【作者】 陈曦；

【导师】 邹立群；

【作者基本信息】 四川大学 ， 肿瘤学（专业学位）， 2021， 博士

【摘要】 背景和目的:结外鼻型NK/T细胞淋巴瘤(Extranodal NK/T-cell lymphoma,nasaltype,ENKTL)与EBV感染密切相关,具有特殊的形态、免疫表型以及生物学行为,其侵袭性高、预后差。近年来,随着治疗方案不断地改进,ENKTL预后得以改善,但仍有部分患者因原发或者继发耐药导致治疗失败或短期复发。对于复发或难治(R/R)ENKTL患者,目前无标准的治疗方案,传统治疗手段疗效差,中位总生存期小于6个月。免疫检查点发挥维持自我耐受从而限制正常组织损伤的作用,而肿瘤细胞可以通过改变免疫检查点蛋白逃避宿主的免疫监视。免疫检查点PD-1和PD-L1已成为包括ENKTL在内的多种恶性肿瘤最具希望的治疗靶点。根据本课题组临床实践及前期文献报道,PD-L1抑制剂在R/R ENKTL中显示出可观的治疗前景,但其疗效存在很大的异质性,原发耐药常见。探索PDL1在ENKTL的调控机制,有利于进一步阻断PD-1/PD-L1信号通路的活化,改善免疫治疗效果。研究表明EBV相关的多类肿瘤PD-L1表达更高,EBV如何改变宿主免疫检查点的研究却罕有涉及。EBV编码的Ⅱ型潜伏膜蛋白2A(Latent membrane protein 2A,LMP2A)在EBV感染相关的多类肿瘤中高表达并诱导细胞增殖,促进肿瘤发生发展。LMP2A具有抗原性,可诱导特异性免疫反应的特点也为免疫疗法提供了新的思路。目前为止,LMP2A在ENKTL中的作用罕见报道。因此,本研究拟探索LMP2A与PD-L1通路之间可能存在的肿瘤免疫学相互作用及相关机制,为揭示ENKTL免疫检查点疗效预测新指标及新靶点提供理论及实践基础。材料和方法:1.评估LMP2A和PD-L1在ENKTL病理组织及细胞系中的表达1)收集由病理科确诊的初治ENKTL及鼻咽炎症病理组织,通过免疫组化对比两组标本LMP2A和PD-L1的表达;2)利用免疫荧光检测分析LMP2A在ENKTL细胞系SNK6和SNT8的表达和分布;3)通过q RT-PCR、Western blot、流式细胞术比较EBV阳性ENKTL细胞系SNK6、SNT8和EBV阴性NK淋巴瘤细胞系KHYG-1、NK92之间LMP2A和PD-L1的表达差异。2.研究LMP2A对ENKTL细胞的生物学作用1)构建靶向LMP2A基因的sh RNA表达载体,基因测序及酶切鉴定;2)慢病毒包装p LKO-sh LMP2A质粒感染ENKTL细胞系SNK6、SNT8敲降LMP2A,通过Western blot检测LMP2A的表达水平,筛选其中2条敲降效率最高的sh RNA,使用嘌呤霉素筛选稳转细胞株;3)通过q RT-PCR、Western blot检测稳转细胞株(SNK6 sh-nc和SNK6 shLMP2A、SNT8 sh-nc和SNT8 sh-LMP2A)LMP2A的敲降效率;4)CCK8细胞增殖实验检测SNK6和SNT8稳转细胞的增殖能力,流式细胞术检测凋亡比例。3.探索LMP2A与PD-L1之间的调控关系1)通过q RT-PCR、Western blot、流式细胞术比较SNK6和SNT8稳转细胞株LMP2A敲降前后PD-L1的表达变化,验证LMP2A与PD-L1之间的调控关系;2)构建p Tomo-LMP2A过表达载体,基因测序及酶切鉴定;3)重组慢病毒p Tomo-LMP2A感染EBV阴性NK细胞淋巴瘤细胞系KHYG-1构建LMP2A过表达细胞株,荧光显微镜观察细胞荧光表达量及流式细胞术检测GFP阳性细胞率鉴定慢病毒感染效率;4)通过q RT-PCR、Western blot、流式细胞术比较LMP2A过表达前后LMP2A和PD-L1的表达,进一步阐明LMP2A与PD-L1之间的调控关系。4.研究LMP2A调控PD-L1表达对T细胞活性的影响1)收集健康志愿者外周血,密度梯度离心法分离T细胞,使用CD3/CD28单抗偶联磁珠激活与扩增T细胞;2)将LMP2A敲降前后的ENKTL细胞SNK6和SNT8与T细胞共培养模拟体内肿瘤微环境,收集上清液,流式细胞术微球阵列法检测上清液中IFN-γ的分泌量以反映T细胞的活化程度。同时由于NK/T细胞淋巴瘤起源于转化的NK细胞或细胞毒性T细胞,本身也是免疫细胞,设置无T细胞共培养组进行比较,明确激活的是T细胞还是转化的NK细胞或细胞毒性T细胞本身;3)在敲降LMP2A的情况下回补PD-L1,Western blot检测LMP2A和PD-L1的表达,再次与T细胞共培养收集上清液检测IFN-γ的变化,验证LMP2A调控PD-L1对T细胞活性的影响。5.LMP2A调控PD-L1表达的机制研究1)提取稳转细胞株SNK6 sh-nc和sh-LMP2A的总RNA,通过mi RNA基因芯片筛选差异表达谱,进行GO和KEGG分析,分析LMP2A敲降前后的差异mi RNA基因以及相关的生物学功能和通路;2)应用生物信息学预测可能靶向调控PD-L1的mi RNA,双荧光素酶报告基因进行靶向性验证;3)通过q RT-PCR检测筛选mi RNA在SNK6和SNT8 sh-nc、sh-LMP2A中的表达,然后分别转染mi RNA mimic或阴性对照NC,通过q RT-PCR、Western blot和流式细胞术检测靶基因的表达,明确ENKTL中LMP2A是否通过筛选的mi RNA调控PD-L1的表达。6.体内研究LMP2A过表达对肿瘤生长、PD-L1表达及肿瘤免疫反应的影响1)重组慢病毒p Tomo-LMP2A感染小鼠T细胞淋巴瘤细胞系EL4,构建EL4LMP2A过表达细胞株;2)通过q RT-PCR、Western blot比较LMP2A过表达前后LMP2A和PD-L1的表达,研究小鼠T细胞淋巴瘤细胞系EL4体外LMP2A与PD-L1之间的调控关系;3)建立C57BL/6小鼠T细胞淋巴瘤LMP2A过表达皮下荷瘤模型,观察肿瘤的生长、体积;4)免疫组化检测肿瘤PD-L1的表达,流式细胞术检测肿瘤组织PD-L1阳性细胞比例、肿瘤浸润性CD8+T细胞的比例及脾脏T细胞IFN-γ分泌。结果:1.LMP2A和PD-L1在ENKTL病理组织及细胞系中呈现高表达1)LMP2A和PD-L1在ENKTL肿瘤组织中表达明显增高:ENKTL组织中LMP2A表达情况为,阴性12.5%,低表达31.25%,高表达56.25%,ENKTL组织PD-L1 100%表达,低表达6.25%,高表达93.75%;鼻咽炎症组织中LMP2A表达情况为,阴性75%,低表达25%,鼻咽炎症组织PD-L1表达阴性占比75%,低表达占比25%;2)在ENKTL细胞系SNK6与SNK8中,LMP2A广泛分布于细胞膜;3)相比于EBV阴性NK细胞淋巴瘤细胞系KHYG-1与NK92,EBV阳性ENKTL细胞系SNK6与SNK8中,LMP2A和PD-L1共表达,且表达明显上调,在m RNA及蛋白水平具有一致性。2.LMP2A促进ENKTL细胞增殖、抑制凋亡1)成功构建EBV阳性细胞SNK6和SNT8 sh-LMP2A稳转细胞株,LMP2A基因敲降效率80%以上;2)SNK6和SNT8 sh-LMP2A与其对照sh-nc比较,细胞增殖能力降低、凋亡增加。