Exploring The Mechanism Of Xiaoliufang In Regulating Lymphoma Tumor Microenvironment And Inhibiting Proliferation Based On MiR-532-3p/β-catenin Signaling Pathway

Objective:The purpose of this study was to verify the clinical efficacy and safety of Xiaoliufang prescription in the treatment of Diffuse large B lymphoma(DLBCL).observe the effects of Xiaonao prescription on lymphoma proliferation,apoptosis and tumor microenvironment,and to reveal the anti-lymphoma mechanism of Xiaonao prescription from the perspective of regulating miR-532-3p/β-catenin signaling pathway and regulating tumor microenvironment in vivo and in vitro.Methods:(1)Sixty DLBCL patients who met the diagnostic criteria,exclusion criteria and exclusion criteria were collected and divided into control group(30 cases)and treatment group(30 cases)according to random number table method.The control group was given R-CHOP chemotherapy regimen,and the treatment group was given Xiaoliufang on the basis of the control group,which was added and subtracted according to the syndrome.After 4 cycles of treatment(21 days/cycle),before and after 4 cycles of treatment,the clinical efficacy,TCM syndrome score,safety related indexes,the expression of peripheral blood serum(IL-2,IL-4,IL-6,IL-10),the expression of miR-532-3p and β-catenin in peripheral blood lymphocytes of 2 groups were observed.(2)The expression of miR-532-3p in lymphoma sample tissues was analyzed using the GEO database and in cell lines by quantitative reverse transcription(QRT)-PCR miR-532-3p mimic and inhibitor cell were transfected using Lipofectamine 2000,and the expression of miR-532-3p was detected by RT-PCR.CCK-8 assay,Annexin V/PI,Western Blotting and ELISA were used to detect the effects of overexpression/silencing of miR-532-3p on the proliferation,apoptosis,apoptosis protein and tumor microenvironment of lymphoma cells U937 and Jurkat,respectively.The downstream target genes of miR-532-3p were predicted by the miRanda database,and β-catenin was verified as the direct downstream target gene of miR-532-3p by luciferase gene reporting assay.β-catenin overexpression plasmid was constructed,and β-catenin m RNA and protein expression were detected by(QRT)-PCR and Western Blotting to verify the construction of β-catenin overexpression plasmid.Proliferation,apoptosis,apoptosis-related proteins,tumor microenvironment related indicators(IL-2,IL-4,IL-6,IL-10)of miR-NC,miR-532-3p mimic,OE-β-catenin mimic and miR-532-3p mimic were detected by CCK-8,Annexin V/PI,Western Blotting,and ELISA.The subcutaneous xenograft tumor model of nude mice with lymphoma was established and divided into miR-NC group and miR-532-3p mimic group.Tumor volume and weight of the two groups were measured,and tissue morphology was observed by HE staining.The expressions of miR-532-3p and β-catenin in the two groups were detected by(QRT)-PCR and Western Blotting,and the expressions of IL-2,IL-4,IL-6 and IL-10 in peripheral blood of the two groups were detected by ELISA.(3)Lymphoma cell lines U937 and Jurkat cells were cultured in vitro,which were divided into control group and Xiao Liu Fang group(20mg/m L,40mg/m L,80mg/m L).CCK-8 and Annexin V/PI were used to detect the effects of Xiao Liu Fang on the proliferation and apoptosis of lymphoma cells at different doses and at different time.The expressions of IL-2,IL-4,IL-6 and IL-10 in the supernatant of lymphoma cell culture were detected by ELISA.The subcutaneous transplantation tumor model of nude mice with lymphoma was established in vivo and divided into control group,Xiao Liu Fang group,CTX group,and Xiao Liu Fang +CTX group.The volume and weight of subcutaneous transplantation tumors in the four groups were measured,and tissue morphology was observed by HE staining.The expressions of miR-532-3p andβ-catenin in the two groups were detected by(QRT)-PCR and Western Blotting,and the expressions of IL-2,IL-4,IL-6 and IL-10 in peripheral blood of nude mice in the two groups were detected by ELISA.