Study Of Down-regulating IFITM3to Inhibit Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is the most common acute leukemia in adults,accounting for about 80%of leukemias,and about 10,000 people die from the disease every year in the United States.AML is a highly heterogeneous disease.It is characterized by transcription disorders that lead to hindered differentiation,reduced normal blood cells,and excessive proliferation of poorly differentiated cell clones,resulting in bone marrow failure,and can be transmitted or infiltrated to other organs of the body(such as liver,lung,spleen,and Lymph nodes).At present,the main treatment method is still the combination of cytosine arabinoside(Ara-C)and anthracyclines with allogeneic stem cell transplantation.Although it can relieve most patients,it can still cause fatal relapses.In addition,the incidence of AML increases with age,and elderly patients generally cannot tolerate such treatments.Therefore,multiple research teams are working to find a new type of treatment to improve the prognosis of AML patients.Interferon-induced transmembrane protein 3(Interferon-induced transmembrane protein 3,IFITM3)is a small protein located in plasma and lysosomal membranes.It is stimulated by type I and type II interferons(IFNs).The expression of the above IFITM3 will quickly rise.In addition to showing the role of IFITM3 related to virus defense,it is highly expressed in many tumors;meanwhile it shows a high correlation with the proliferation,metastasis and invasion of solid tumors.Iron Oxide Nanoparticles(IONPs)are one of the novel discovered nanozymes(nanozyme)family members in recent years.Without any modification on the surface,they are similar to property of horseradish peroxidase(HRP).Under acidic conditions,it can generate hydroxyl radical through the reaction of hydrogen peroxide,which increases the level of reactive oxygen species(ROS).ROS can lead to increased oxidative stress and cell death.In order to find a new and efficient treatment strategy to kill AML cells in this study,we used IONPs combined with Ara-C to incubate the KG-1a cells and treat AML model mice by silencing the expression of IFITM3 in AML cell line KG-1a,and to explore the role of IFITM3 in the KG-1a cells and the effect mechanisms of IONPs combined with Ara-C on KG-1a cells in vitro and in vivo.ObjectivesTo explore the effect of down-regulation of IFITM3 expression on human-derived AML cell line KG-1a and its molecular mechanism,and to provide experimental evidence by verifying IFITM3 as an effective therapeutic target.To explore the inhibitory effects and possible mechanisms of IONPs combined with Ara-C on KG-1a cells in vitro and in vivo,and to provide new ideas for improving the survival rate of AML patients and ultimately curing AML disease.Methods and Contents:1.We used p LV-sh IFITM3 and p LV-sh NC recombinant lentivirus to infect human-derived AML KG-1a cells,and then screened positive cells with puromycin to obtain positive monoclonal cells that stably down-regulated the IFITM3 molecular expression.Real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)and Western blot were used to detect the expression of IFITM3 molecule at the m RNA and protein levels.2.To identify the effect of down-regulating IFITM3 molecules on several biological characteristics of KG-1a cells:CCK8 and soft agar cloning assays were used to detect the ability of proliferation and cloning formation of KG-1a cells after down-regulating IFITM3 expression;Flow cytometry(FCM)was used to analyze the sensitivity of KG-1a cells to Ara-C after down-regulation of IFITM3 expression;q RT-PCR and Western blot were used to detect the expression of nuclear factor k B (NF-k B)related protein p65.KG-1a cells,Scrambled cells and Lv-sh IFITM3-KG1a cells were inoculated into NOD/SCID mice via the tail vein at 5×10~6 ells/mouse,and the survival status and weight changes of the mice were monitored carefully.The expression levels of CD11b and CD33 in the bone marrow of tumor-bearing mice were analyzed by FCM,and the ratio of tumor cells in the bone marrow cells was observed by fluorescence microscope.3.We set up the control group(Control),Ara-C group(0.4μM),IONPs group(150μg/ml)and Ara-C+IONPs(Ara-C 0.4μM+IONPs 