.Results1. The expression level of PTEN mRNA was (0.723±0.059) in 55 patients of MM and (0.984±0.359) in the control group. The expression level of PTEN mRNA was significantly lower in MM patients than that in control group (P<0.05). The expression level of FAK mRNA was (1.072±0.626) in MM patients and (0.433±0.240) in control group. There was a statistically significant difference in the expression level between these two groups (P<0.01). Spearman bivariate correlation analysis showed that the expression levels of PTEN and FAK were significantly negatively correlated in MM patients (r =- 0.560, P <0.01).2. All MM patients were divided into two groups according to Durie-Salmon stage. The expression level of PTEN mRNA was (0.909±0.429) in MM patients of stageⅠ+Ⅱ(15 cases), and (0.653±0.347) in MM patients ofⅢphase (40 cases). When the expression level of PTEN mRNA in MM patients of stageⅠ+Ⅱcompared with control group, the difference was not statistically significant (P>0.05). When the expression level of PTEN mRNA in stageⅢof MM patients compared with control group, the differenc was statistically significant (P<0.01). The difference of PTEN mRNA expression level between stageⅠ+Ⅱof MM patients and stageⅢof MM patients was statistically significant(P<0.05).The expression level of FAK mRNA was (0.726±0.317) in MM patients of stageⅠ+Ⅱand (1.224±0.667) in MM patients of stageⅢ. Compared with the control group, the differenc was statistically significant (P <0.01). Furthermore, the expression level of FAK mRNA was statistically significant difference when MM patients of stageⅠ+Ⅱcompared with MM patients of stageⅢ(P<0.01).3. The MM patients were divided into two groups according to whether having extramedullary infiltration or not. The expression level of PTEN mRNA was (0.656±0.296) in 12 MM patients with extramedullary infiltration. Compared with the control, the differenc was statistically significant (P<0.05). The expression levels of PTEN mRNA were (0.742±0.407) in 43 MM patients without extramedullary infiltration. Compared with the control, the differenc was statistically significant (P<0.05). When MM patients with extramedullary infiltration compared with MM patients without extramedullary infiltration, the difference between these two group was not statistically significant (P> 0.05).FAK mRNA expression level was (1.791±0.618) in MM patients with extramedullary infiltration, and (0.878±0.468) in MM patients without extramedullary infiltration, and the difference between these two group was statistically significant (P<0.01). And the expression level of FAK mRNA in each patients group was significantly higher than that in control group (P <0.01).4. PTEN and T-FAK protein expressed in the bone marrows of 6 selected normal controls, and the average expression level of PTEN protein was (0.332±0.119) and T-FAK protein was (0.106±0.090) respectively. The expression of p-FAK protein was not detected. 5. PTEN- and FAK- protein expression level was measured in 12 MM patients of stageⅢby Western blot. Amoung these 12 patients, 6 had no extramedullary infiltration while the other 6 had. PTEN protein expression was detected in 9 patients and the average protein expression level was (0.081±0.087), which was statistically lower than that in control group (P<0.01). T-FAK protein were expressed in all thses 12 patients, and the average protein expression level was (0.257±0.119), which was statistically significant higher than that in control group (0.010.05).Conclusions There was statistically significant difference of PTEN and FAK mRNA expression between multiple myeloma patients and normal controls. The expression level of PTEN protein was lower and p-FAK protein was higher in MM patients of stageⅢthan that of in controls. The abnormal expression level of PTEN and FAK may be associated with the progression of the disease and extramedullary infiltration in MM patients. PTEN and FAK may be mutually antagonistic in the occurrence and development of MM.Part two: The effect of wild-type PTEN gene transfection on proliferation, apoptosis, invasion ability of multiple myeloma cells and its mechanismObjective RPMI 8226 cells and purified myeloma cells from MM patients were transfected with a recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP) or an adenovirus vectors only expressing green fluorescent protein (Ad-GFP), explore the effects of wild-type