(4)CCK-8,Annexin V/PI and Wester Blotting were used to detect the cell proliferation,apoptosis and apoptotic proteins of lymphoma cells in the control group,Xiao Liu Fang group,Xiao Liu Fang +miR-532-3p inhibitor group and miR-532-3p inhibitor group,respectively.The expressions of miR-532-3p,β-catenin,IL-2,IL-4,IL-6 and IL-10 in each group were detected by(QRT)-PCR,Western Blotting and ELISA,respectively.Results:(1)Clinical observation showed that after 4 cycles of treatment,the expression of miR-532-3p in peripheral blood lymphocytes of both the control group and the treatment group was up-regulated(P <0.0001).However,compared with the control group,the expression level of miR-532-3p in peripheral blood lymphocytes of the treatment group was more significantly increased(P <0.0001).After 4 cycles of treatment,the expression level of β-catenin in peripheral blood lymphocytes of both control group and treatment group decreased(P <0.0001),but the decrease level ofβ-catenin in treatment group was more obvious(P <0.05).After 4 cycles of treatment,the expression levels of IL-4,IL-6 and IL-10 in the two groups were all down-regulated before and after treatment(P <0.05),while the expression levels of IL-2 were up-regulated(P <0.0001).However,after treatment,the down-regulated levels of IL-4,IL-6 and IL-10 in the treatment group were significantly better than those in the control group(P <0.05),and the up-regulated levels of IL-2 were significantly different from those in the control group(P <0.05).Clinical curative effect,15 cases were effective,9 cases stable,6 cases ineffective in the control group,23 cases were effective,5 cases stable,2 cases ineffective in the treatment group.The effective + stable number of cases were 28 in the treatment group,and that were 24 in control group,but the total response rate did not see obvious difference in treatment group compared with control group(p = 0.089).In terms of curative effect of TCM syndromes,9 cases were clinically cured,9 cases were significantly effective,7 cases were effective,and 5 cases were ineffective in the control group,with a total response rate of 83.33%.19 cases were clinically cured,7 cases were significantly effective,3cases were effective,and 1 case was ineffective in the treatment group,with a total response rate of 96.67%.The curative effect of TCM syndromes in the treatment group was significantly better than that in the control group(P <0.05).In terms of safety,there were 12 cases(40%)of digestive tract symptoms in the treatment group,including 8 cases(26.67%)of nausea and vomiting,and 4 cases(13.33%)of decreased appetite,and 4 cases(13.33%)of digestive tract reactions in the control group,including 3 cases(10%)of nausea and vomiting,and 1 case(3.33%)of decreased appetite.The side effects of digestive tract reactions in the treatment group were significantly less than those in the control group(P <0.05).In terms of skin and mucosa,there were 7 cases(23.33%)of oral ulcer in the control group and 2 cases(6.67%)in the treatment group.The side effects of skin and mucosa in the treatment group were significantly lower than those in the control group(P <0.05).There was no obvious liver and kidney function in the control group and the treatment group.(2)The expression of miR-532-3p was low in lymphoma tissues and lymphoma cells(P <0.01).After transfection with miR-532-3p mimic and miR-532-3p inhibitor,the expression of miR-532-3p was significantly increased in the overexpressed group detected by RT-PCR(P <0.001),while the expression of miR-532-3p was decreased in the silent group(P <0.0001).Overexpression of miR-532-3p inhibited lymphoma cell proliferation and induced apoptosis(P<0.0001);Inhibited the expression of proliferating protein c-myc,PCNA and Survivin,and promoted the expression of apoptotic protein Cleaved caspase3.The expression of IL-4,IL-6 and IL-10 was inhibited,and the expression of IL-2 was promoted.The opposite result was obtained by silencing miR-532-3p.The results of luciferase gene reporting assay showed that the activity of wild-type luciferase was decreased(P <0.001),while the activity of mutant luciferase was increased(P <0.001).Overexpression of β-catenin reversed the effects of miR-532-3p mimic on proliferation(P <0.001),apoptosis(Cleaved caspase3),proliferation protein c-myc,PCNA,Survivin,apoptosis protein Cleaved caspase3,and IL-2,IL-4,IL-6,IL-10 expression(P <0.001)of lymphoma cells.In vivo results showed that the tumor volume and weight in miR-532-3p mimic group were significantly decreased compared with the control group(P <0.0001).Compare with the control group,the expression of miR-532-3p in the tumor tissues of miR-532-3p mimic group was increased(P <0.0001),and the expression of β-catenin was decreased(P <0.01).