Regulatory Mechanism Of PTEN And Akt In RA FLS Abnormal Proliferation

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease. RA early symptoms usually include red and swelling joints, burning pain and action obstacles. As the disease became severe, the joints appear stiffness and deformed, and show vary degrees of skeletal muscle atrophy, which increase the risk of disability. RA can also produce diffuse inflammation in the heart, lung, kidney and other subcutaneous tissue. In extreme cases RA can be fatal. About 1%～2% of the world's population is afflicted by RA, approximately 20 million patients were suffering from it. RA seriously threatens the human healthy and living quality. In China the inflicted RA incidence approaches 0.4%, almost 70% of the patients become disability two years after diagnosis. Therefore, illustrating RA synovial proliferation pathology process shows great importance in improving the effectiveness of treatments and drugs usage, so that enhance the survival rate and improve the qualities of life of RA patients.The pseudo-tumoral proliferation of RA fibroblast-like synoviocytes (FLS) is considered to be the major mechanism for the hyperplasic growth of the RA synovium. In recent years, phosphatidyl inositol 3-kinase (phosphatidylinositol 3-kinase, PI3K) signal pathway was shown to be activated in RA synovial tissue, and which has been recognized as one of the important mechanisms to regulate the proliferation and apoptosis of RA FLS, thus play key roles in the pathologic process of RA. Moreover, The activity of PI3K/Akt signaling pathways is correlated with distinct proinflammatory cytokines, including IL-1, TNF, NF-κB, and etc. PTEN is the central negative regulator of PI3K/Akt signal pathway. PTEN acts as protein phosphatase and lipid phosphatase, antagonizing the PI3K/Akt signaling pathway by dephosphorylating phosphoinositides, down regulating its activity. Despite of influencing PI3K/Akt pathway, PTEN also involved in inhibiting cell motility, regulating cell cycle and maintaining chromosome stability, etc. The activation of PI3K/Akt signaling pathways maybe associated with the down expression or dysfunction of PTEN. Therefore, induction of PTEN or inhibition of Akt specifically could block PI3K/Akt signaling pathways, thus contributing to study the function of PI3K/Akt pathway in regulation the proliferation and apoptosis in RA FLS.RNAi is a new technique discovered in recent years and it is useful for studying the function of specific genes. Exogenous or endogenous double-stranded RNA (dsRNA) can bind to its complementary sequence of mRNA in vivo, and then selectively degrades targeted mRNA, interferes specific gene expression and lead to gene silence. Meanwhile, RNAi is high specific, high stable and high efficiency and acts as an important tool in studying the function of specific gene.The Methods to transfer exogenous gene into target cells includes either virus expression vector or non-virus expression vector systems. The transfection efficiency of non-virus expression vector is lower than virus expression vector. In general, virus expression vectors include lentivirus, adenoviruses, retrovirus, etc. Among them the lentivirus expression vectors have an important advantage over other types of expression systems, it can transfect dividing and non-dividing cells and elicit less immune response. In this study we constructed two lentivrus expression vectors (pLenti6/V5-PTEN and pLenti6/shAkt), the former was used to induce the expression of PTEN, while the latter was used to inhibit the expression of Akt. Each vector was packed into lentivirus, and then was used to infect RA FLS individually. After infection, MTT assay and scratch test were performed to study the effect of these vectors to inflect the proliferation and migration of RA FLS, and to further explore the function and mechanism of PTEN and Akt in the pathology of RA. ObjectiveThe purpose of this study was to characterize the proliferation and growth of RA FLS, and to investigate the protein expression levels and location of PTEN, Akt and p-Akt.MethodsParaffin-embedded and formalin-fixed RA synovium were cut into 5μm sections, which were then processed for immunohistochemistry (IHC) to monitor the amount and location of PTEN and Akt.RA/OA FLS were isolated by adheration of synovial tissues obtained from RA patients undergoing total joint replacement surgery or knee synovectomy. After FLS were isolated, flow cytometry was used to define the purity, MTT assay was performed to inspect the proliferation of the cells and the growth curve was drawn according to the data. Western blot were performed to detect the protein expression of PTEN, Akt and p-Akt.Results1. IHC showed that PTEN and Akt mainly located in the nuclear of the cells, partially in the plasma.2. In the 4th generation, the purity of RA FLS reached 99% and were suitable to perform further experiment.3. MTT assay showed significant difference in the proliferation rate of the 4th generation of RA FLS and the 3rd generation of OA FLS. OD value was 0.31 of RA against 0.259 of OA, and each time point showed significance in statistic (p<0.05).4. Compared with OA FLS, the protein expression of PTEN was lower, while Akt and p-Akt were higher In RA FLS.ConclusionThe expression of PTEN in RA FLS was lower than in OA FLS