PTEN gene on proliferation, apoptosis, cell cycle and invasion activity of RPMI 8226 cells and purified myeloma cells from MM patients and the possible molecular mechanism.Methods Ad-PTEN-GFP or Ad-GFP was transfected into RPMI 8226 cells, purified myeloma cells from MM patients and the bone marrow mononeuclear cell of healthy controls. The growth inhibition rate was measured by MTT assay after the cells were transfected with PTEN gene. The transfection efficiency, apoptosis rate and cell cycle distribution were assessed by flow cytometry (FCM). Cell morphology was detected by light microscope and electron microscope. Cell proliferation and apoptosis were detected by Hoechst33342 staining. Transwell chamber test was used to meacure MM cell invasion activity. The mRNA expression levels of PTEN, FAK, MMP-2 and MMP-9 were detected by FQ-PCR. The protein expression levels of PTEN, FAK, p-FAK, MMP-2 and MMP-9 were detected by western blot.Results1. At the condition of multiple of infection (MOI)=100, at the second days of transfection, the transfection efficiency with adenovirus reached the top level: (83.1±6.4)% for RPMI 8226 cells, (26.4±11.90% for purified fresh myeloma cells and (29.3±10.5)% for the BMMNC of controls.2. After RPMI 8226 cells had been transfected with Ad-GFP or Ad-PTEN-GFP for 72 h, the expression level of PTEN mRNA and protein in RPMI 8226 cells from each group was measured by FQ-PCR and western blot. PTEN mRNA level was 19.244±5.065 vs 1.031±0.259 (p<0.01), and protein level was 1.426±0.201 vs 0.783±0.176 (p<0.01) respectively in Ad-PTEN-GFP transfected RPMI 8226 cells vs Ad-GFP transfected RPMI 8226 cells.3. Ad-PTEN-GFP, Ad-GFP were transfected into RPMI8226 cells, purified myeloma cells from MM patients and BMMNCs from healthy control, maximal growth inhibition rate was (42.01±7.92) % and (33.37±7.12) % respectively in Ad-PTEN-GFP transfected RPMI 8226 cells and the Ad-PTEN-GFP transfected purified myeloma cells from MM patients at the 4th day. Maximal growth inhibition was (8.42±3.09) % in Ad-PTEN-GFP transfected BMMNCs from healthy control at the 4th day, which was statistically significant lower compared with that of RPMI 8226 cells and purified myeloma cells from MM patients (p<0.01).4. After transfected with Ad-PTEN-GFP for 3d, the untransfected cells and Ad-GFP transfected MM cells displayed excellent growth state. The non-apoptotic cells were rounded and light blue. The chromatin was delicate and homogeneous, the nucleolus was clear, the organelles complete and the microvilli on the cells were clear and visible under transmission electron microscope. Ad-PTEN-GFP transfection induced typical apoptosis of MM cells, the apoptotic cells were bright blue and exhibited cell shrinkage and detachment, with lobular nuclear, debris-like, nuclear margination, nuclear condensation and fragmentation. Under the transmission electron microscope, it was shown that the microvilli on the cells disappeared and structure of the organelles was incomplete.5. After transfected with Ad-PTEN-GFP for 72 h, the apoptosis rate of RPMI8226 cells was (4.56±2.02)% in untransfected group, (5.51±2.43)% in Ad-GFP group, (35.02±6.80)% in Ad-PTEN-GFP group, and the difference of apoptosis rate between Ad-PTEN-GFP group and Ad-GFP group was significantly different (P<0.01). Accordingly, after the transfection for 72 hours, the apoptosis rate of the purified primary myeloma cells was (3.62±1.99) % in untransfected group, (3.45±2.87)% in Ad-GFP group and (20.97±7.93)% in Ad- PTEN-GFP group, and the diference of apoptosis rate between Ad-PTEN-GFP group and Ad-GFP group was significantly different (P<0.01). Samely, after the transfection for 72 hours, the apoptosis rate of the BMMNCs of control was (1.32±1.01) % in untransfected group, (1.04±0.79) % in Ad-GFP group and (4.01±2.48) % in Ad-PTEN-GFP group. And the diference of apoptosis rate between RPMI 8226 cells, or BMMNCs of control, and between the purified primary myeloma cells and BMMNCs of control was significantly different (P<0.05). 