| ObjectiveAfter subcutaneous injection of B16 cells into C57/B6J male mice,a mouse melanoma model was established to verify the therapeutic effect of quercetin on melanoma.Using B16 and A375 cell line as the main research object,through the cell proliferation,cell migration,invasion,and apoptosis experiment to verify the effect of quercetin at a cellular level.By western blot,q-PCR,and luciferase reporter gene experiment,RIG-? and their downstream molecules were quantitatively analysed,in order to find and verify the mechanism of quercetin in the treatment of melanoma.Methods1.Study on the therapeutic effect of quercetin on mouse melanoma model(1)Establishment and evaluation of mouse melanoma modelTwenty-four 7-8-week-old C57/B6J male mice were divided into 4 groups by random number table method,6 in each group.After adaptive feeding for 3 days,back hair was removed and 1×106 B16 cells were injected subcutaneously into the shaving area to establish a melanoma model.After three days,the success of the model was confirmed,and the mice that failed in modeling were removed,and quercetin intragastric therapy was started immediately.They were divided into the control group(CMC-Na),the low-dose group(3mg/kg/d),the medium-dose group(15mg/kg/d)and the high-dose group(75mg/kg/d).(2)Efficacy evaluation of quercetin in the treatment of melanomaThe weight of mice in each group was recorded every 3 days from modeling,and the weight change curve was made.Tumor size was measured and recorded every 3 days from the start of administration,and tumor growth curve was made.After the administration,the mice were weighed and the tumor size and weight were measured.After the tumor was removed,the tumor tissue was photographed and stored in liquid nitrogen for further experiment.The inguinal lymph nodes of the mice were placed in liquid nitrogen for further experiment.The effect of quercetin on melanoma was evaluated by measuring tumor volume and tumor weight of mice.2.Effect of quercetin on melanoma cell lines(1)Effect of quercetin on mouse melanoma cell line B16To evaluate the inhibitory effect of quercetin on mouse melanoma cells,the effects of quercetin at different concentrations on B16 cells were detected by cell proliferation,cell migration,invasion experiment,scratch healing experiment and apoptosis experiment,etc.(2)Effect of quercetin on human melanoma cell line A375To evaluate the inhibitory effect of quercetin on human melanoma cells,the effects of quercetin at different concentrations on A375 cells were detected by cell proliferation and apoptosis experiment,etc.3.Study on the mechanism of quercetin in treating melanoma(1)Mechanism of quercetin in B16 cellsConfocal was used to detect the changes in the expression of RIG-? in B16 cells after quercetin treatment,and double Luciferase reporter gene assay was used to analyze the mechanism of quercetin affecting the expression of RIG-?.The expression levels of RIG-? and its downstream signaling molecules TBK1,IRF3,IRF7,IFN-?,IFN-?,STAT1 and IFN-? induced ISGs were verified by western-blot and q-PCR to verify the effect of quercetin on the RIG-?/IFN-? signaling pathway.SiRNA silencing was used to knock down the expressions of RIG-? and STAT1 in B16 cells,respectively,to verify the mechanism of quercetin,and q-PCR was used to detect mRNA expression levels of relevant signaling molecules under the condition of simultaneous silencing of RIG-? and STAT1,to further clarify the mechanism of quercetin.(2)Study on the mechanism of quercetin in miceThe tumor tissues and lymph node tissues of mice were treated with ultra-low temperature tissue lapping apparatus to extract the protein.The expression levels of RIG-? and its downstream signaling molecules IRF3,IRF7 and IFN-? in tumor tissues and lymph node tissues were detected by western-blot experiment to verify its mechanism of action in mice.Results1.Quercetin inhibited the growth of melanoma in miceAfter two weeks of continuous administration,quercetin at 75mg/kg/d significantly inhibited the growth of melanoma in mice compared with the control group,showing that the tumor growth rate of mice in the 75mg/kg/d group was significantly lower than that in the control group,and the tumor volume and tumor weight at the end of administration were significantly lower than those in the control group,with statistically significant differences(P<0.05).2.Effects of quercetin on B16 and A375 cell lines(1)Quercetin inhibited B16 cell proliferation and promoted apoptosisB16 cells were treated with quercetin at different concentrations for 24h,and the proliferation of cells was detected with CCK-8 reagent.Starting from 2.5 ?M,the proliferation of cells was significantly inhibited compared with the control group,with statistical difference(P<0.05).When quercetin was treated for 48h,cell growth was significantly inhibited starting from 2.5 ?M,with significant difference(P<0.05).The inhibition level leveled off when the concentration of quercetin was greater than 10 ? M.Using the CTV dye markers B16 cells,24h after treatment with different concentration of quercetin,found that the concentration can has the different degree of inhibition effect on the proliferation of B16 with dose-dependent inhibition degree.Concentration from 2.5 ?M began its inhibition effect compared with control group with significant difference(P<0.05).The inhibition degree also leveled off after more than 10 ?M.In the colony formation experiment,quercetin could inhibit the formation of B16 cell colonies in a concentration-dependent manner,and the difference between the 5 ?M group and the 10 ?M group with the control group was statistically significant(P<0.05).After 24h of quercetin treatment on B16 cells,annexin-v/PI staining showed that quercetin promoted B16 apoptosis,and the proportion of apoptotic cells gradually increased with the increase of administration concentration.The difference between the control group and the 1 ?M group was statistically significant(P<0.05).