The Effects Of Astragalus Polysaccharides On Proliferation And Differentiation Of Human Hepatocellular Carcinoma Cells SMMC-7721 And Huh-7

Objective: In China,cancer is the leading cause of death.Additionally,cancer accounts for about a quarter of cases and deaths globally,while liver cancer accounts for more than 50% of new cases and deaths globally.Hepatocellular carcinogenesis?HCC?is the most common cancer of liver cancer,which accounts for approximately 90 % of primary liver cancers.HCC is mainly treated by surgery resection,while radiotherapy and chemotherapy are the adjuvant therapies.However,HCC cannot be completely cured,and the toxic side effects caused by surgical trauma and chemotherapy lead to poor quality of life,and there is currently a lack of specific treatment for HCC.Therefore,it become the focus and difficulty to find an effective way to cure HCC in the medical field.In recent years,traditional Chinese medicine plays its unique advantages in the treatment of cancer.Astragalus stilaceus?AM?has the effect of invigorating qi and dispersing blood stasis.Astragalus polysaccharides?APS?is the main pharmacological component of AM.The modern pharmacological studies have demonstrated that APS can enhance the sensitivity of some tumor cells to chemotherapy drugs and have anti-tumor efficacy.More and more studies have reported that APS can inhibit the growth of malignant tumor cells such as breast cancer,cervical cancer,gastric cancer and lung cancer,but it is still unknown whether APS can affect the proliferation and differentiation of HCC cells in SMMC-7721 and Huh-7.In this study,we explored the effect of APS on the proliferation and differentiation of in SMMC-7721 and Huh-7 cells.Besidse,the role and feasibility of APS in the treatment of HCC were preliminarily discussed.Methods: 1.Cell culture: SMMC-7721 and Huh-7 cells were revived in DMEM?high glucose?medium containing 1% non-essential amino acids and 10 % neonate bovine serum,then inoculated in 100 ml culture bottle and cultured at37?in 5%CO₂ incubator.2.Determine the concentration of APS were divided into 0 mg/L,50mg/L,100 mg/L,200 mg/L and 400 mg/L by using Cell Count kit-8?CCK-8?experiment.Cells were inoculated on 96-well plates and cultured in a 5%CO2incubator at 37?for 24 h?48 h and 72 h.OD values were calculated to determine the concentration and time point of APS.3.Transcription levels of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 gene in SMMC-7721 cells were detected.Trizol one-step method was used to extract total RNA from SMMC-7721cells.GAPDH was used as internal reference,and mRNA expression of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 were detected by Real-time PCR.4.The contents of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 gene in SMMC-7721 cells were detected.After extracting the total protein by RIPA lysis,the protein content was determined by Lowry assay,and the PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 gene protein content in SMMC-7721 cells were detected by Western-Blotting.5.Transcription levels of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 gene in Huh-7 cells were detected.Trizol one-step method was used to extract total RNA from Huh-7 cells.GAPDH was used as internal reference,and mRNA expression of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 were detected by Real-time PCR.6.The contents of PCNA and c-Myc,CD133 and EpCAM as well as HNF4?and HNF-6 gene in Huh-7 cells were detected.Results:1.Determine the concentration of APS.Through CCK-8 experiment,OD value was calculated,and the concentration of APS was successfully determined to be 200 mg/L and the time point was 48h.2.APS can inhibit the proliferation and promote the differentiation of SMMC-7721 tumor cells.2.1 Effects of APS on mRNA and protein expression levels of PCNA and c-Myc in SMMC-7721 tumor cells.Real-time PCR results showed that the mRNA expression levels of PCNA and c-Myc were significantly lower than those of the control group?P<0.05?.The results of WB showed that the protein expression levels of PCNA and c-MYC were basically consistent with the mRNA expression levels.2.2 Effects of APS on mRNA and protein expression levels of CD133 and EpCAM in SMMC-7721 tumor cells.Real-time PCR results showed that mRNA expression levels of CD133 and EpCAM were significantly lower than those of the control group?P<0.01?,while WB results showed that protein expression levels of CD133 and EpCAM were basically consistent with mRNA expression levels.2.3 Effects of APS on the mRNA and protein expression levels of HNF4?and HNF-6 in SMMC-7721 tumor cells.Real-time PCR results showed that the mRNA expression levels of HNF4?and HNF-6 were significantly higher than those of the control group?P<0.01?.The results of WB showed that the protein expression levels of HNF4?and HNF-6 were basically consistent with the mRNA expression levels.3.APS can inhibit the proliferation and promote the differentiation of Huh-7in tumor cells.3.1 Effects of APS on mRNA and protein expression levels of PCNA and c-MYC in Huh-7 tumor cells.Real-time PCR results showed that the mRNA expression levels of PCNA and c-Myc were significantly lower than those of the control group?P<0.01?.The results of WB showed that the protein expression levels of PCNA and c-Myc were basically consistent with the mRNA expression levels.3.2 The effect of APS on mRNA expression levels and protein expression levels of CD133 and EpCAM in tumor cells of Huh-7.Real-time PCR results showed that the mRNA expression levels of CD133 and EpCAM were significantly lower than those of the control group?P<0.05?.The results of WB showed that the protein expression levels of PCNA and c-Myc were basically consistent with the mRNA expression levels.3.3 Effects of APS on the mRNA and protein expression levels of HNF4?and HNF-6 in Huh-7 tumor cells.Real-time PCR results showed that the mRNA expression levels of HNF4?and HNF-6 were significantly higher than those of the control group?P<0.01?.The results of WB showed that the protein expression levels of HNF4?and HNF-6 were basically consistent with the mRNA expression levels.Conclusion:1.The cell activity of SMMC-7721 and Huh7 cells can be inhibited by APS in a concentration-dependent manner.2.APS can inhibit the expressions of proliferation-related and stem-related genes in both SMMC-7721and Huh7 cells.3.APS can promote the expressions of differentiation-related genes in both SMMC-7721 and Huh7 cells.