6. After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, cell cycle distribution showed the percentage of cells in G2/M phase increased from 16.20% to 51.10%. Cell cycle of Ad-PTEN-GFP transfected RPMI8226 cells was arrest in G2/M phase and there was an apoptotic sub G1 peak in cell cycle diagram. Cell cycle diagram showed that the apoptosis rate was (21.6±2.35) % in Ad-PTEN-GFP, which was significantly higher than the (4.8±0.74)% in Ad-GFP group and the (4.2±0.51) % in untransfected group (P<0.05).7. For the transwell chamber test, after the cells had been transfected with Ad-PTEN-GFP for 24 h, the average number of fluorescent RPMI8226 cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the Ad-GFP group and the Ad-PTEN-GFP group was 50.16±7.60, 52.65±7.39 and 23.50±6.12 respectively. The difference between the Ad-GFP group and the Ad-PTEN-GFP group was statistically significant (P<0.01). Samely, The average number of fluorescent purified myeloma cells from MM patients which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the Ad-GFP group and the Ad-PTEN-GFP group was 57.66±11.63, 56.01±8.45 and 30.84±7.81 respectively. And the difference between the Ad-GFP group and the Ad-PTEN-GFP group was statistically significant (P<0.01).8. The expression level of target mRNAs: After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, the expression level of PTEN mRNA was 1.020±0.228, 1.031±0.259, 19.244±5.065 in untransfected group, Ad-GFP group and Ad-PTEN-GFP group respectively.In untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the expression level of Survivin mRNA was 1.045±0.273, 1.017±0.269 and 0.380±0.059 respectively. Caspase-3 mRNA level was 1.018±0.227, 0.980±0.254 and 2.029±0.420 respectively. Caspase-7 mRNA level was 1.005±0.191, 1.013±0.205 and 1.614±0.311 respectively. The expression level of FAK mRNA was 0.987±0.300, 0.958±0.258 and 0.446±0.110 respectively. The expression level of MMP-2 mRNA was 0.992±0.251, 0.984±0.270 and 0.469±0.147 respectively. The expression level of MMP-9 mRNA relative expression level was 0.983±0.238, 0.972±0.257 and 0.366±0.104 respectively. The difference was statistically significant between the Ad-PTEN-GFP group and Ad-GFP group (P<0.01, P<0.05).9. The expression level of target proteins: After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, the expression level of PTEN protein was 0.732±0.159, 0.783±0.176 and 1.426±0.201 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. Survivin protein level was 0.348±0.081, 0.356±0.090 and 0.082±0.023 in untransfected group, Ad-GFP group and Ad-PTEN-GFP group (P<0.01).T-FAK protein level was 1.178±0.302, 1.137±0.282 and 0.730±0.197 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. The expression level of p-FAK protein was 0.289±0.083, 0.304±0.095 and 0.068±0.027 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. MMP-2 protein level was 0.622±0.220, 0.691±0.238 and 0.155±0.076 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. MMP-9 protein level was 0.358±0.094, 0.339±0.143 and 0.098±0.060 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. The difference was statistically significant between the Ad-PTEN-GFP group and Ad-GFP group (P<0.01, P<0.05).10. After transfection with Ad-PTEN-GFP for 72 h, the activitay of caspase-3/7 (0.786±0.081) in Ad-PTEN-GFP transfected RPMI 82226 cells was increased significantly compared with that of in the Ad-GFP transfected cells (0.373±0.039) and the Untransfected cells(0.370±0.034)(P<0.01).Conclusions Over expression PTEN gene in RPMI8226 cells and purified myeloma cells from MM patients could inhibit the proliferation, induce apoptosis. With the transfection of wild type PTEN gene, cell cycle of the transfected cells was arrested in G2/M phase, apoptosis occurred, the invasion ability decreased, the expression levels of many apoptosis related genes were changed, which might be related to the inhibition of FAK / MMP signal pathway by the high expression of PTEN.Part three: Effect of Silencing PTEN gene expression on the ability of proliferation, invasion of RPMI8226 cells and its mechanismObjective RPMI 8226 cells were transfected with mouse PTEN specific siRNA (PTEN-siRNA) to down regulated the expression of wild type PTEN, to investigate the effect of PTEN gene on the proliferation, cell cycle and invasion activity of RPMI8226 cells, and explore its molecular mechanism.Methods RPMI 8226 cells were transfected with PTEN-siRNA or non-specific siRNA (NS-siRNA) to knockdown the expression of wild type PTEN. Cell growth curve was measured by MTT assay. Cell cycle distribution was assessed by flow cytometry (FCM). Transwell chamber test was used to meacure MM cell invasion activity. The mRNA expression levels were detected by FQ-PCR. The protein