Studies On Apoptosis Mechanism Of HepG2 Cells Induced By Bitter Melon Seed Extract

Bitter melon seed(BMS),which is rich in oil,protein,polyphenols,saponins and other active ingredients,has antibacterial,antioxidant,anti-cancer and other medicinal values.MiRNAs,present in all organisms,have a negative regulatory effect on post-transcriptional levels and are the key regulatory factors in the activities of various organisms.There is much evidence,including preclinical,epidemiological,and clinical,that diet is one of the most important modifiable determinants of health and disease.Therefore,from the point of miRN A to explore the anti-tumor mechanisms of BMS,not only provide new natural anti-cancer food,but also a new therapeutic targets for the treatment of cancer.In this paper,the main components of BMS were analyzed,including the purification and identification of anti-cancer activity proteins in bitter gourd seeds.The proliferation,cell cycle,ROS,mitochondrial membrane potential,protein expression and nuclear morphological of HepG2 cells were analyzed after treated with water extract of Bitter melon seeds.We found that the anti-tumor mechanisms of Bitter melon seeds are resulted from the change of miR-421 and relevant target genes.The main contents and results are as follows:1.Analysis of the main components of bitter melon seeds and purification of anti-cancer activity proteinThe content of main components of the bitter gourd seeds were tested and analyzed.The content of oil and protein are 44.44 ± 0.17% and 34.83 ± 0.47%,respectively,the total sugar,saponinis and Polyphenols are 11.88±0.04%,2.27±0.17% and 1.03±0.08%.When the concentration of ammonium sulfate is 30%-60%,the protein extraction rate and the inhibitory activity on cancer cells of crude protein were highest.Then we purified crude protein the by CM-Sepharose FF and collected active protein peaks.There are seven protein peaks but only the peak1 has significant antitumor activity by CCK-8 analysis and protein molecular weight is about 30 KD by SDS gel electrophoresis.Further qualitative analysis of the peak 1 by Q exactive mass spectrometry showed that the major protein components of peak 1 were ribosome inactivating proteins and trypsin inhibitors.2.Studies on Antitumor Activity of Water Extract from bitter melon seedsThe water extract of bitter gourd seeds could significantly inhibit the proliferation of HepG2,SGC-7901 and HT-29 cells in a dose-dependent manner,and the IC50 values were 586.27 ± 0.07 ?g / mL,771.09 ± 0.08 ?g / mL and 1206.17 ± 0.08 ?g / mL.The water extract of bitter gourd seeds could block the cell cycle of HepG2 in S phase and promote the production of ROS.At the same time,mitochondrial membrane of Hep G2 cells was reduced which affected the protein expression and resulting in cell nucleus lax or even broken and a large number of apoptotic bodies.3.The mechanism of apoptosis of Hep G2 induced by bitter melon seed extract by down-regulating miR-421The expression of miR-421 and miR-23b-3p were significantly down-regulated,and the expression of miR-138-1-3P and miR-190 b were significantly up-regulated in Hep G2 cell with bitter melon seed water extract.By analysis on target genes of miR-421 and miR-138-1-3p via bioinformatics and target gene chip technology,the results showed that there were 9 target genes of miR-421 in Hep G2 cell with bitter gourd seed water extract.For further verification,we detected the expression of target gene Caspase-3 and DUSP6 in Hep G2 cell transfected with miR-421 agonists and inhibitors.The results showed that when the expression of miR-421 was overexpressed,the expression of Caspase-3 gene expression and protein expression were significantly down-regulated,while the expression of DUSP6 protein did not change,which indicated that bitter melon seed water extract may be down-regulated miR-421 and up-regulated downstream expression of Caspase-3 to induced HepG2 cell apoptosis.