150μg/ml)group to treat KG-1a cells in vitro respectively.After that,we observed the apoptosis of each group,analysing related molecular expression levels and ROS level of each group,as well as its anti-AML effect and mechanism.In vivo KG-1a cells were administered with 5×10~6cells/mouse,the PBS-only injection group was used as the normal control group(Control),and KG-1a cells were inoculated into non-obese diabetic/severe combined immunodeficiency(NOD/SCID)mice via the tail vein,and the Ara-C group(75mg/kg/mouse/once),IONPs group(15mg/kg/piece/once)and Ara-C+IONPs(Same dose as before)group were treated separately,once every 3 days for 3 weeks.The treatment effect and mechanism of each group were observed and analyzed.After the 20th day of treatment,the mice were sacrificed,bone marrow and peripheral blood were taken for related examination and analysis.Results:1.KG-1a cells infected with lentivirus(p LV-sh IFITM3 or p LV-sh NC),and then was screen out p LV-sh IFITM3 stably infected strain(named LV-sh IFITM3-KG1a cells)with puromycin.The results of q RT-PCR and Western blot showed that after p LV-sh IFITM3 lentivirus infected KG-1a cells,the expression of IFITM3 molecule was significantly reduced compared with the control group,the difference was statistically significant(P<0.05).After p LV-sh NC lentivirus infected KG-1a cells(named Scrambled cells),there was no significant difference between the expression of IFITM3 molecule and the uninfected groups(P>0.05).2.The results of in vitro experiments showed that,compared with wild-type KG-1a cells and Scrambled cells,LV-sh IFITM3-KG1a cell proliferation and colony-forming ability were reduced;FCM results showed that LV-sh IFITM3-KG1a were more sensitive to Ara-C incubation.The results of q RT-PCR showed that the expression of caspase 3 molecule related to apoptosis in LV-sh IFITM3-KG1a cells was increased,and the expression of NF-k B p65 molecule was decreased,and the difference was statistically significant(P<0.05).3.The results of vivo tumor development experiment showed that,compared with Scrambled cells,the AML-bearing mouse bone marrow cells with injection of LV-sh IFITM3-KG1a cells significantly reduced KG-1a cells,and AML-related molecules CD45,CD33,and CD11b were respectively decreased,and the difference was statistically significant.4.The vivo mouse animal experiment results showed that the cells treated with IONPs+Ara-C group had significantly higher intracellular ROS levels and more obvious apoptosis compared with the Control,Ara-C,and IONPs treatment groups;intracellular pro-apoptotic genes Caspase 3 was also increased in protein levels,and the difference was statistically significant(P<0.05).5.The results of vivo treatment experiments showed that the number of peripheral blood white blood cells were increased and the hemoglobin level was decreased in the IONPs+Ara-C group compared with the Control,Ara-C,and IONPs groups.The difference was statistically significant(P<0.05).The number of immature granulocytes in blood smears and bone marrow smears were simultaneously decreased,and the test results showed alleviation and relief of signs of AML-bearing mice,the difference was statistically significant(P<0.05).Conclusions:1.The IFITM3 molecule is related to the promotion of KG-1a cell proliferation,cloning,anti-apoptosis and tumorigenic ability in NOD/SCID mice.After down-regulating the IFITM3 molecular expression,the above biological characteristics are all weakened.2.In vitro IONPs combined with Ara-C can promote the apoptosis of KG-1a cells stronger than Ara-C or IONPs alone.This effect may be related to the increase of intracellular ROS levels by IONPs.3.The results of vivo mouse animal experiments showed that after down-regulating the IFITM3 molecule,the signs of leukemia in AML-bearing mice can be alleviated.The Ara-C+IONPs combination group has a stronger therapeutic effect on AML-bearing mice,and can more effectively kill KG-1a cells,alleviate and reduce the signs of leukemia.The above findings indicate that the IFITM3 molecule plays an important role in KG-1a cells and IFITM3 may be an effective therapeutic target for AML.The combination of IONPs+Ara-C may be a new type of treatment strategy for highly effective treatment of AML.