| Objective:Diabetes mellitus is an important risk factor for liver cancer,but its pathogenesis remains unclear.Corosolic acid(CA)has been proved to have both hypoglycemic and anti-tumor effects,so revealing the function of CA will help us to understand the relationship between diabetes and liver cancer.In previous studies,we confirmed that CA can effectively inhibit the expression of YAP,an important oncoprotein in HCC cells,and inhibit the proliferation of HCC cells.In addition,we also found that O glycosylation plays an indispensable role in the occurrence and development of liver cancer.However,it is not clear whether CA can inhibit the effect of O-GlcNAcylation in HCC cells induced by high glucose.In this study,the anti-tumor effect and mechanism of CA were explored by inhibiting the level of O-GlcNAcylation acylation in liver cancer cells under the effect of high glucose,so as to provide a more reliable theoretical and experimental basis for the clinical application of the preparation for the treatment of liver cancer.Methods:(1)HCC cells and normal hepatocytes HL-7702 were treated with different concentrations of CA(0 μM/ ml,10 μM/ ml,20 μM/ ml,40 μM/ ml,80 μM/ ml,160 μM/ml)for 24 h.Then CCK8 was used to detect the effect of CA on cell viability,and the two most sensitive cells were screened out.Two kinds of liver cancer cell lines were treated with 5.5 m M of normal glucose and 15 m M and 25 m M of increasing D-glucose,and the cell activity was detected by CCK-8.The proliferation ability of HCC cells was detected by soft AGAR tumorigenesis assay.In addition,CCK-8 detection and soft AGAR tumorforming experiment were used to detect the proliferation of the two liver cancer cells in the culture medium with different concentration gradient of CA in normal(glucose 5.5m M),high(glucose 25 m M)and high(glucose 25 m M).(2)The expressions of YAP,OGT,SLC5A3 and Nudt9 in liver cancer tissues were predicted by TCGA online visualization website,and the expressions of YAP,OGT,SLC5A3 and Nudt9 were detected by immunohistochemical staining assay in matched paracancerous and liver cancer samples.The effects of different glucose concentrations and CA treatments on the expression of YAP,OGT,SLC5A3,Nudt9 and O-Glc Acylation were studied by Western-blot,RT-q PCR,subcutaneous tumor transplantation in nude mice and immunofluorescence experiments.(3)CDK19,a kinase that may be related to CA’s effect on O-Glc Acylation,was screened by RT-q PCR.The effects of different sugar concentrations and CA treatments on CDK19 were detected by Western-blot and RT-q PCR.CDK19 knockout vector was constructed.After successful transfection,the effects of overexpression and knockout of CDK19 on O-GlcNAcylation in cells were detected by Western-blot.CCK-8 and soft AGAR oncogenic experiments were used to detect the changes in cell proliferation.(4)Through online analysis of GEPIA database,it was found that there was a significant correlation between the expression of CDK19,OGT and YAP in liver cancer tissues.To prove whether CDK19 promotes O-GlcNAcylation of HCC cells through OGT.Overexpression of CDK19 also knocked down OGT,and the expression of CDK19,OGT,and o-Glc Acylation in cells was detected by Western-blot.Meanwhile,in order to prove whether CDK19 promotes the over-expression of CDK19 and YAP knockdown of HCC cells through YAP,the expression of CDK19,YAP and O-Glc Acylation in HCC cells was detected by Western-blot.Senexin B,an inhibitor of CDK19,was used to detect the expression of CDK19,OGT,YAP and O-GlcNAcylation proteins in cells by Westernblot analysis.CCK-8 and soft AGAR tumor-formation experiments were used to detect the changes in cell proliferation,and immunofluorescence assay was used to detect the changes in intracellular YAP and CDK19.(5)The expressions of CDK19,OGT,YAP,and O-Glc Acylation in normal and successfully transfected CDK19 SG cells treated with CA were detected by Western-blot.Meanwhile,CCK-8 and soft AGAR oncoformation experiments were also used to detect the changes in cell proliferation,and to explore whether CA suppressed OGT through CDK19 and thus reduced the O-Glcacylation level of HCC cells.The expressions of CDK19,OGT,YAP,and O-Glcacylation in cells with CDK19 overexpression and normal CDK19 expression were detected by Western-blot.Meanwhile,CCK-8 test and soft AGAR oncoformation test were used to detect cell proliferation,further confirming that CDK19 is the role of CA in regulating the level of O-Glcacylation in HCC cells.Results:(1)CA inhibited the proliferation of HCC cells in a dose-dependent manner.Compared with other cells,the growth inhibition of Bel-7402 and Bel-7404 cells by 40 μm CA was about 50%,so these two hepatoma cell lines were selected as the study subjects.CA significantly inhibited the proliferation of Bel-7402 and Bel-7404 cells,while the proliferation activity of Bel-7402 and Bel-7404 cells increased with the increase of glucose concentration.The effect of high glucose on hepatoma cell viability was independent of osmotic pressure.