| Background In recent years,more and more studies have found that the prevalence of breast cancer in women with diabetes is higher than that of non-diabetic patients,suggesting that diabetes is one of the risk factors for breast cancer.Long-term persistent high blood glucose causes environmental disturbances in the body,resulting in low levels of inflammation and low adiponectin(APN)levels,which may be associated with increasing risk of breast cancer.Clinical studies have found negative correlation between serum APN level and malignant tumor,and adiponectin can suppress the proliferation of breast cells through multiple pathways,as well as induce the apoptosis of breast cancer cell.Autophagy is a process of self-digestion and degradation of proteins and organelles in eukaryotic cells,which can lead to programmed cell death.Excessive autophagy is also responsible for the death of tumor cells,known as”autophagy death”.Therefore,autophagy is considered to be an important target for the prevention and treatment of tumors.Microtubule-associated protein l light chain3(LC 3),as a kind of autophagy-related protein,is one of the specific methods to determine the expression level of autophagy.Previous studies have found that low APN levels are an important factor in the risk of breast cancer in patients with T2 DM,and APN may promote the autophagy of MCF-7cells in breast cancer.But there is not a specific relationship between the expression of APN and LC3 in breast cancer tissues in diabetes group,as well as it is unknown what are the specific mechanisms.Objective:(1)To study the expression of APN and LC3 in breast cancer tissues of patients with type 2 diabetes mellitus,and to explore the correlation between them and their clinical significance.(2)To investigate the effects of full-length adiponectin(Acrp30)on the proliferation,apoptosis and autophagy of human breast cancer MCF-7 cells in high-sugar environment,and the relationship between autophagy induced by adiponectin and the NF-?B signaling pathway.Methods:Part One:48 breast cancer patients with type 2 diabetes mellitus were included,and 53 cases of non-diabetic as control,while biochemical indicators and serum adiponectin levels were detected.The m RNA expression of APN and LC3 in breast cancer tissues was detected by real-time RT-PCR.APN and LC3 expression in breast cancer tissues were detected by immuno-histological method.Part Two:1)MCF-7 cells were cultured In high sugar environment firstly,then treated with different concentrations of Acrp30(25,50,100,200 ng/ml)for 24 to 48 hours,and un-adiponectin group as the control.The cell growth situation was observead by inverted microscope,and the cell proliferation activity determined,total cell apoptosis rate and the expression of LC3,NF-?B protein had been tested.2)Acrp30,PMA and PDTC(activator and inhibitor of NF-?B signal transduction pathway)were selected as the grouping factors.The MCF-7 cells were divided into six groups: control group,Acrp30,PMA,PMA + Acrp30,PDTC and PDTC+ Acrp30.The expression of LC3 protein of all cells was detected by Western blot method.Result:Part 1:1)The level of FBG(P<0.01),Hb A1C(P<0.01)and TG(P<0.05)in T2 DM group was higher than the control group;the serum adiponectin level was lower than the control group(P<0.05);the detection rate of tumor diameter greater than > 2 cm and rate of lymph node metastasis in T2 DM group was higher than the control group.There was no statistically significant difference in menstrual condition,BMI,WHR,TCH,age and histology.2)The ? Ct APN m RNA and ? Ct LC3 m RNA values of diabetes group are higher than the control group,so APN and LC3 m RNA expression of diabetes group is lower than the control group(P<0.05).3)The positive rate of APN expression in breast cancer tissue with diabetes was18.8%,lower than 35.9% in the control group(P<0.05).The positive rate of LC3 protein expression was only 14.5%,compared with 32.0% in the control group(P<0.05).4)The expression of LC3 in the APN(-)group was high,and the expression of the LC3 in APN(-)group was significantly lower than that in APN positive group.Whatever T2 DM or not,the difference was statistically significant(P<0.01),and APN was positively correlated with the expression of LC3.Part2:1)Inverted microscope observed the growth of each cell.After 48 hours of intervention with Acrp30,the cell volume gradually shrank,the number of living cells decreased,the floating cells increased and the cell growth was inhibited.2)After 24 hours of intervention,compared with the control group,the OD490 nm