The Biological Effect And Molecular Mechanism Of Lumican In NK/T-cell Lymphoma

Extranodal NK/T-cell lymphoma(nasal type)is a type of non-Hodgkin's lymphoma(NHL),accounting for about 2% to 10% of NHL,which is very rare in western populations but rather common among Asians,Mexicans,and South Americans.Nasal NK/T-cell lymphomas are a distinct clinicopathological entity,which is rare and aggressive,with poor outcome,but the exact pathogenesis and the optimal therapy of this disease still remain unclear.Further study of the etiology and molecular mechanisms in NK/T-cell lymphomas helps to evaluate the early diagnosis,treatment and prognosis of NKTCL and provide an important theoretical basis for exploring a new therapeutic target.Lumican(LUM)is located on human chromosome 12q21.3-q22,whose product is a leucine-rich prototype protein polysaccharide with keratan side chain,which is the main component of the stratum corneum,skin and muscle connective tissue.LUM is widely expressed in tissues such as skin,blood vessels,lungs,breasts,pancreas,colorectal,articular cartilage and other tissues.LUM is an important component of the extracellular matrix,and some studies have shown that the abnormal expression of LUM gene or interaction with integrins can not only indirectly affect the invasion of tumor,but also regulate the formation of tumor bloodvessels by participating in the formation of epithelial cell tube-like structure,thus affecting tumor proliferation.The study of LUM in NK/T cell lymphoma has not been reported yet.At present,the biological effect and molecular mechanism of LUM on development of NK/T cell lymphoma are needed to be further studied.In this study,the expression of LUM in extranodal nasal NK/T cell lymphoma and reactive proliferative lymphnode was detected by immunohistochemistry.The relationship of positive expression of LUM in NK/T cell lymphoma with clinical pathological parameters/clinical efficacy was analyzed.Then,we constructed LUM overexpression vector and recombinant lentivirus carried LUM,and NK/T cell lymphoma YTS cells were infected with recombinant lentivirus to obtain stable expression YTS-LUM cell line.The effect of LUM overexpression on the colony formation,proliferation,apoptosis,cell cycle,cell invasion and chemosensitivity of NK/T cell lymphoma cells were examined by cell and molecular biological methods.The effect of LUM overexpression on proliferation-and apoptosis-related proteins as well as NF-?B and JAK/STAT3 signaling pathways was detected by Western blot.In vivo,we constructed the tumor animal models,and further studied the effect of LUM overexpression on the growth of NK/T cell lymphoma.This study was designed to explore the biological function and molecular mechanism of LUM in NK/T cell lymphoma and provide a vital candidate molecular marker and target gene for the clinical treatment of NK/T cell lymphoma.Purpose:1.To detect the expression of LUM in extranodal nasal type NK/T cell lymphoma and reactive hyperplasia lymph nodes,and analyze the relationship between LUM and clinicopathological parameters and curative effect.2.To study the effect of LUM overexpression on the colony formation,proliferation,apoptosis,cell cycle,cell invasion and chemosensitivity of NK/T cell lymphoma cells,and the effect of LUM overexpression on the expression of proliferation-and apoptosis-related proteins as well as NF-?B and JAK/STAT3 signaling pathways.3.To study the effect of LUM on growth of xenograft tumor and the the expression of proliferation-and apoptosis-related proteins as well as the NF-?B and JAK/STAT3 signaling pathways in xenograft tumor.Main Content:The first part: The study on expression of LUM in NK/T cell lymphoma Methods1.Immunohistochemical method was used to detect the expression of LUM and PCNA in exogenous nasal type NK/T cell lymphoma and reactive hyperplasia lymph nodes.2.The relationship of the positive expression rate of LUM and clinicopathological parameters in the NK/T cell lymphoma was analyzed.3.We analyzed the relationship of positive expression rate of LUM and therapeutic effect.Results1.Immunohistochemical result exhibited that the positive expression rate of LUM in NK/T cell lymphoma(25%)was significantly lower than that in reactive hyperplasia lymph nodes(79.2%),P<0.001.2.There was no significant relationship between the positive expression rate of LUM and age,sex,place of birth,clinical stage as well as lactate dehydrogenase(LDH)in NK/T cell lymphoma(P>0.05).The positive expression rate of LUM in tumor tissues of patients with PCNA weak expression was significantly higher than that in tumor tissues of patients with PCNA strong expression,which indicated that LUM was closely related to PCNA