| Lymphoma is one of the most common hematological malignancies.Although they are potentially curable diseases,the survival rates of lymphoma depend on mutiple factors.Burkitt lymphoma(BL)is a highly proliferative B-cell tumor.People who have a poor survival prognosis need intensive chemotherapy.So far,no effective targeted drug has been approved for the treatment of BL.Therefore,lots of researches pay attention to finding effective targets for the treatment of BL.Sperm-associated antigen 6(SPAG6)was first detected in human testis tissue.Its main function is to participate in the maturation of germ cells and maintain sperm vitality and fertility.Current researches have found that SPAG6 is defined as a new cancer-testis antigen(CTA)in solid tumors such as lung cancer and breast cancer.and the CTA gene is considered a tumor marker and a potential drug candidate for cancer treatment.And in the latest study,SPAG6 was highly expressed in CALM/AF10-positive leukemia and childhood acute myeloid leukemia(AML),and it was related to the poor prognosis of AML.However,the role and molecular mechanisms of SPAG6 in the other hematological malignancies,especially lymphomas,are less studied.ObjectiveIt was found that there was a correlation between the expression of SPAG6 and the prognosis of patients with lymphoma by TCGA database analysis.This study aimed to clarify the roles of SPAG6 expression in the occurrence and development of BL and explore its molecular mechanism.Therefore to open up new ideas for the exploration of new targets for clinical BL intervention.MethodsTCGA database data analysisTCGA database data analysis was used to compare the relationship between the levels of SPAG6 and the prognosis of patients with lymphoma.In vitro study1.Human peripheral blood normal B lymphocytes(IM-9)and different human BL cell lines(CA46,NAMALWA,Daudi,Raji)were cultured.The m RNA levels and protein levels of SPAG6 in different cell lines were performed by quantitative PCR and Western Blot assays.2.Daudi and Raji SPAG6 knockdown stable cell lines were constructed by transfecting with lentivirus.The knockdown efficiency of SPAG6 was detected by quantitative PCR and Western Blot methods.CCK8,Annexin V-PI flow cytometer and Western Blot assays were used to verify the effects of the proliferation and apoptosis in Daudi and Raji cells after knocking down SPAG6 expression.3.CA46 and NAMALWA cells were transfected with SPAG6 overexpression plasmids,and the stable overexpressed SPAG6 cells were constructed.The overexpression efficiency of SPAG6 was detected by quantitative PCR and Western Blot methods.The effects of SPAG6 overexpression on the proliferation of CA46 and NAMALWA cells were verified by CCK8 assay.4.Western Blot assay was used to detect the protein leves of PTEN/PI3K/AKT pathway related proteins(AKT,p-AKT,PTEN)in the SPAG6 depleted or controlled Daudi and Raji cells.The purpose of the experiment is to verify the effects of SPAG6knockdown on PTEN/PI3K/AKT pathway in Daudi and Raji cells.5.The PI3K inhibitor LY294002 was used to treat CA46 and NAMALWA overexpressing SPAG6 cells.The CCK8 assay was used to detect the cell proliferation of three groups(pcDNA,pcDNA-SPAG6,pcDNA-SPAG6/LY294002).CA46 and NAMALWA stable overexpressing SPAG6 cells were transfected with PTEN overexpression plasmids.The proliferation of three groups(pcDNA,pcDNA-SPAG6,pcDNA-SPAG6/PTEN)was measured by CCK8 assay.6.PTEN inhibitor SF1670 was used to treat Daudi and Raji SPAG6 depleted cells.The effectes on cell proliferation and apoptosis of three groups(nc-sh RNA,sh SPAG6,sh SPAG6/SF1670)were performed by CCK8,Annexin V-PI flow cytometry and Western Blot assays.siRNA was used to knock down PTEN expression in Daudi and Raji SPAG6 depleted cells.The roles of proliferation and apoptosis in three groups(nc-sh RNA,sh SPAG6,sh SPAG6/si PTEN)were checked using CCK8,Annexin V-PI flow cytometry and Western Blot assays.In vivo studyA subcutaneous tumor formation model of SCID mice was established.15 four-week-old male SCID mice(average weight 17.5g)were randomly divided into three groups(5 in each group).Raji-nc-sh RNA cells(5×10~6cells,50?l)were injected subcutaneously into the left armpit of 5 mice,and one week later DMSO(50?l,daily injection)was injected intraperitoneally into mice of the Raji-nc-sh RNA group.Raji-sh SPAG6 cells