3.LMP2A靶向调控PD-L1的表达,两者呈正相关1)ENKTL细胞系SNK6和SNT8 sh-LMP2A与其对照sh-nc比较,PD-L1表达下调,m RNA和蛋白水平具有一致性;2)成功构建EBV阴性细胞KHYG-1 LMP2A过表达细胞株,LMP2A表达增高,PD-L1的表达也随之上调,m RNA和蛋白水平具有一致性。4.下调LMP2A激活T细胞,促进IFN-γ的分泌1)ENKTL细胞系SNK6和SNT8 sh-nc、sh-LMP2A稳转细胞株与T细胞共培养,LMP2A表达下调后IFN-γ的分泌量增多,而无T细胞共培养组LMP2A表达下调后IFN-γ的分泌量无明显变化;2)LMP2A表达下调的同时过表达PD-L1的回补实验结果显示,与T细胞共培养上清液中IFN-γ的分泌量增加的情况被逆转。5.ENKTL中LMP2A通过mi R-200b-3p调控c Jun/PD-L1的表达1)ENKTL细胞系SNK6 sh-LMP2A与SNK6 sh-nc比较,mi RNA基因芯片发现差异表达基因共90个,其中上调48个,下调42个;2)GO富集分析显示,差异mi RNAs主要与细胞生物发育、生物学过程调控、蛋白质结合、转录活性调节、跨膜转运蛋白活性调节等生物学功能相关;KEGG信号通路分析显示涉及到B细胞受体信号通路;3)生物信息学分析表明上调表达差异基因mi R-200b-3p与c Jun的3’-UTR区域具有靶向结合位点,双荧光素酶报告基因验证了mi R-200b-3p直接靶向PDL1基因增强子的元件序列c Jun,与PD-L1之间不存在直接靶向作用;4)SNK6和SNT8细胞中,敲降LMP2A后mi R-200b-3p表达上调;5)LMP2A正常表达的情况下,转染mi R-200b-3p mimic后c Jun下调,敲降LMP2A后转染mi R-200b-3p mimic,c Jun无变化,表明mi R-200b-3p对c Jun的调控依赖于LMP2A;6)转染了mi R-200b-3p mimic后,同时检测PD-L1的表达,PD-L1的表达与c Jun的变化趋势相同。6.体内研究进一步揭示LMP2A促进肿瘤生长,上调PD-L1表达,抑制肿瘤免疫1)成功构建小鼠T细胞淋巴瘤细胞EL4的LMP2A过表达细胞系,体外显示LMP2A表达增高,PD-L1表现出同向协同变化;2)小鼠LMP2A过表达模型中,LMP2A过表达组肿瘤的体积大于对照组;3)LMP2A过表达组与对照组相比,肿瘤细胞PD-L1表达增高;4)肿瘤内浸润性CD8+T细胞减少,脾脏T细胞分泌的IFN-γ降低。结论:1.LMP2A和PD-L1在ENKTL组织及细胞系呈现高表达;2.LMP2A促进ENKTL细胞增殖、抑制凋亡;3.在ENKTL细胞中,敲降LMP2A可以下调PD-L1的表达,同时过表达LMP2A可以上调PD-L1的表达,两者协同正向表达,证实了LMP2A对PD-L1的靶向调控作用;4.敲降LMP2A后能够激活T细胞而非NK/T淋巴瘤细胞本身,促进IFN-γ分泌,即ENKTL细胞中的高表达的LMP2A通过上调PD-L1表达抑制T细胞活性;5.LMP2A通过mi R-200b-3p负性调控c Jun的表达,进一步调节PD-L1的表达,形成LMP2A/mi R-200b-3p/c Jun/PD-L1信号轴;6.体内实验中,LMP2A过表达促进肿瘤细胞PD-L1高表达,降低肿瘤内CD8+T的浸润,减少脾脏T细胞功能性IFN-γ的分泌,进一步揭示LMP2A高表达能够抑制抗肿瘤免疫反应,从而促进肿瘤生长。更多还原

【Abstract】 Background and objective The development of extranodal natural killer/T cell lymphoma,nasal-type(ENKTL)is closely associated with EBV infection.ENKTL possesses specific morphology,immunophenotype and biological behavior,leading to high aggressiveness and poor prognosis.Although the clinical treatment of ENKTL has improved in recent years accompanied with improved prognoses,some patients failed the response to treatment,or relapsed within a short time due to primary or secondary drug resistance.In refractory or relapsed patients,there is no standard treatment,and the efficacy and prognosis of conventional treatments are poor with a median overall survival of less than 6 months.Immune checkpoints play an important role in maintaining self-tolerance and thus limiting the damage of normal tissue,yet tumor cells can evade host immune surveillance by altering immune checkpoint proteins.The immune checkpoints PD-1 and PD-L1 have emerged as the most promising therapeutic targets for a variety of malignancies,including ENKTL.According to our group’s clinical practice and previous reports,PD-L1 inhibitors showed promising results in R/R ENKTL,but heterogeneity in efficacy has also been observed,and primary drug resistance was common.Exploring the regulatory mechanism of PD-L1 in ENKTL is beneficial to further block the activation of PD-1/PD-L1 signaling pathway and improve the immunotherapeutic effect.Moreover,studies have shown