Effect of Fuzitang on Proliferation of Human Rheumatoid Arthritis Synovial Fibroblast Cell Line MH7A and Expression of miR-155 / 中国实验方剂学杂志

ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.

Effect of SHIP1 on viability and apoptosis of leukemia Jurkat cells by reg-ulating STAT3 signaling pathway / 中国病理生理杂志

AIM:To investigate the effect of SH2 domain-containing inositol 5-phosphatase 1(SHIP1)on the viability and apoptosis of leukemia cells by regulating signal transducer and activator of transcription 3(STAT3)signaling pathway.METHODS:Human leukemia Jurkat cells were transfected with null vector and SHIP1 overexpression vector in negative control(NC)group and SHIP1 group,respectively.The cells without treatment served as control group.The transfection efficiency was detected by real-time PCR and Western blot.The changes of the cell activity were measured by MTT assay.The apoptosis was detected by flow cytometry.The protein levels of cleaved caspase-3,STAT3 and p-STAT3 were determined by Western blot.The STAT3 signaling pathway inhibitor AG 490 was applied to the cells in control group and SHIP1 group as control +AG490 group and SHIP1+AG490 group,respectively,and the above indexes were also ana-lyzed.RESULTS:Compared with the control group and NC group ,the mRNA and protein expression levels of SHIP 1 in SHIP1 group were both upregulated ,the cell viability was increased ,the apoptotic rate was decreased ,the protein level of cleaved caspase-3 was upregulated ,and the protein level of p-STAT3 was decreased(P<0.05 ).Compared with control group and control +AG490 group,the cell viability in SHIP1+AG490 group was decreased ,the protein level of cleaved caspase-3 was increased the protein level of p-STAT3 was decreased ,and the apoptotic rate was increased(P<0.05 ). CONCLUSION:SHIP1 inhibits the viability and promotes the apoptosis of human leukemia Jurkat cells by inhibiting the STAT3 signaling pathway.

Preliminary study of role of post-transcription regulation on SH2 domain-containing inositol 5'-phosphatase 1 gene expression by miR-155 in the pathogenesis of acute myeloid leukemia / 白血病·淋巴瘤

Objective To investigate the role of microRNA-155 (miR-155) on post-transcription regulation of SH2 domain-containing inositol 5'-phosphatase 1 (SHIP1) gene expression in the pathogenesis of acute myeloid leukemia (AML).Methods Quantitative real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of miR-155 and SHIP1 mRNA in the AML patients and controls.miR-155 mimics was transfected into U937cells (U937m) by using X-treme GENE siRNA transfection reagent.Cells without transfection (U937c) and cells with negative transfection (U937mc) were used as controls.RT-PCR was performed to detect the expression of miR-155 and SHIP1 mRNA in these cells.The expression of SHIP1,TAKT and pAKT were detected by Western blot in U937 cells.Apoptosis was studied by flow cytometry (FCM).Results The average level of SHIP1 protein content in 15 samples of patients with AML-M4 or AML-M5 from 30 AML patients was significantly lower compared with that of patients with the other types of AML,and the levels of miR-155 were significantly higher in the same group of patients (P ＜ 0.05).Significantly decreased levels of SHIP1 protein were found in U937m cells compared that of U937c and U937mc (P ＜ 0.05).Significantly decreased rate of apoptosis was observed in U937m cells compared with that of U937mc and U937c.U937m cells also exhibited no alteration in total AKT content,while increased p-AKT levels were found (P＜ 0.05).Conclusion SHIP1 was also a primary target of miR-155 in AML.Overexpression of miR-155 could activate PI3K-AKT pathway to depressing SHIP1 and decrease the apoptosis rate of AML cells.