suspension(5×10~6Raji-sh SPAG6 cells,50?l)were injected subcutaneously into the left armpit of 5 mice,and one week later DMSO(50?l,daily injection)was injected intraperitoneally into mice of the Raji-sh SPAG6 group.Raji-sh SPAG6 cells(5×10~6,50?l)were injected subcutaneously into the left armpit of 5 mice,and one week later SF1670(10?mol/kg,50?l,daily injection)was injected intraperitoneally into mice of Raji-sh SPAG6/SF1670 group.The length and width of the tumors were measured every 3 days.According to the formula V=1/2ab~2(a:length of tumor,b:width of tumor),the volume of tumors in each group were calculated.After 3weeks treatment,mice were euthanized and tumors were harvested and weighed.The expression of proliferation-associated antigens Ki67 and PCNA in tissues was measured by Immunohistochemistry assay.TUNEL staining was used to detect the apoptosis in tumor tissues.Statistical analysisData analysis was performed using SPSS Statistics 20.0.Data are expressed as mean±standard deviation((?)±s)and from at least three independent experiments.The results were calculated using Graphpad Prim 7.0.When the data conformed to the normal distribution and the variances were similar,student's t test was used.One-way ANOVA statistical method was used in multiple groups(3 or more).Kaplan-Meier curve was performed and analyzed by log-rank test.P<0.05 was considered statistically significant.ResultsTCGA database data analysisThe TCGA database shows that the expression of SPAG6 is correlated with the prognosis of patients with lymphoma.The higher expression of SPAG6 has the worse prognosis in lymphoma patients.Conversely,the lower expression of SPAG6 has the better prognosis in lymphoma patients.This results show that the expression of SPAG6may be closely related to the occurrence and development of lymphoma.In vitro study1.The expression of SPAG6 is higher in different lymphoma cell lines than in normal B-lymphoid IM-9 cellsThe SPAG6 expression in human BL cell lines(CA46,NAMALWA,Daudi,Raji)was 1.80±0.26;2.88±0.31;6.17±0.65;7.70±1.14 times higher than that of normal B-lymphoid IM-9 cells by quantitative PCR assay.There were statistically significant differences(all P<0.05).At the same time,the expression of SPAG6 in protein levels of BL cells(CA46,NAMALWA,Daudi,Raji)was higher than in IM-9 cells by western Blot assay.The levels of SPAG6 were as follows:IM-9<CA46<NAMALWA<Daudi<Raji.There was statistically significant difference between BL cells and IM-9 cells(all P<0.05).2.Silencing SPAG6 inhibits the proliferation and promotes the apoptosis in the BL cellsWe first stably constructed SPAG6 knockdown Daudi and Raji cells and found that the expression of SPAG6 significantly reduced in SPAG6 knockdown cells by quantitative PCR and western Blot assay(all P<0.001).Furthermore,CCK8 assay showed that SPAG6 knockdown group(SPAG6-sh RNA)had a significantly reduced proliferation ability after 72 hours compared with control group(nc-sh RNA)(all P<0.05).In addition,Annexin V-PI flow cytometry also showed that the apoptotic ratio in the sh SPAG6 group was significantly increased compared with nc-sh RNA group(all P<0.01).Finally,we also found that the expression of anti-apoptotic protein Bcl-2 was significantly down-regulated and the expression of pro-apoptotic protein Bax was significantly up-regulated,and the expressions of Cleaved-caspase-8,Cleaved-caspase-3and Cleaved-PARP were significantly increased in sh SPAG6 group compared with the nc-sh RNA group by western Blot assay.3.SPAG6 overexpression,promotes the proliferation of BL cellsThe SPAG6 overexpressed or controlled plasmids were transfected into CA46 and NAMALWA cells.The results showed that the expression of SPAG6 showed a significant increase in SPAG6 overexpression group compared with control group by quantitative PCR and western Blot assay(all P<0.01).Next,to investigate the role of SPAG6 overexpression on cell proliferation,we performed CCK8 assay and the results showed that the OD values of CA46 and NAMALWA cells were significantly higher starting from 48 hours in pcDNA-SPAG6 group than in pcDNA group(all P<0.05).These data suggested that SPAG6 overexpression promotes the proliferation ability of CA46 and NAMALWA cells.4.The relationship between SPAG6 knockdown and PTEN/PI3K/AKT pathway-related proteins expressionWe tested the expression