| Part I Copy number variation (CNV) and its clinicopathological significance of subunits of PI3K/AKT in diffuse large B-cell lymphoma (DLBCL)Objective:To investigate the PI3K/AKT activation mechanism and its association with clinicopathological information in DLBCL; and to elucidate the etiological mechanism of DLBCL from the angle of copy number variation (CNV), therefore providing the new molecules for the diagnosis and prognosis of DLBCL.Methods:The 60 cases of fresh DLBCL tissues were archived from the department of pathology in Fudan University Shanghai Cancer Center from 2005 to 2011 year. Meanwhile,10 cases of lymph reactive hyperplasia tissue were as negative control. Of 60 cases, prognoses were available for 57 cases. In 60 cases of fresh DLBCL tissues, high-throughput Nanostring nCounter approach was employed to detect the CNV of 12 members of PI3K/AKT signal pathway, including the chief catalytic subunits, regulatory subunits and downstream subunits of AKT. Meanwhile, prognosis and clinic-pathological information of the 4 different chief catalytic subunits, that is PIK3CA, PIK3CB, PIK3CD and PIK3CG, were also statistically analyzed.Results:(1) Among all the PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3C2A, PIK3C2B, PIK3C2G, PIK3R1, PIK3R2, AKT1, AKT2, AKT3 that were enrolled as genes of interest and detected, the CNVs were all detected using Nanostring nCounter in the form of amplification or deletion in all the subunits of PI3K and AKT enrolled, with the exception of PIK3R1. The variation rate ranges from 8.3% to 20.0%. In addition, the majority of genes have occurred copying number amplification, accounting for 64%-100% of CNV rates. Only the copy number was mainly deleted in PIK3CD, accounting for 70% of CNV rates. The frequency of CNV in PI3K/AKT was as higher as 58.3%. (2) Meanwhile, in DLBCL and BL cell lines, it was found that DOHH2, LY1, LY8 and Toledo whose copying number were amplified. (3) There were no statistically significant association of CNVs of PIK3CA, PIK3CB, PIK3CD, PIK3CG with ages, gender, clinical stage, B symptom, tumor size and LDH (P>0.05). It was found that PIK3CA and PIK3CB gene CNVs were significantly associated with decreased overall survival (P=0.029 and P=0.019, respectively).Conclusions:Therefore, CNVs of components of the PI3K/AKT pathway play an important role in the tumorigenesis of DLBCL. CNV of PIK3CA and PIK3CB are of great important value for predicting inferior prognosis in DLBCL.Ⅱ The expression and clinicalpathological significance of p110 catalytic subunits and pAKT in DLBCLObjective:It has been reported that the PI3K/AKT signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL), however, the underlying mechanism is still largely unknown.Methods:The 60 cases of DLBCL tissues were archived from the department of pathology in Fudan University Shanghai Cancer Center from 2005 to 2011 year. Immunohistochemistry (IHC) was performed to examine the expression of p110α, p110β, p110γ, p110δ, and pAKT in 60 de novo DLBCLs and 10 reactive hyperplasia specimens as controls.Results:(1) To confirm these findings at the protein level, IHC for p110α, p110β, p110γ, p110δ, and pAKT was performed on tissue microarrays. The positive rate of four main catalytic subunits and pAKT is 80%,81.6%,81.6%,81.6% and 75% respectively. The strong positive expression is 26.7%,25.0%,18.3%,25.0% and 16.7% respectively. (2) Clinic pathologically, except strong positive expression of p110δ was associated with lower IPI, there is no significant association of the four subunits above mentioned as well as pAKT expression with ages, gender, clinical stage, B symptom, tumor size and LDH (P>0.05). (3) Strong positive expression of p110α (26.7%) was significantly associated with CNVs of PIK3CA (P=0.003), and positively correlated with strong positive expression of pAKT (P=0.026). Strong positive expression of p110α, p110β, p110γ, and p110δ was found to be associated with decreased survival (P=0.022, P=0.015, P=0.015, and P=0.008, respectively). Mediate to strong positive expression of pAKT was shown decreasd survival(P=0.165).Conclusions:The aberrant protein expression of catalytic isoforms of PI3K are of great important value for predicting inferior prognosis in DLBCL. Strong positive expression of p110α was associated with expression of pAKT, indicating p110α plays important role in activating AKT pathway.Part Ⅲ Effect after gene silencing of PI3K catalytic subunits on activation of AKT and biological behaviors in DLBCL cell