Sericin Targeting Akt Induces Autophagy In TNBC MDA-MB-468 Cells

According to the Global Cancer Statistics Report 2020 released by the International Agency for Research on Cancer(IARC),breast cancer has overtaken lung cancer to become the world’s highest incidence of cancer by2020.Triple negative breast cancer(TNBC)is a type of breast cancer with negative expression of estrogen receptor,progesterone receptor and human epidermal growth factor receptor 2.It is characterized by strong invasion ability,high metastasis rate and poor prognosis.Due to the lack of specific targeted therapeutic drugs and the high recurrence rate of TNBC,clinical treatment methods are limited.Therefore,the exploration of efficient therapeutic targets and effective therapeutic drugs has become a hot spot in the research of anti-breast cancer treatment.Sericin is a kind of natural polymer protein extracted from silk,which has anti-inflammatory,antioxidant,antibacterial and other biological functions,as well as anti-tumor properties.Autophagy is a degradation pathway unique to eukaryotes,which is closely related to the occurrence and development of many diseases,including tumors.Our previous study found that sericin can inhibit the proliferation of TNBC cells,but the specific mechanism remains to be further explored.The purpose of this study was to observe the changes of autophagy related indexes of TNBC MDA-MB-468 cells under the effect of sericin,to study whether sericin induces autophagy through PI3K/Akt/m TOR signal transduction pathway,and to explore the target of sericin-induced autophagy of MDA-MB-468 cells.Objective:To observe the effects of sericin on PI3K/Akt/m TOR signal transduction pathway related indicators and autophagy related indicators of TNBC MDA-MB-468 cells,and to explore whether sericin induces autophagy of MDA-MB-468 cells by targeting Akt.Methods:1.MDA-MB-468 cells were randomly divided into three groups: blank control group,8mg/ml sericin group(sericin group),and 8mg/ml sericin+Akt activator SC79 group(sericin +SC79 group).Blank control group was cultured with DMEM medium for 24 h.Sericin group was treated with8mg/ml sericin for 24h;Sericin +SC79 group was treated with 8mg/ml sericin +10μM Akt agonist SC79 for 24 h.2.The m RNA expression levels of PI3 K,Akt,m TOR,LC3,P62,ATG5 and Beclin1 in MDA-MB-468 cells of each group were detected by real-time quantitative PCR.3.The protein expression levels of PI3 K,Akt,p-Akt,m TOR,p-m TOR,P62,ATG5,Beclin1 and the changes of LC3II/I in MDA-MB-468 cells of each group were detected by western blot.4.The localization of LC3 and P62 and the changes of fluorescence intensity of MDA-MB-468 cells in each group were detected by immunofluorescence staining.Results:1.Effect of sericin on PI3 K expression in MDA-MB-468 cellsThe m RNA expression level of PI3 K was significantly decreased in sericin group compared with blank control group(P<0.05);There was no significant difference in m RNA expression level of PI3 K between sericin+SC79 group and sericin group(P> 0.05).The protein expression level of PI3 K was significantly decreased in sericin group compared with blank control group(P<0.05);There was no significant difference in PI3 K protein expression between sericin +SC79group and sericin group(P> 0.05).2.Effects of sericin on the expression levels of Akt and m TOR in MDA-MB-468 cellsThe m RNA expression levels of Akt and m TOR in sericin group were significantly decreased compared with blank control group(P<0.05);m RNA expression levels of Akt and m TOR were significantly increased in sericin+SC79 group compared with sericin group(P<0.05).The protein expression levels of Akt,p-Akt,m TOR and p-m TOR in sericin group were significantly decreased compared with blank control group(P<0.05);The protein expression levels of Akt,p-Akt,m TOR and p-m TOR in sericin +SC79 group were significantly increased compared with sericin group(P<0.05).3.The effects of sericin on the expression levels of P62,Beclin1,ATG5 and LC3II/I in MDA-MB-468 cellsm RNA expression levels of LC3,Beclin1 and ATG5 in sericin group were significantly increased compared with blank control group(P<0.05);m RNA expression levels of LC3,Beclin1 and ATG5 in sericin +SC79 group were significantly decreased compared with sericin group(P<0.05).The m RNA expression level of P62 was decreased in sericin group compared with blank control group(P<0.05);The m RNA expression level of P62 in sericin +SC79 group was significantly increased compared with sericin group(P<0.05).The protein expression levels of Beclin1,ATG5 and LC3II/I in sericin group were significantly increased compared with blank control group(P<0.05);The protein expression levels of Beclin1 and ATG5 and LC3II/I of sericin +SC79 group were significantly decreased compared with sericin group(P<0.05);The protein expression level of P62 in sericin group was significantly decreased compared with blank control group(P<0.05);The protein expression level of P62 in sericin +SC79 group was significantly increased compared with sericin group(P<0.05).4.Localization and fluorescence intensity of LC3 and P62 proteinsThe results of LC3 staining showed that the nucleus was light blue after DAPI staining,and the LC3 protein was green fluorescent.The average fluorescence intensity of sericin group was significantly higher than that of blank control group(P<0.05);The average fluorescence intensity of sericin+SC79 group was significantly decreased compared with sericin group(P<0.05).The results of P62 staining showed that the nucleus was light blue after DAPI staining,and the P62 protein was green fluorescence.The average fluorescence intensity of sericin group and blank control group decreased significantly(P<0.05);The average fluorescence intensity of sericin +SC79group was significantly increased compared with sericin group(P<0.05).Conclusions: Sericin induces autophagy in TNBC MDA-MB-468 cells by regulating PI3K/Akt/m TOR signaling pathway with Akt as the target.