(3)Xiaoliufang inhibited lymphoma cell proliferation and promoted apoptosis in a time-and concentration-dependent manner.Western Blotting results showed that,compared with the control group,the expressions of proliferating protein C-Myc,Survivin and PCNA were significantly down-regulated,and the expression of apoptotic protein Cleaved caspase-3 was significantly up-regulated in the Xiaoliufang group.Compared with the control group,the expressions of IL-4(P <0.05),IL-6(P<0.01)and IL-10(P <0.05),in Xiaoliufang group were down-regulated,and the expression of IL-2 was significantly up-regulated(P <0.05).Compared with the control group,the expression of miR-532-3p in Xiaoliufang group was up-regulated(P <0.001),and the expression of β-catenin m RNA and protein was down-regulated(P<0.001).The results of in vivo experiment showed that compared with the control group,tumor volume and weight of nude mice in the Xiaoliufang group,CTX group and Xiaoliufang +CTX group were all decreased,and the decrease was more obvious in the Xiaoliufang +CTX group(P <0.0001).Compared with CTX group,there was no significant difference in tumor volume and weight.Compared with the control group,the expressions of IL-4,IL-6 and IL-10 in the Xiaoliufang group,CTX group and Xiaoliufang +CTX group were all down-regulated,and the down-regulation was more obvious in the Xiaoliufang +CTX group(P <0.0001).There was no significant difference in the expression of IL-4 and IL-10 between the Xiaoliufang and CTX group,while the expression level of IL-6 in the CTX group was higher compared to Xiaoliufang group(P <0.05).Compared with the control group,the expression of IL-2in the Xiaoliufang Xiaoliufang,CTX group and Xiaoliufang +CTX group was up-regulated,and the up-regulation was more obvious in the Xiaoliufang group and CTX group,and the up-regulation of IL-2 in the Xiaoliufang group was more obvious than that in the CTX group(P <0.01).Compared with the control group,the expression level of miR-532-3p in the Xiaoliufang group,CTX group and Xiaoliufang+CTX group was up-regulated,and the expression level of β-catenin was down-regulated.(4)CCK-8 results showed that compared with the control group,the cell proliferation rate of the Xiaoliufang group was decreased(P <0.0001),and the cell proliferation rate of the miR-532-3p inhibitor group was increased(P <0.0001),while no significant difference was found in the Xiaoliufang +miR-532-3p inhibitor group.Compared with the Xiaoliufang group,the cell proliferation rate of the Xiaoliufang+miR-532-3p inhibitor group increased(P <0.0001).Annexin V/PI results showed that compared with the control group,the apoptosis rate of the Xiaoliufang group increased and miR-532-3p inhibitor apoptosis rate decreased,while no significant difference was found in the Xiaoliufang +miR-532-3p inhibitor apoptosis rate.Western Blotting showed that,compared with the control group,the expression of c-myc,Survivin,PCNA was decreased and the expression of apoptosis protein Cleaved caspase3 was increased in the Xiaoliufang group,while the expression of c-myc,Survivin,PCNA was increased and the expression of apoptosis protein Cleaved caspase3 was decreased in the miR-532-3p inhibitor group.Compared with the Xiaoliufang group,the expression of proliferative proteins(c-myc,Survivin,PCNA)in the Xiaoliufang +miR-532-3p inhibitor group increased,and the expression of apoptosis protein Cleaved caspase3 decreased.Therefore,it can be concluded that miR-532-3pinhibitor can reverse the effect of anti-tumor prescription on the proliferation and apoptosis of lymphoma cells.ELISA results showed that compared with the control group,the expression levels of IL-4,IL-6 and IL-10 in the Xiaoliufang group were decreased(P <0.01),the expression levels of IL-2 were increased(P <0.001),and the expression levels of IL-4,IL-6 and IL-10 in the miR-532-3p inhibitor group were increased(P <0.05),and the expression level of IL-2 was decreased(P <0.05).Therefore,it could be concluded that miR-532-3p inhibitor could inhibit the regulation of Xiaoliufang on the lymphoma tumor microenvironment.Western Blotting results showed that compared with the control group,the expression of β-catenin protein in the Xiaoliufang group was down-regulated,and the expression of β-catenin protein in the Xiaoliufang group was up-regulated,while the expression of β-catenin protein in the Xiaoliufang group was up-regulated compared with the Xiaoliufang group +miR-532-3p inhibitor group.Conclusions:1.Xiaoliufang combined with chemotherapy in the treatment of DLBCL has good clinical efficacy and was safe to use.2.Xiaoliufang can inhibits lymphoma proliferation,induces apoptosis and regulates tumor microenvironment in vivo and in vitro.3.MiR-532-3p inhibited lymphoma proliferation,induced apoptosis and regulated tumor microenvironment by targeting β-catenin.4.Xiaoliufang prescription inhibited lymphoma proliferation,induced apoptosis and regulated tumor microenvironment through miR-532-3p/β-catenin signaling pathway.