of PTEN/PI3K/AKT pathway-related proteins by western Blot assay.The results showed that the protein levels of PTEN were significantly up-regulated and the protein levels of p-AKT protein were significantly down-regulated in the sh SPAG6 groups compared with the nc-sh RNA groups.5.PTEN overexpression or LY294002(PI3K inhibitor)reverse the effect on SPAG6overexpression mediated proming proliferation of BL cellsFirst,we used CCK8 assay to detect the proliferation of three groups(pcDNA group,pcDNA-SPAG6 group and pcDNA-SPAG6/PTEN group)and the results showed that the proliferation ability of pcDNA-SPAG6 groups was significantly enhanced compared with pcDNA groups(all P<0.05)in CA46 and NAMALWA cells..However,there was no significant difference between pcDNA groups and pcDNA-SPAG6/PTEN groups in proliferative capacity(all P>0.05).There was a significantly lower proliferation ability in pcDNA-SPAG6/PTEN groups than in pcDNA-SPAG6 groups(all P<0.05).Furthermore,LY294002,a PI3K inhibitor,was used to treat with stable SPAG6overexpressed CA46 and NAMALWA cells.The proliferation in pcDNA,pcDNA-SPAG6,pcDNA-SPAG6/LY294002 groups was detected by CCK8 test.The results showed that there was no statistical difference between pcDNA-SPAG6/LY294002groups and pcDNA groups(all P>0.05).While the proliferative capacity in pcDNA-SPAG6/LY294002 group significantly reduced compared with pcDNA-SPAG6 group(all P<0.01).6.PTEN knockdown or SF1670(PTEN inhibitor)reverse the effects of SPAG6knockdown on the proliferation and apoptosis of BL cellsThe CCK8 and Annexin V-PI flow cytometry assays were used to detect the proliferation and apoptosis after knocking down PTEN expression in SPAG6 knockdown cells.The results showed that sh SPAG6 group was significantly decreased in cell proliferation and increased in apoptosis compared with nc-sh RNA group.The effect of knocking down SPAG6 on cell proliferation was reversed after knocking down PTEN by siRNA.Specifically,the cell proliferation in the sh SPAG6/si PTEN group significantly increased,and the apoptotic ratio significantly decreased compared with the sh SPAG6group(all P<0.05).However,there was no statistical difference in proliferation and apoptosis between nc-sh RNA group and sh SPAG6/si PTEN group(all P>0.05).We further treated Daudi and Raji cells with the SF1670(PTEN inhibitor),and also found that SF1670 reversed the effects of SPAG6 knockdown on the proliferation and apoptosis of BL cells.In vivo study1.Knockdown of SPAG6 inhibits tumor growth and PTEN inhibitor reverse the effect on knockdown SPAG6 mediated supressing tumor growth in vivoAnimal experiment results showed that the tumor sizes and weights of the Raji-sh SPAG6 group were significantly reduced compared with the Raji-nc-sh RNA group(all P<0.05).However,there was no statistical difference between Raji-nc-sh RNA group and Raji-sh SPAG6/SF1670 group(all P>0.05).Compared with the Raji-sh SPAG6 group,the tumor sizes and weights in Raji-sh SPAG6/SF1670 group significantly increased(all P<0.05).2.Knockdown SPAG6 induces tumor growth via PTEN/PI3K/AKT pathway in vivoWe next used immunohistochemistry assay to measure the proliferation-related proteins(Ki67 and PCNA).The results showed that the expression of Ki67 and PCNA in the Raji-sh SPAG6 group was significantly reduced compared with the Raji-nc-sh RNA group(all P<0.05).However,there was no statistical difference between Raji-nc-sh RNA group and Raji-sh SPAG6/SF1670 group(all P>0.05).Compared with the Raji-sh SPAG6 group,the expression of Ki67 and PCNA in the Raji-sh SPAG6/SF1670 group significantly increased(all P<0.05).Furthermore,we used TUNEL staining assay to check the apoptosis ratio in tumor tissues.The positive percentage of TUNEL in the Raji-sh SPAG6 group was significantly higher compared with the Raji-nc-sh RNA group(P<0.05).However,there was no statistical difference between Raji-nc-sh RNA group and Raji-sh SPAG6/SF1670 group(P>0.05).Compared with the Raji-sh SPAG6 group,the TUNEL positive percentage of the Raji-sh SPAG6/SF1670 group was significantly reduced(P<0.05)..ConclusionThe expression of SPAG6 gene was negatively correlated with the prognosis of lymphoma patients.SPAG6 promotes the proliferation and inhibits the apoptosis in Burkitt lymphoma cells via PTEN/PI3K/AKT pathway. |
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