(2)Quercetin inhibited B16 cell migration,invasion and wound healingAfter 24h of different concentrations of quercetin were added to the transwell compartment,it was found that quercetin could inhibit the migration and invasion of B16,and the degree of inhibition gradually increased with the increase of the dose concentration.The differences between the 5 ?M group and 10 ?M group with the control group were statistically significant(P<0.001).After 24 treatment with quercetin,it was found that the degree of cell scratch healing gradually decreased with the increase of administration concentration,and the degree of cell scratch healing at 10 ?M was significantly different compared with control group(P<0.001).(3)Quercetin inhibited A375 cell proliferation and promoted apoptosisA375 cells were treated with different concentrations of quercetin for 24h,and the proliferation of cells was detected with CCK-8 reagent.Starting from 20 ?M,the proliferation of cells was significantly inhibited compared with the control group,with significant difference(P<0.05).When quercetin was treated for 48h,it also started from 5 ?M,and cell growth was significantly inhibited,with significant difference(P<0.05).The inhibition level leveled off when the concentration was greater than 60?M.A375 cells were treated with different concentrations of quercetin for 24h,and the proportion of apoptotic cells gradually increased with the increase of administration concentration.Starting from 2.5 ?M,the proportion of apoptotic cells was significantly different from the control group(P<0.001).3.The therapeutic mechanism of quercetin in vivo and in vitro(1)Quercetin promoted the expression of RIG-? by activating the promoter of RIG-?Immunofluorescence was used to detect the expression of RIG-? in B16 cells after quercetin treatment for 24h,and it was confirmed that quercetin could upregulate the expression of RIG-? in cells.Through dual-Luciferase report assay,it was found that quercetin could activate the promoter sequence of RIG-?,and activate the production of RIG-? to induce the expression of IFN-?.With the increase of the dose concentration,the production of IFN-? increased gradually,starting from 5 ?M with statistically significance compared with the control group(P<0.001).(2)Quercetin induced IFN-? and other signaling molecules by activating RIG-?The addition of quercetin to B16 and A375 cells could promote the expression of RIG-? at protein and mRNA level,and the promoting effect is dose-dependent.At mRNA level,increased RIG-? expression could promote the production of downstream TBK1,IRF3,IRF7,IFN-? and some ISGs,such as MX1,PML,TRAIL and STAT1,and the expression levels gradually increased-with the increase of drug concentration,with statistically significance compared with the control group(P<0.05).At the protein level,quercetin significantly upregulated the expression levels of RIG-? in B16 and A375 cells,as well as the expression levels of downstream signaling pathway molecules represented by IRF7 and STAT1,with statistically significance compared with the control group(P<0.05).(3)The mechanism of quercetin in miceIn the tumor tissues and lymph node tissues of mice,the protein expression levels of RIG-?,IRF3,p-IRF3,IRF7,p-IRF7,STAT1 and p-STAT1 gradually increased with the increase of administration concentration,showing statistically significance compared with the control group(P<0.05).(4)The mechanism of quercetin was verified by siRNA silencing experimentSiRNA was used to silence the expression of RIG-? in B16 cells,and it was found that the expression levels of downstream IRF7 and STAT1 protein were no longer increased with the increase of administration concentration,and the mRNA levels of TBK1,IFN-?,IFN-? And ISGs induced by RIG-? were not significantly changed,with no statistically significance compared with the control group(P>.05).Moreover,there was no significant difference in the apoptosis of B16 cells induced by quercetin after silencing RIG-?compared with the control group(P<0.05).When siRNA was used to silence the expression of STAT1 in B16 cells,the protein expression levels of RIG-? and IRF7 were significantly decreased with the increase of administration concentration.Silencing both RIG-? and STAT1 expression in B16 cells resulted in further down-regulation of mRNA levels of TBK1,IFN-?,IFN-? and ISGs induced by RIG-?,compared with silencing RIG-? alone.Conclusion1.Quercetin inhibited melanoma growth in miceQuercetin inhibited the growth of melanoma in mice,and showed the best inhibitory effect at the concentration of 75mg/kg/d.The difference in tumor growth rate,tumor volume and tumor weight were statistically significant compared with control group(P<0.05).2.Quercetin could inhibit melanoma growth in vitroQuercetin inhibited the proliferation of B16 and A375 cells and promoted apoptosis in a dose-dependent manner.Quercetin could inhibit B16 cell migration,cell invasion,cell scratch healing and cell colony formation.3.Quercetin inhibited tumor growth by activating the expression of RIG-? and inducing the expression of IFN-?Quercetin could significantly up-regulate the expression of RIG-? in B16 and A375 cells,and it was confirmed that quercetin promotes the expression of RIG-? by activating the promoter of RIG-?.After the up-regulation of RIG-?,the expression levels of downstream IRF3,IRF7,STAT1 and other molecules increased gradually with the increase of the concentration of quercetin.In mice,protein expression levels of RIG-?,IRF3,IRF7,STAT1 and other molecules in tumor and lymph node tissues increased gradually with the increase of drug concentration.This suggests that quercetin has consistent efficacy and mechanism in vivo and in vitro.4.Quercetin formed a positive-feedback loop of RIG-?/IFN-?/STAT1/RIG-? and amplified the efficacyBy silencing the expression of RIG-? gene in B16 cells,it was confirmed that quercetin played an antitumor role by up-regulating the expression of RIG-? and inducing the production of downstream products.The dose dependence of RIG-?,IRF7 and STAT1 on quercetin was weakened by silencing the expression of STAT1 gene.Simultaneously silencing RIG-? and STAT1 further weakened the dose dependence of the RIG-? and downstream signaling molecules by quercetin.This indicated that under the treatment of quercetin,a positive-feedback loop was formed between RIG-?/IFN-?/STAT1/RIG-?,thus inducing a series of antitumor molecules. |
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