expression levels were detected by western blots.Results1. Detection of PTEN mRNA and protein to evaluate the efficency of PTEN-siRNA transfection. After RPMI 8226 cells had been transfected with PTEN-siRNAs or NS-siRNAs for 48 h, the expression level of PTEN mRNA and protein in RPMI 8226 cells from each group was measured by FQ-PCR and western blot. PTEN mRNA level was 1.107±0.306 vs 0.143±0.045 (p<0.01), and protein level was 0.699±0.130 vs 0.089±0.025 (p<0.01) respectively in NS-siRNAs transfected RPMI 8226 cells vs PTEN-siRNAs transfected RPMI 8226 cells.2. RPMI8226 cells were divided into three group: untransfected group, NS-siRNA transfected group and PTEN-siRNA transfected group. After RPMI 8226 cells had been transfected with PTEN-siRNAs for 4 days, cell survival rate in PTEN-siRNA transfected group was (141.55±8.34)%. The OD 490 values were significant difference between the NS-siRNA transfected group and the PTEN-siRNA transfected group (P<0.01).3. After RPMI8226 cells had been transfected with PTEN-siRNA for 48 h, cell cycle analysis showed that the percentage of RPMI8226 cells in G0/G1 phase was increased and the percentage of RPMI8226 cells in G2/M phase and S phase was decreased. Sub-diploid apoptotic peak showed that the percentage of apoptotic cells was decresed.4. For transwell chamber test, after RPMI8226 cells had been transfected with PTEN-siRNA for 24 h, the average number of RPMI8226 cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the NS-siRNA group and the PTEN-siRNA group was 49.33±7.63, 47.17±7.76 and 79.50±11.89 respectively. The difference in the cell number between NS-siRNA group and the PTEN-siRNA group were statistically significant (P<0.01).5. The expression level of target mRNA: After RPMI8226 cells had been transfected with PTEN-siRNA for 48h, the expression level of PTEN mRNA was 1.049±0.248, 1.107±0.306 and 0.143±0.045 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group.In untransfected group, NS-siRNA group, and PTEN-siRNA group, the expression level of Survivin mRNA was 1.013±0.207, 1.008±0.191 and 2.534±0.438 respectively. Caspase-3 mRNA level was 1.008±0.198, 0.979±0.174 and 0.473±0.089 respectively. Caspase-7 mRNA level was 1.015±0.196, 1.003±0.204 and 0.510±0.090 respectively. Accordingly, in untransfected group, NS-siRNA group, and PTEN-siRNA group, the expression level of FAK mRNA was 0.979±0.308, 0.940±0.301 and 2.176±0.612 respectively. The expression level of MMP-2 mRNA was 1.002±0.213, 0.998±0.206 and 2.793±0.740 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group. The expression level of MMP-9 mRNA was 1.086±0.219, 1.029±0.280 and 1.980±0.733 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group. The difference was statistically significant between the PTEN-siRNA group and NS-siRNA group (P<0.01, P<0.05).6. The expression level of target protein: After RPMI8226 cells had been transfected with PTEN-siRNA for 48h, the expression level of PTEN protein was 0.647±0.124, 0.699±0.130 and 0.089±0.025 respectively in the untransfected group, NS-siRNA group and PTEN-siRNA group. Survivin protein level was 0.312±0.089, 0.324±0.086 and 1.247±0.321 (P<0.01). T-FAK protein level was 0.983±0.217, 0.945±0.226 and 1.637±0.380 respectively. The expression level of p-FAK protein was 0.345±0.086, 0.390±0.091 and 1.054±0.272 respectively. MMP-2 protein level was 0.233±0.080, 0.295±0.092 and 1.265±0.383 respectively. MMP-9 protein level was 0.237±0.083, 0.221±0.084 and 0.544±0.161 respectively. The difference was statistically significant between the PTEN-siRNA group and NS-siRNA group (P<0.01, P<0.05).7. After transfection with PTEN-siRNA for 72 h, the activitay of caspase-3/7 (0.073±0.008) in PTEN-siRNA transfected RPMI 82226 cells was decreased significantly compared with that of the NS-siRNA transfected cells (0.369±0.038) and the Untransfected cells(0.370±0.034)(P<0.01).Conclusions Down regulated the expression of wild type PTEN by using PTEN-siRNA can significantly enhance the proliferation of RPMI8226 cells, decrease cell apoptosis, increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in G2/M and S phase, enhance the cell invasion ability, indicate that the deceased expression of wild type PTEN might further regulate apoptosis related genes, and increase the expression level of FAK, MMP-2 and MMP-9 mRNA and protein, and stimulate the proliferation and invasion of myeloma cells.