Uniflower Swisscentaury Root Water Extract Impact Vitality Of HepG2 Cells By Regulating PAK4

Background and objectiveAccording to the World Health Organization,cancer is the second leading cause of death in the world,with about 14 million new ca ncer cases in 2012 and 8.8 million deaths in 2015,with liver cancer taking the second place(788000 deaths)[1].In C hina,the number of new cases of liver cancer every year is 366000,the annual death rate is 321000,ranking second in the country cancer deaths.About 80% to 90% of HCC is primary hepatocellular carcinoma(HCC).There are: surgical resection and minimally invasive interventional therapy,radiotherapy,chemotherapy and biological cell immunotherapy,traditional C hinese medicine and other comprehensive treatment.Chinese medicine has great development space in the treatment of primary liver cancer.According to clinical observation and report,C hinese medicine combined with C hinese medicine in the treatment of liver cancer patients can prolong the life span and improve the quality of life.The discovery of effective anti-tumor cell proliferation and invasion components and the elucidation of its molecular mechanism in C hinese herbal medicine and its extracts have become one of the research and development directions of some new drugs.As one of the representative drugs of antipyretic and detoxifying C hinese medicine,Lilu has been widely used in clinical Chinese medicine treatment of primary liver cancer and obtained some experimental support from relevant experience.The thiophene compounds contained in the leucodes can reverse the multidrug resistance of the tumor.It can induce apoptosis of tumor cells and improve the effect of chemotherapy.However,the intrinsic molecular mechanism of its anti-tumor cell proliferation and invasion is still unclear.Our previous studies have shown that the Uniflower Swisscentaury ROOT water extract for flila-EGFP can inhibit the angiogenesis of flila-EGFP transgenic zebrafish,and its mechanism is related to the promotion of TIE1 gene expression and negative feedback regulation of ANGPT-TIE signaling pathway [2].Hepatocellular carcinoma(HCC)is a progressive process with multi-gene,multi-locus and multi-pathway changes.PAK family is a serine / threonine protein kinase,which is conserved by evolution.It is an important target protein of Rho family,especially CDC42 TPA and Rho downstream.Can be activated by extracellular signals,involved in regulation of cytoskeleton,cell regulation Apoptosis,cell cycle and mitosis,gene transcriptional regulation and angiogenesis play an important role in tumorigenesis,invasion and metastasis.PAK4p21-activated protein kinase 4 belongs to the PAK family.High expression or abnormal activation in hepatocellular carcinoma,colorectal cancer,thyroid carcinoma and choriocarcinoma [3-7].In this study,the human hepatoma cell line HepG2 was used to treat the cell with the water extract of Reed.The growth of HepG2 cells and the proliferation of HepG2 cells were observed.Apoptosis,migration,invasion,and associated genomic bioinformatics,high-throughput transcriptome sequencing,or RNA-seq,was analyzed to analyze the differential expression of related genes.The tumor-related gene PAK4 was screened and its intrinsic molecular mechanism was studied.Experimental methods:1.Preparation of Water extract from Uniflower Swisscentaury Root.2.Inhibition of HepG2 cells proliferation by water extract.3.High throughput RNA-seq analysis of genes.4.q RT-PCR and Western Blot of PAK4 gene.Results of the study:1.The vitality of HepG2 cells increased with the increase of drug concentration(p < 0.01),and the 50 percent inhibitory concentration(IC50)on HepG2 was 0.29 mg/ ml at 72 h.2.Cell Titer-Glo fluorescence cell activity assay was used to detect the inhibition rate of the water extract on HepG2 cells,and the cell growth curve was plotted.The results showed that the water extract could inhibit the viability of HepG2 cells.3.High throughput RNA-seq sequencing analysis showed that the number of gene expression was 10726 and the number of transcripts was 23242 in the control group.In treatment group,the number of gene expression was 10922 and the number of transcripts was 23184.4.The difference of differentially expressed PAK4 gene between high-throughput sequencing gene and transcripts was significant log2(fold change)=-1.742405725;p-value =0.00037379.5.The results of q RT-PCR and Western Blot experiments of PAK4 gene showed that the mechanism of the inhibition of HepG2 vitality by water extract was related to the down-regulation of PAK4 gene expression.Conclusion:1.The aqueous extract could inhibit the proliferation of human hepatoma cell line HepG2.2.The molecular mechanism of inhibiting the proliferation of human hepatoma cell line HepG2 and promoting its apoptosis is related to the down-regulation of the expression of PAK4 gene.