a higher PD-L1 expression in a variety of EBVassociated tumors,but the effects of EBV on host immune checkpoints are seldomly explored.The EBV-encoded type II latent membrane protein 2A(LMP2A)was reported to express in a variety of tumors associated with EBV infection and it possesses the ability to induce cell proliferation and promote tumor progression.The antigenic properties of protein LMP2 A,which induce specific immune responses,also provide it as a promising target for immunotherapy.However,the role of LMP2 A in ENKTL has rarely been reported.Therefore,the aim of this study is to explore the possible tumor immunological interactions between LMP2 A and PD-1/PD-L1 pathway and related mechanisms,and to provide a theoretical and practical basis for revealing new indicators and targets for predicting the efficacy of immune checkpoint therapy in ENKTL.Materials and methods 1.Evaluating LMP2 A and PD-L1 expression in ENKTL tissues and cell lines 1)Pathological specimens from patients with ENKTL before treatment and inflammation of the nasopharynx were diagnosed in the pathology department were collected,the expression of LMP2 A and PD-L1 in both groups were detected via immunohistochemical staining.2)The expression and distribution of LMP2 A in ENKTL cell lines SNK6 and SNT8 were dected by immunofluorescence.3)The expression of LMP2 A and PD-L1 were compared in the EBV positive ENKTL cell lines SNK6 and SNT8,and the EBV negative NK cell lymphoma cell lines KHYG-1 and NK92,via quantitative real-time polymerase chain reaction(q RT-PCR),western blot and flow cytometry.2.Exploring the biological functions of LMP2 A on ENKTL cells 1)Four sh RNA expression vectors for the LMP2 A gene were constructed and subjected to gene sequencing and enzyme digestion for identification.2)The lentiviral packaging p LKO-sh LMP2 A plasmid was used to infect ENKTL cell lines SNK6 and SNT8 to knock down gene LMP2 A,and the expression levels of LMP2 A were detected via western blot.Two sh RNAs with high knockdown efficiency were screened to identify construct-stable transfected cell lines by using puromycin.3)The knockdown efficiency of LMP2 A in stable cell lines(SNK6 sh-nc,SNK6 shLMP2 A,SNT8 sh-nc and SNT8 sh-LMP2A)was detected by q RT-PCR and western blot.4)The CCK8 cell proliferation assay was used to detect the proliferation of SNK6,SNT8 sh-nc and sh-LMP2 A cells.Flow cytometry was used to detect the apoptosis of SNK6,SNT8 sh-nc and sh-LMP2 A cells.3.Exploring the regulatory relationship between LMP2 A and PD-L1 1)The expression of PD-L1 before and after LMP2 A knockdown in stably transfected cell lines(SNK6,SNT8 sh-nc and sh-LMP2A)were compared via q RT-PCR,western blot,and flow cytometry,in order to investigate the regulatory relationships between LMP2 A and PD-L1.2)A p Tomo-LMP2 A overexpression vector was constructed for gene sequencing and enzyme digestion identification.3)The recombinant lentivirus p Tomo-LMP2 A was used to infect the EBV negative NK cell lymphoma cell line