(2)The expressions of YAP,OGT,SLC5A3 and Nudt9 in liver cancer tissues were upregulated compared with those in normal liver tissues.After Bel-7402 and Bel-7404 cells were treated with different concentrations of glucose,the expression of O-GlcN acylation increased with the increase of glucose concentration,and the higher glucose concentration,the higher YAP and OGT levels also increased.Moreover,the expressions of SLC5A3 and Nudt9 were significantly increased in 25 m M glucose,and the HBP pathway was activated in HCC cells cultured with high glucose.Further study found that high glucose had no effect on LATS1,LATS2,MST1,and MST2,suggesting that the regulation of YAP by High-glucose was not due to the effect of hippo pathway.(3)CA down-regulates the m RNA and protein levels of OGT,Nudt9 and SLC5A3 under high glucose,suggesting that CA can inhibit the activity of the HBP pathway.In addition,CA can regulate the protein expression of YAP,but has little effect on the m RNA level of YAP,suggesting that CA regulates the expression of YAP through the protein level.CA can inhibit the expression of High-glucose-induced O-GlcNAcylation and enhance the expression of O-GlcNAcylation,YAP and OGT in A dose-dependent manner.Immunofluorescence results showed that increasing glucose concentration could stimulate the nuclear accumulation of YAP,but CA treatment could promote YAP from the nucleus to the cytoplasm.Nude mouse transplantation tumor experiment results show that the STZ xenograft tumor can be induced to grow significantly in the body,but the CA or insulin treatment can significantly reduce the size of the tumor and the weight,IHC staining showed that the STZ treatment induced OGT,YAP and the increase of the expression of O-GlcNAcylation,SLC5A3 HBP key genes and significantly increased the expression level of Nudt9,and CA treated both expression were significantly lower in the organization.(4)The protein expression of CDK19/CDK8 increased with the increase of glucose concentration,while CA could significantly inhibit the expression of CDK19 under high glucose,but had little effect on the expression of CDK8.Compared with normal glucose concentration,cell proliferation and colony formation were significantly enhanced in high glucose culture,while in CDK19-deficient HCC cells,high glucose did not induce enhanced transformed phenotypes.Overexpression of CDK19 resulted in increased expression of O-GlcNAcylation in Bel-7402 and Bel-7404 cells,while knockout of CDK19 was the reverse.The only endogenous enzyme OGT catalyzing OGlcNAcylation was positively regulated by CDK19.Online analysis of GEPIA database showed a significant correlation between CDK19 and OGT expression in HCC tissues.OGT knockout significantly reduced YAP and global O-GlcNAcylation levels,while overexpression of OGT increased YAP and global O-GlcNAcylation levels.However,overexpression or knockout of OGT had no effect on CDK19 expression.The increase in YAP and global O-glycation induced by CDK19 overexpression can be reversed by simultaneous OGT knockout.Consistent results were also confirmed in the ability of cell proliferation and colony formation.(5)The overexpression of CDK19 resulted in significantly upregulated YAP,while CDK19 knockout had the opposite effect.The overexpression or knockout of YAP did not affect the expression of CDK19.In addition,YAP knockout prevented the increase of O-GlcNAcylation induced by CDK19 overexpression.CDK19 overexpression significantly upregulated YAP,OGT and O-GlcNAcylationlevels,enhanced cell proliferation and clone formation ability,while simultaneous YAP knockout could reverse these effects.In addition,Senexin B significantly inhibited the levels of OGT,YAP and O-GlcNAcylation,cell proliferation and colony formation in Bel-7402 and Bel-7404 cells.In HCC cells treated with Senexin B,high glucose did not induce an enhanced transformed phenotype.Immunofluorescence assay showed that both CDK19 knockout and Senexin B treatment reduced nuclear localization of YAP.(6)The levels of OGT,YAP and O-GlcNAcylation in HCC cells treated with CA were higher than those treated with CA alone when CDK19 gene was knocked out.In addition,the levels of YAP,OGT and O-GlcNAcylation in HCC cells treated with CA or CDK19 SG were significantly decreased,and CDK19 overexpression could partially reverse the levels of YAP and O-GlcNAcylation and transformed phenotypes damaged by CA treatment.Conclusion:High glucose can promote the proliferation of HCC cells,while CA can inhibit the growth of HCC cells and xenograft tumor in nude mice.CA inhibited the activation of HBP pathway under high glucose,decreased the expression of YAP and OGT,and inhibited the level of O-GlcNAcylation in HCC cells under high glucose culture.CA could significantly inhibit the expression of CDK19 under high glucose,and CDK19 could regulate the level of O-GlcNAcylation in HCC cells through YAP and OGT.It was demonstrated for the first time that CA can reduce the proliferation ability of high-glucose cells through the inactivation of CDK19/YAP/O-GlcNAcylationn pathway,and thus inhibit tumor growth,suggesting that CA may be a candidate drug for anti-diabetic hepatocellular carcinoma. |
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