and the expression of Akt in RA FLS was higher than in OA FLS. ObjectiveTo construct pLenti6/V5-PTEN lentivirus expression vector; To screen effective Akt siRNA targeting sequences and construct lentivirus expression vector carrying shRNA targetting Akt.Methods1. Human PTEN complementary DNA was cloned into pLenti6/V5 vector, named pLenti6/V5-PTEN. pLenti6/V5-NC served as the control viruses.2. The siRNA sequences targeted human Akt transcript were designed using the software developed by Invitrogen, Inc. Two candidate sequences in human Akt gene, classified as Akt-1 and Akt-2, were selected for RNA interference (RNAi). The control RNA duplex (named as NC), was non-homologous to any human genome sequences. A scrambled sequence (NC), which is nonhomologous to any human DNA sequence, served as a negative control and was classified as NC. The three sequences were cloned into BLOCK-iTTM U6 RNAi Entry Vector (named as pU6/shAkt-1, pU6/shAkt-2, pU6/sh NC). The entry vector containing the U6 RNAi cassette was used to transfer the U6 RNAi cassette into the lentiviral expression plasmid (named as pLenti6/shAkt-1,pLenti6/shAkt-2 and pLenti6/sh NC) using Gateway(?) Technology. Each construct was sequenced to confirm the right sequence of insert. pLenti6/V5-PTEN,pLenti6/shAkt-1 and pLenti6/shAkt-2 were transfected into LOVO cells to determine their efficiencies.3. The successfully constructed lentivirus expression vectors were transfected into 293T cells with ViraPowerTM Packaging Mix, After culture for 48h and 72h, the supernatant contains virus was collected, aliquoted and then stored at-80℃.Results1. In comparison with the parental LOVO cells, pLenti6/V5-NC transfectants showed a significant increase of PTEN in both mRNA and protein levels. While pU6/shAkt-1, pU6/shAkt-2, pLenti6/shAkt-1,pLenti6/shAkt-2 transfectants showed a significant decrease of Akt in both mRNA and protein levels, indicating the successful knockdown of Akt in these derived clones. Furthermore, there was no difference in OPN expression level between the mock cells and the parental LOVO cells. The siRNA sequence Akt-2 shown a better silencing efficiency than Akt-1.2.The recombinant plasmid of pLenti6/V5-PTEN,pLenti6/V5-NC pLenti6/shAkt-1,pLenti6/shAkt-2 and pLenti6/sh NC were packaged into virus with ViraPowerTM Packaging Mix in 293T cells respectively. The virus titers were 1.5×107TU/ml,8×106TU/ml,4×106TU/ml,4.3×106TU/ml and 7.4×106TU/ml separately.ConclusionBoth pLenti6/V5-PTEN and pLenti6/shAkt-2 letivirus expression vectors were successfully constructed.Part 3 The effects of pLenti6/V5-PTEN and pLenti6/shAkt on RA FLS in proliferation, migration and protein expressionObjectiveThe aim of this study was to examine the function of PTEN and Akt gene in RA FLS, to study the effects of these genes on RA FLS proliferation and mortality, to further elucidate the function and relation of PTEN and Akt in the pathogenesis of RA.Methods1. RA FLS were infected with distinct virus. In various time point after infection, the mRNA and protein were extracted from RA FLS, RT-PCR was used to detect the mRNA expression of PTEN and Akt, while western blotting were performed to analyze the protein expression of PTEN, Akt and p-Akt.2. MTT assay and scratch test were performed to determine the proliferation and mortality of RA FLS after infection.Results1. To elucidate the underlying mechanism for the induction of PTEN or silencing of Akt in RA FLS, we examined the kinetic expression of PTEN and Akt mRNA and protein in RA FLS over a 96 h time course.24h,48h,72h,96h after infection, the pLenti6/V5-PTEN transfectants showed higher PTEN mRNA expression than the NC group, the increment was 76.02%,63.67%,67.24% and 72.83% respectively (p<0.05). The pLenti6/sh Akt-2 transfectants showed significant decrease of the Akt subtypes, the decrement of Aktl was 76.13%,65.1%,79.12% and 70.18%(p<0.05). Moreover, the expressions of Akt2 and Akt3 (the other Akt subtypes) were also significantly decreased. In agreement with Part 2, pLenti6/sh Akt-2 was better than pLenti6/sh Akt-1 to silence the expression of Akt.2. We further examined the effects of PTEN induction and Akt inhibition on the protein expressions.48h after transfected with pLenti6/V5-PTEN, western blot showed that PTEN protein was increased by 107.85%(p<0.05), while p- Akt was decreased by 48%(p<0.05) compared to NC. In pLenti6/sh Akt-2 transfectants, Akt protein decreased by 6.19% and p-Akt decreased by 58.75%(p<0.05). And we found that pLenti6/sh Akt-1 had few influences on the Akt and p-Akt expression.3. MTT assay results displayed that pLenti6/V5-PTEN inhibited the proliferation of RA FLS, the OD value was decreased by 4.46%,25.14%,41.84% and 52.46% at 24h,48h,72h,96h post transfection. And we also found the same effects of pLenti6/sh Akt-2, which showed a decline of OD value by 27.5%,44.69%,56.80% in 24h,48h,72h,96h respectively compared with NC.4. The scratch test showed the mortality of RA FLS was dramatically decreased after infected with pLenti6/V5-PTEN and pLenti6/shAkt-2. Furthermore, In comparison with NC, the transfectants displayed high mortality in both cell counts and invasiveness 72 h after infection.ConclusionpLenti6/V5-PTEN and pLenti6/shAkt-2 lentiviral expression vectors can down-regulate p-Akt protein expression and inhibit RA FLS proliferation and migration.