values of 25?50ng/ml group were decreased but the difference was not statistically significant.The value of OD490 nm in the 100 and 200 ng/ml group was significantly lower than that in the control group(P<0.01~ 0.05).After 48 hours,the difference of OD490 nm in 25ng/ml group was not statistically significant compared with the control group.The value of OD490 nm in the 50,100,200 ng/ml group was significantly reduced(P < 0.01~ 0.05).The value of OD490 nm in different concentration groups gradually decreased,and the difference between groups was statistically significant(P<0.01).3)After 24,48 hours,the growth inhibition rate of 50,100,200ng/ml group was increased(P < 0.01~ 0.05)compared with the 25ng/ml group.Compared with50ng/ml group,the growth inhibition rate of 100,200ng/ml group was increased(P<0.01).Compared with 100ng/ml group,the growth inhibition rate of 200ng/ml group was increased(P<0.01).The inhibition rate of group 48 h was higher than that of 24 h group.Acrp30 can reduce the activity of MCF-7 cells,inhibit cell growth,depending the dose and time.4)After 24 hours,the apoptosis rate of the four experimental groups increased with the group concentration,but the difference was not statistically significant(P >0.05).After 48 hours,the apoptosis rate of group 25 and 50 ng/ml was no significant change compared with control group(P>0.05),and the apoptosis rate of group 100 and 200 ng/ml group was significantly increased(P < 0.01).The apoptosis rate of 200ng/ml group was higher than 100ng/ml group(P<0.01),and the difference between groups was statistically significant(P < 0.01).The apoptosis rate of the 24 h group was more than 48 h group.Acrp30 can promote apoptosis of MCF-7 cells,which depend on the dose and time.5)After 24 hours,compared with the control group,NF-?B expression in 25,50ng/ml group is lower,but there was no statistically significant difference(P >0.05);NF-?B expression in 100,200 ng/ml group is significantly lower(P<0.05),there was no statistically significant difference between the two groups(P>0.05).After 48 hours,compared with the control group,NF-?B expression in 25 ng/ml group is reduced,but there was no statistically significant difference(P>0.05),decreased significantly(P < 0.01 ~ 0.05)in 50,100,200 ng/ml group.Compared with 24 h,the expression of NF-?B in 48 h group was more.6)After 24 hours of intervention,compared with the control group,LC3-II/LC3-I expression in 25,50ng/ml group increased but has no statistical significance(P>0.05);was significantly increased in 100ng/ml,200ng/ml group(P<0.05).After48 hours,compared with the control group,LC3-II/LC3-I expression in 25ng/ml group was elevated but there was no statistically significant difference(P >0.05).The levels of LC3-II/LC3-I in 50,100,200ng/ml group were significantly higher(P<0.05).The levels of LC3-II/LC3-I in 100 and 200ng/ml were higher than that in 50ng/ml group(P < 0.05),and there was no statistically significant difference between the two groups(P<0.05).7)After the PMA activation of NF-?B pathway,the expression of NF-?B was increased,and the Acrp30 could still inhibit the NF-?B pathway,eventually increasing the autophagy level of the cells,and the autophagy level was lower than that of PMA intervention.8)After the suppression of NF-?B pathway by PDTC,the expression of NF-?B decreased and the autophagy level increased.In the case of Acrp30 intervention,the autophagy levels did not change significantly due to the blocking of the pathway.Conclusion:(1)Compared with non-diabetic breast cancer patients,the level of serum adiponectin in patients of T2 DM group was low.The expression of APN and LB3 in T2 DM group was lower than those of non-diabetic patients,and the APN and LB3 were positively correlated.Low adiponectin expression and low autophagy may be involved in the progression of breast cancer in patients with type 2 diabetes.The Acrp30 can inhibit not only the proliferation but the apoptosis of MCF-7 cells,and the effect is dependent on concentration dependence.(2)Adiponectin inhibites the growth,cell proliferation and promotes apoptosis of human breast cancer MCF-7 cells.These effects show a concentration-time dependence.(3)Adiponectin inhibites the expression of NF-?B in the high-sugar environment and increased LC3-II/LC3-I,which depend on the concentration and time.(4)Adiponectin reduces the expression of NF-?B by inhibiting the NF-?B pathway,reduces its suppression of autophagy and ultimately induces the autophagy enhancement of MCF-7 cells. |
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