index,P<0.05.3.The results exhibited that the positive rate of LUM in NK/T cell lymphoma in the effective group(CR+PR)(48.1%)was significantly higher than that in the ineffective group(SD+PD)(15.6%),P<0.05.The second part : The study on the biological function and molecular mechanism of LUM in YTS cells Methods1.LUM gene was combined with lentiviral vector pCDH-CMV-MCS-EF1-Puro to construct lentiviral overexpression vector plenti-LUM,and then sequenced.Lentivirus package was performed and then YTS lymphoma cells were infected with lentivirus,followed by the puromycin selection.2.The expression of LUM,proliferation-and apoptosis-related proteins and the NF-?B and JAK/STAT3 signaling pathway were detected by Western blot.3.The effect of LUM overexpression on colony formation of YTS lymphoma cells was detected by cell cloning assay.4.The effect of LUM overexpression on proliferation of YTS cells was detected by CCK-8 assay.5.The effect of LUM overexpression on cell cycle and apoptosis of YTS cells was detected by flow cytometry assay.6.The effect of LUM overexpression on the invasive ability of YTS cells was detected by transwell assay.7.The effect of LUM overexpression on chemotherapy sensitivity of purinomycin in YTS cells was measured by CCK-8 assay.Results1.The result of pLenti-LUM lentiviru expression vector colony PCR and gene sequencing revealed that the molecular size and sequence of LUM were in line with the information from NCBI,which showed that LUM overexpressing lentiviral vector plasmid(pLenti-LUM)was successfully constructed.2.Cell cloning assay and CCK-8 assay revealed that LUM overexpression significantly inhibited the colony formation and the proliferation of YTS cells.3.Flow cytometry analysis showed that LUM overexpression could significantly promote the apoptosis in YTS cells,and prevent cell cycle progression.4.Transwell assay revealed that LUM overexpression could significantly inhibitthe invasion of YTS cells.5.Western blot analysis showed that LUM overexpression could significantly promote the expression of pro-apoptotic protein p53,Bax and caspase-3,and inhibit the expression of anti-apoptosis protein Bcl-2 and proliferation-related proteins CCND1 and myc.LUM overexpression significantly inhibited the expression of NF-?B and JAK/STAT3 signaling pathway-related proteins NF-?B p65,p-JAK1,p-JAK2 and p-STAT3 in YTS cells.6.LUM overexpressing could enhance the sensitivity of YTS cells to chemotherapeutic drug adriamycin.The third part: The effect of LUM overexpression on the growth of NK/T cell lymphoma in animals Methods1.YTS,YTS-Con and YTS-LUM cells were inoculated subcutaneously with the right anterior limb of BABL/nude female mice(nude mice)to construct lymphoma animal models.The transplanted tumor volume was observed every 3 day,and the transplanted tumor was weighed after 32 days.2.The expression of LUM in transplanted tumor was detected by Western blot.3.The expression of proliferation-and apoptosis-related proteins and NF-?B and JAK/STAT3 related protein NF-?B p65,p-JAK1,p-JAK2,p-STAT3 and STAT3 was detected by Western blot in the tumor tissues.Results1.The weight and volume detection of YTS tumor in each group showed that LUM overexpression significantly inhibited tumor growth.2.The expression of LUM protein in YTS-LUM group was significantly increased than that in control group by Western blot.3.Compared with the control group,the expression of pro-apoptotic protein p53,Bax and caspase-3 in the YTS-LUM group were significantly increased,and the expression of anti-apoptosis protein Bcl-2 and proliferation-related proteins CCND1 and myc was significantly decreased.4.Compared with the control group,the expression of NF-?B and JAK/STAT3 signal pathway related proteins NF-?B p65,p-JAK1,p-JAK2 and p-STAT3 were significantly decreased in YTS-LUM group,which indeicated that LUM overexpression inhibited the activity of the NF-?B and JAK/STAT3 signaling pathways.Conclusion1.The expression of LUM in NK/T cell lymphoma was significantly decreased,and there were not significantly relationship between the positive expression rate of LUM and age,sex,location,clinical stage,as well as lactate dehydrogenase,but LUM expression was closely related with the therapeutic effect and PCNA expression.2.Cell experiments showed that LUM overexpression may inhibit lymphoma cell proliferation,clone formation,invasion,block cell G1 phase,promote apoptosis and enhance the sensitivity of the cells to the chemotherapy drug adriamycin by regulating the NF-?B and JAK/STAT3 signaling pathway.3.In vivo experiments showed that LUM overexpression may inhibit the growth of transplanted tumor in mice by regulating the NF-?B and JAK/STAT3 signaling pathway.