linesObjective:To explore the effect after knocking-down of PI3K 4 catalytic subunits using RNA interference (RNAi) approach on the AKT signal transduction pathway and cellular proliferation in vitro in DLBCL cells.Methods:Lentiviral short hairpin RNA (shRNA) vectors against PIK3CA, PIK3CB, PIK3CG, PIK3CD and its control vectors were stably transfected into LY8 cells after construction with success. Positive cells having green fluorescent protein (GFP) were screened using flow cytometry (FCM). Knock-down efficiency of different vectors was detected using western-blot, by which the best stable transfect strain was screened out. Cell apoptosis and cell cycle were detected using FCM; proliferation curve was monitored using CCK-8 assay. In addition, AKT and p-AKT, which were downstream of PI3K pathway as well as some other cell cycle/apoptosis-related proteins, were also detected by western-blot.Results:It was found that the proportion of G1 stage increased as compared with its control after knocking-down of PIK3CA, PIK3CB, PIK3CG, PIK3CD [control: 37.06(36.56±2.01)%, experimental group:51.4(50.77±1.45)%,40.82(46.04±5.58)%, 47.16(47.12±2.02)% and 60.40(59.10± 1.03)%, respectively]; and that proportion of S stage wasn’t changed significantly after knocking-down [control: 48.96(46.47±2.34)%, experimental group:40.36(42.43±2.57)%,49.84(45.83±3.93)%, 44.53(44.13±0.83)% and 30.90(32.87±2.17)%, respectively]. While, the G2 stage was wholly decreased in comparison with control [control:13.98(16.96±2.79)%, experimental group:8.23(6.80±1.34)%,9.34(7.91±1.30)%,8.31(9.37±0.96)% and 8.68(7.99±2.04)%, respectively]. The cells were significantly arrested at G0/G1 after knocking-down of PIK3CA, PIK3CD and PIK3CG (P<0.05) with the exception of PIK3CB. What’s more, the apoptosis rate was 11.5(10.30±1.11)%,9.3(7.83±1.15)%, 7.0(6.67±0.35)% and 4.7(5.80±1.11)%, with control being 5.7(5.13±0.38)%. There was significant difference of cell apoptosis after knocking-down of PIK3CA, PIK3CB and PIK3CD other than PIK3CG compared with its control (P<0.05). The proliferation was decreased after silencing of PIK3CD, and there was significant difference at 72nd and 96th hours versus control group (P<0.05). Western-blot results showed that, the expression level of AKT didn’t change but level of p-AKT decreased significantly, with cell cycle-related protein p27 being increased and cyclinDl being decreased after silencing of PIK3CA, PIK3CB, PIK3CD and PIK3CG.Conclusions:In all, gene silencing of PI3K catalytic subunits can lead to cell cycle arrest and(or) cell apoptosis. Of the 4 catalytic subunits, knock-down of PIK3CD can significantly inhibit the proliferation of DLBCL cell line LY-8, which may facilitate and synergize the effect of PIK3CD selective inhibitor to suppress the growth of DLBCL and therefore may act as an ideal therapeutic target for DLBCL.Part IV Study on the biological effect after knock-down of PIK3CD in human DLBCL xenografted nude mice modelObjective:To establish the nude mice model xenografted with human DLBCL cell line LY-8; and to explore the biological effect after knocking-down of PIK3CD on the proliferation of xenografted tumor in vivo.Methods:The nude mice were randomly grouped into two, with each group being 10 mice. Each group were subcutaneously injected with LY-8 cell line whose PIK3CD has been stably knocked-down and LY-8 cell line as control group, respectively. Xenografted tumor rate, histological morphology, tumor volume and cell apoptosis were observed. Apoptosis-related proteins were detected using IHC method; cell apoptosis after homogenizing xenografted tumor tissues was assayed using FCM.Results:Xenografted tumor rate was 40%(4/10) in experimental group, which was significantly lower than that in control group whose tumor rate was 70%(7/10) in 2th week(P<0.05); the both the tumor volume and nude mice weight in experimental group were smaller than those in control group, but there weren’t significant differences (P>0.05); however, late/total apoptosis rate in experimental group [18.1%(16.07±1.76)/20.8(17.00±1.49)%] were significantly higher than that [6.5%(5.87±0.76)/10.0(10.70±1.04)%] in control group (P<0.05).Conclusions:Our results demonstrated that knock-down of PIK3CD can inhibit the tumor volume to a certain extent in nude mice model xenografted with human DLBCL cell line LY-8, mainly through inducing cell apoptosis, which may provide novel theoretical evidence and thought for the treatment of DLBCL. |
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