| Background and Objective:Non-Hodgkin’s lymphoma(NHL)is a common hematologic malignancy that originates from relatively mature lymphocytes and is highly heterogeneous.In recent years,its morbidity has been rising continuously.Therapeutically,novel monoclonal antibodies,targeted therapies,antibody drug conjugators(ADCs),bispecific antibodies,immune checkpoint inhibitors,and chimeric antigen receptor(CAR)T cell therapies have all been developed.However,many lymphoma patients develop resistance to these therapies,leading to treatment failure or relapse.There is currently a need for new treatments to address relapsed or refractory lymphomas.Kindlin-3,belonging to the family of adhesive plaque proteins known as kindlins and containing the FERM domain,binds to the C-terminal of integrin-β cytoplasmic tails and acts as a co-activator of Talin which mediates integrin activation,leading to cell adhesion,migration,cell-cell interaction,and differentiation.Recently,increasing studies have shown that kindlin-3 plays both inhibitory and promoting roles in breast cancer,melanoma and lung cancer.The role of kindlin-3 in the development of lymphoma has not been studied.Since kindlin-3 is mainly expressed in the hematopoietic system,whether its role and mechanism are same to that in other solid tumors,and whether it still plays a role in lymphoma as an adhesion-related factor are unknown.The purpose of this study is to explore the expression level of kindlin-3 in NHL cells and its impact on survival and prognosis in NHL patients,to study the influence of kindlin-3 on adhesion,migration,proliferation,apoptosis and cytocycle as well as sensitivity to chemotherapy drugs,of NHL cell lines,and to probe its possible mechanism.It will ignite new ideas for the diagnosis,treatment and prognosis assessment of malignant lymphoma.Methods:(1)Paraffin-embedded pathological specimens from confirmed NHL patients were collected,and the kindlin-3 expression was detected by immunohistochemical staining.Pathological specimens of lymph nodes from patients with reactive hyperplasia were used as normal control.NHL patients were divided into highexpressing group and low-expressing group.The correlation between kindlin-3expression level and clinical data,such as age,sex,clinical stage,leukocyte,hemoglobin,tumor length and diameter,and overall survival were analyzed.(2)Kindlin-3 conditionally knockout mice were established.The cell count,adhesion and spreading of peripheral leukocytes from kindlin-3 knockout mice were tested.(3)The m RNA and protein expression of Kindlin-3 in indolent lymphoma cell lines,SJC,Dohh2,and SC1,and aggressive lymphoma cell lines,Jeko-1 and Raji,were tested using reverse transcription quantitative polymerase chain reaction(RTqPCR)and Western blotting(WB).Raji cell lines with high expression of kindlin-3were selected for RNA interference assay through lentivirus transfection.Si-Kindlin3-Raji cell lines were established.(4)The proliferation,adhesion,migration,apoptosis,cytocycle and sensitivity to chemotherapy drugs of Si-Kindlin3-Raji cell line were tested through Cell Counting Kit-8(CCK-8),adhesion experiment,transwell migration assay,Annexin V/propidium iodide apoptosis assay,cell cycle flow analysis.The Si-Control-Raji cell line was served as a control.Results:(1)Kindlin-3 expression was variable in all lymphoma tissues.The score of every specimen was obtained by the staining intensity and the proportion of positive cells.Depending on the score,the lymphoma patients were divided into highexpressing group and low-expressing group.The Kindlin-3 expression of lymph nodes from the high-expressing group was only comparable to that from patients with reactive hyperplasia.Kindlin-3 expression levels were related to lymphoma subtypes.Kindlin-3 expression level from patients with aggressive lymphoma was higher than that from indolent lymphoma.The median length diameter of the masses from the low-expressing group was 3.1cm,while that from high-expressing group was 5cm.There was a strong positive correlation between Kindlin-3 expression and tumor length diameter of lymphoma,and the subtypes of lymphoma.Through COX proportional risk regression model analysis,age(P= 0.017),hemoglobin(P= 0.01),platelet number(P= 0.007),LDH(P = 0.000),number of extranodal invasion(P=0.014),clinical stage(P=0.004),ECOG score(P= 0.001),IPI risk group(P= 0.000)and mass length diameter(P =0.023)were nine factors affecting overall survival,among which age(P = 0.002),LDH(P = 0.003)and mass length diameter(P = 0.001)were independent risk factors for prognosis in lymphoma patients.(2)The number of mononuclear cells in peripheral blood of Kindlin3-/-mice was significantly larger than that of wild-type mice.The adhesion of Kindlin3-/-mouse leukocytes to fibronectin(Fn)was significantly damaged,when compared with wild-type control,and the ability to spread was lost.(3)The expression of Kindlin-3 in three follicular lymphoma cell lines(SJC,Dohh2,SC1)was significantly lower than that in mantle cell lymphoma(Jeko-1,Mino)and Burkitt lymphoma cell lines(Raji).Kindlin-3 knockdown cell line,SiKindlin-3-Raji,and the control cell line,Si-Control-Raji,were successfully established.(4)The adhesion of Si-Kindlin-3-Raji cells on Fn,ICAM-1 and endothelial cells was significantly weaker than that of Si-Control-Raji cells.Transwell assay showed that the migration of Si-Kindlin-3-Raji cells was decreased,compared with SiControl-Raji cells.By CCK-8 assay,the proliferation of Si-Kindlin3-Raji cells was significantly lower than that of Si-Control-Raji cells on the third day.In the cell apoptosis assay,the apoptosis of Si-Kindlin-3-Raji cells in serum-free culture increased,compared with Si-Control-Raji cells,but the difference was not statistically significant.While,24 h after CTX stimulation,the apoptosis of Si-Kindlin-3-Raji cells was higher than that of Si-Control-Raji cells,and the difference was statistically significant.The number of Kindlin3-Raji cells in G2/M phase was significantly smaller than that of Si-Control-Raji cells.Conclusion:In this paper,the differential expression of Kindlin-3 has been verified for the first time in indolent and aggressive lymphomas.Kindlin-3 may promote tumor growth.Knockdown of Kindlin-3 causes decreased cell adhesion and migration,reduced proliferation,and increased sensitivity to cyclophosphamide,as well as stagnated cytocycle in G0/G1 phase or S phase.In conclusion,Kindlin-3 may play a promoting role in tumor growth in non-Hodgkin’s lymphoma. |
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