Overexpression Of Lunasin Gene In Soybean And The Anti-proliferation Mechanism Of Lunasin Peptide On Human Breast Cancer Cells

Lunasin,a novel natual peptide with molecular mass of 5.0 KDa,containing 44 amino acids,was initially isolated from soybean.Because of various physiological functions,Lunasin exhibited a great application value.However,the content of Lunasin is low in natural raw materials,and it is costly through chemical synthesis,how to obtain Lunasin efficiently is a difficult problem to be solved.With the development of molecular biology and genetic engineering,Lunasin biosynthesis through DNA recombination technology makes this possible.Transgenic soybean,as a genetically modified crop,is widely planted in the world.The method to obtain the optimal transgenic soybean through genetic engineering technology is becoming more and more mature.In this study,through agrobacterium-mediated soybean genetic transformation,Lunasin coding gene was transferred into soybean genome under the control of CaMV 35S promoter.Lunasin-overexpression soybean was obtained,and further screening with high Lunasin content was performed.And then the functional activity evaluation system of transgenic soybean was established.Correlation analysis of transcriptome and proteome was conducted to explore the anti-cancer molecular mechanism of Lunasin peptide.The following experimental progress was made:1.The acquisition of transgenic soybean:Through agrobacterium-mediated soybean transformation,15 T1 independent transgenic lines were obtained.The method of UPLC-MS/MS to identify Lunasin in soybean was established,Lunasin exhibited a peak at the retention time of 10.606min,TOFMS and TOF-MS/MS scan related to this peak exhibited multiple parent ion signals and multi-charge states.2.Molecular biology analysis of transgenic soybean:The results of FQ-PCR and Western blot showed that Lunasin peptide was successfully expressed in soybean.Lunasin expression was significantly enhanced in transgenic soybean lines L12,L43,and L45.Lunasin content was determined through ELIAS assay for the further screening,its contents in transgenic soybean were 1.47 mg·g^-1?L12?,1.32 mg·g^-1?L43?,and 1.97 mg·g^-1?L45?,significantly higher than receptor material(0.94mg·g^-1).It is feasible and effective to improve Lunasin content through transgenic technology.3.Antioxidant,anti-inflammatory,and anti-cancer activity evaluation of transgenic soybean:Overexpression of Lunasin coding gene significantly increased the antioxidant activity of Lunasin enrichments in transgenic soybean.LET?Lunasin enrichments from transgenic soybean?exhibited a higher DPPH,ABTS⁺,and oxygen radical scavenging activity than LEW?Lunasin enrichments from wild soybean?.There was no cytotoxic effect on LPS-induced mouse RAW264.7 macrophage cells within a range of concentration gradient(0.25-4 g·L^-1)of LET and LEW.Compared to LEW,LET showed a superior inhibitory activity on the release of NO and cytokines including IL-1 and IL-6 in RAW264.7 cells.Overexpression of Lunasin coding gene can significantly increase the anticancer activity of Lunasin enrichments in transgenic soybean.Cell immunofluorescence staining analysis showed that Lunasin peptide could internalize into mammalian cells,located in the nucleus and then functioning.In the anti-proliferation assay,the cell proliferation of MDA-MB-231 was significantly inhibited by LET and LEW,and LET exhibited a higher inhibition rate than LEW.4.Research on the anti-proliferation function and molecular mechanism of Lunasin peptide on human breast cancer cells:Correlation analysis of transcriptome and proteome was conducted to comprehensively explore the intracellular gene and protein regulation in MDA-MB-231 cells after Lunasin treatment,screening the differentially expressed genes and proteins for GO and KEGG enrichment analysis,revealed its anti-cancer function and possible molecular mechanism.The results showed that DNA replication and lysosomal pathway were significantly inhibited after Lunasin treatment,and apoptosis pathway was highly activated.It was suggested that Lunasin peptide can inhibit DNA replication and cell mitosis,suppress autophagy as well as induce cell apoptosis through the lysosome-mitochondrial axis,thereby inhibiting cell proliferation.