KHYG-1,to construct a LMP2A-overexpressing cell line.4)The efficiency of lentiviral infection was determined via fluorescence microscopy and flow cytometry of green fluorescent protein(GFP)-positive cells.The expression of LMP2A and PD-L1 before and after the LMP2 A overexpression were compared via q RT-PCR,western blot and flow cytometry,for further investigate the regulatory relationships between LMP2 A.4.Analyzing the effect of LMP2 A regulation of PD-L1 expression on T cell activity 1)Peripheral blood was collected from healthy volunteers.T cells were isolated by using density gradient centrifugation,and activated and amplified by using CD3/CD28 monoclonal antibody-coupled magnetic beads.2)The LMP2A-silenced ENKTL cell lines SNK6 and SNT8 were co-cultured with T cells to simulate the in vivo tumor microenvironment.The IFN-γ secretion in the supernatant was detected via a flow cytometry microsphere array method,to reflect the level of T cell activation.Notably,NK/T cell lymphoma originates from transformed NK cells or cytotoxic T cells,which are immune cells themselves.Thus,in order to confirm whether the activation involving the transformed NK cells or cytotoxic T cells,a non-T cell co-culture group was set up for comparison.3)After back-supplementation of PD-L1 in the presence of LMP2 A knockdown cells,western blot was employed to detect the changes in the expression of LMP2 A and PD-L1 protein,and then the cells were co-culture with T cells again to detect in the secretion of IFN-γ,to verify the effects of LMP2 A on T cell activity.5.Exploring the mechanism of LMP2 A regulates PD-L1 expression 1)Total SNK6 sh-nc and SNK6 sh-LMP2 A RNA were extracted from stable transient cell lines,and differential expression profiles were screened via micro RNA gene microarray.Differential expressed genes involved in biological functions after LMP2 A silencing were analyzed based on databases of gene ontology(GO)and KEGG.2)Bioinformatics were used to predict the possible mi RNA regulating PD-L1,and verification of targeting was conducted via the dual luciferase reporter gene assay.3)The q RT-PCR was performed to detect the screened mi RNA expression in SNK6,SNT8 sh-nc and sh-LMP2 A cells.Expression levels of target molecules in the screened mi RNA mimic or negative control were detected via q RT-PCR,western blotting,and flow cytometry to verify whether LMP2 A in ENKTL regulates PD-L1 expression through the screened mi RNA.6.Investigating the effects of LMP2 A overexpression on tumor growth,PDL1 expression and tumor immune response in vivo 1)The mouse T-cell lymphoma cell line EL4 was infected with recombinant lentivirus p Tomo-LMP2 A and the EL4 LMP2 A overexpressing cell line was constructed.2)The regulatory relationship between LMP2 A and PD-L1 was clarified in vitro via comparing the expression of LMP2 A and PD-L1 before and after LMP2 A overexpression by using q RT-PCR and western blot.3)A C57BL/6 mouse T cell lymphoma LMP2A-overexpressing tumor model was established via subcutaneous injection,and the growth and volume of tumor were monitored.4)PD-L1 expression in tumor was determined via immunohistochemical analysis,and the proportions of PD-L1 positive cells in tumor tissues were determined via flow cytometry,as well as the proportions of tumor-infiltrating CD8+ T cells and the secretion of IFN-γ in spleen T cells.The effects of LMP2 A overexpression on PDL1 expression and tumor immune responses were verified in vivo.Results 1.LMP2 A and PD-L1 were high expression in ENKTL pathological tissues and cell lines 1)The expressions of LMP2 A and PD-L1 were significantly increased in ENKTL tumor tissues.LPM2 A expression in ENKTL tissues was 12.5% negative,31.25% low and 56.25% high;PD-L1 expression in ENKTL tissues was 100% expression,6.25% low and 93.75% high.LPM2 A expression in nasopharyngeal inflammatory tissues was 75% negative and 25% low;PD-L1 in nasopharyngeal inflammatory tissues was 75% negative and 25% low.2)LMP2A was widely distributed on the surface of cell membrane in the ENKTL cell lines SNK6 and SNK8.3)LMP2A and PD-L1 expressions were also increased in the EBV positive ENKTL cell lines SNK6 and SNT8,compared with the EBV negative NK cell lymphoma cell lines KHYG-1 and NK92,in consistent with the m RNA level and the protein level.2.LMP2 A promoted ENKTL cell proliferation and inhibited apoptosis 1)The ENKTL cell lines SNK6 and SNT8 sh-LMP2A-stable cell lines were successfully constructed.2)SNK6 and SNT8 sh-LMP2 A showed slowed cell proliferation and increased apoptosis compared with their control.3.LMP2 A regulated PD-L1 expression,and the two were positively correlated 1)In consistent with m RNA and protein levels,the PD-L1 expression in ENKTL cell lines SNK6 and SNT8 sh-LMP2 A were down-regulated compared with their control sh-nc.2)The EBV-negative KHYG-1 LMP2 A overexpression cell line was successfully constructed,and the LMP2 A expression was increased and PD-L1 expression was up-regulated,in consistent with the m RNA and protein levels.4.Down-regulation of LMP2 A expression activated T cells and promoted the secretion of IFN-γ 1)ENKTL cell lines SNK6 and SNT8 sh-nc,sh-LMP2 A stable-transformed cell lines co-cultured with peripheral blood primary T cells,exhibited increased secretions of IFN-γ after down-regulation expression of LMP2 A,whereas the IFN-γ secretion of the non-T cell co-cultured group did not exhibit significantly differences.2)The increased secretion of IFN-γ of cells co-cultured with T cells was reversed by back-supplementation of PD-L1 in LMP2 A knockdown cells.5.LMP2 A in ENKTL regulated c Jun/PD-L1 expression via mi R-200b-3p 1)Compared with the SNK6 sh-nc cell line,there were 90 differentially expressed genes in total in the ENKTL cell line SNK6 sh-LMP2 A.Thereinto,48 genes were upregulated and 42 genes were downregulated as determined via mi RNA gene microarray.2)GO enrichment analysis indicated that the differential expressed mi RNAs were mainly involved in cell development,regulation of biological processes,protein binding,regulation of transcriptional activity and transmembrane transporter protein activity.Analysis of the KEGG signaling pathway indicated that they mainly involved in the B cell receptor signaling pathway.3)Bioinformatics analysis indicated that the upregulated expression of the DEG mi R-200b-3p had a target binding site to the 3’-UTR of c Jun,which is a component sequence of the PD-L1 gene enhancer.The dual luciferase reporter gene verified that c Jun was the direct target of mi R-200b-3p,and there is no direct targeting interaction with PD-L1.4)In ENKTL cell lines SNK6 and SNT8,the expression of mi R-200b-3p was upregulated after knockdown of LMP2 A.5)In ENKTL cells with normal LMP2 A expression,c Jun was downregulated after transfection with mi R-200b-3p mimic,whereas in the case of LMP2 A knockdown,c Jun was unchanged after transfection with mi R-200b-3p mimic.This result confirmed that mi R-200b-3p was dependent on LMP2 A to regulate c Jun.6)The expression of PD-L1 exhibited similar variation tendency with that of c Jun after transfection with mi R-200b-3p mimic under normal expression or knockdown of LMP2 A in ENKTL cells.6.LMP2 A promoted tumor growth in vivo,regulated PD-L1 expression and inhibited tumor immune response 1)The LMP2A-overexpressing EL4 mouse T cell lymphoma cell line was successfully constructed and exhibited an increased LMP2 A expression in vitro,followed by an increased expression of PD-L1.2)In the mouse T cell lymphoma transplantation tumor model the volume of tumors in the LMP2A-overexpressing group were significantly higher than those in the control group.3)The LMP2A-overexpressing group exhibited higher PD-L1 expression,a lower percentage of infiltrating CD8+ T cells in the tumor,and lower IFN-γ in spleen T cells compared with the control group.Conclusions 1.LMP2 A and PD-L1 were highly expressed in both ENKTL pathological tissues and cell lines.2.LMP2 A promoted the proliferation and reduced the apoptosis in ENKTL cells.3.In ENKTL cells,the knockdown of LMP2 A could downregulate PD-L1 expression,whereas the overexpression of LMP2 A could upregulate PD-L1 expression,indicating that LMP2 A could target and regulate PD-L1 expression.4.Knockdown LMP2 A in ENKTL cells activated T cells but not NK/T lymphoma cells per se,contributing to the increased IFN-γ secretion.High expression of LMP2 A in ENKTL cells inhibited T cell activity by upregulating PD-L1 expression.5.LMP2 A promoted the expression of mi R-200b-3p,which negatively regulating the expression of c Jun,consequently forming the LMP2A/mi R-200b-3p/c Jun signaling axis and ultimately regulating PD-L1 expression.6.In in vivo experiments,the overexpression of LMP2 A increased the PD-L1 expression in tumor cells,decreased the infiltration of CD8+ T in tumors,and significantly reduced the secretion of functional IFN-γ by splenic T cells,further revealed that high LMP2 A expression could suppress anti-tumor immune responses and thus promote the tumor growth.更多还原