| Background:Rapid developed medical technology makes our life better these days:a variety of HCC diagnostic markers have been found;immunotherapy,surgical resection,radiotherapy and chemotherapy for different liver cancer therapy are also widely used in clinical practice.However,the incidence of HCC in the world is still on the rise,and it becomes a serious threat to human health and well-being.Many types of liver damages eventually develop into HCC,including:viral infection,fatty liver,liver fibrosis,cirrhosis,etc.It seems that at present,our understanding of HCC is still flawed,and the diagnosis and treatment of HCC are not comprehensive enough.Researchers need to continue with the investigation into HCC to discover unknown mechanisms.Our task is currently the first to provide a study of HCC associated with TRPML1.Perhaps a more in-depth understanding of the function of TRPML1 might provide some new clues to the complicated mechanism of HCC and provide a new potential new target for the clinical treatment of HCC.Objective:To investigate the presence of TRPML1?ML-1?in liver tissue,and the relationship between TRPML1?ML-1?and hepatocellular carcinoma.Then,at the cellular level,the mechanism of TRPML1?ML-1?affecting hepatocellular carcinoma was preliminary studied.Methods:First,the presence of ML-1 in human liver cancer tissue microarrays was detected by immunohistochemical staining.If it exists,use the multi-spectral quantitative system to analyze its distribution law;and further verified in Kunming mice,C57 BL/6 mice liver tissue.Then,the presence of ML-1 protein in HepG2 cells and HepG2 cells responded to the ML-1 agonist ML-SA1 were detected by Western Blotting.Under the above conditions,we used intracellular calcium chelators,extracellular calcium chelators,cAMP downstream protein inhibitors,and ML-SA1 alone or in combination to stimulate HepG2 cells to detecte ML-1,ERK1/2,P-ERK1/2 proteins expression;and explored TRPML1 involved in the process of HCC.Results:First,we detected the presence of ML-1 in human hepatocellular carcinoma,and its expression showed a certain pattern:ML-1 was less expressed in the cancer areas;while in the Para cancer area was more expressed.Using multispectral statistics of 120 samples of human hepatocellular carcinoma tissue chips,we found that the distribution of total ML-1signal in the sample varied greatly,but the total ML-1 signal in the sample was relatively low.Among them,no signal of ML-1 was detected in 10.83%of the samples,and more than 49%of the samples'ML-1 signal for 1-100.Then,we verified the expression of ML-1 in liver tissue in animal samples.The presence of ML-1 protein in the liver tissue of Kunming mouse was found by immunohistochemical staining;and the expression of ML-1 in Kunming mouse hepatocellular carcinoma was similar to that in human hepatocellular carcinoma's.In Kunming mouse,there was almost no ML-1 expression in the cancer area;but relatively high in precancerous and fatty areas.We administered to stimulate the 5 weeks,10 weeks,15 weeks and 20 weeks after a total of four groups of Kunming mouse liver tissue immunohistochemical staining,the use of multispectral quantitative statistics found:compared with the expression of ML-1 at 5 weeks after administration,ML-1 expression increased significantly after 10 weeks of administration?P<0.001?;and the expression of ML-1 was significantly decreased after 15 weeks of administration?P<0.001?,this was same too about 20 weeks after administration?P<0.001?.In addition,there was also a statistically significant difference?P<0.01?in the expression of ML-1 between the 15th week of administration and the 20th week of administration.We found that ML-1 also existed in C57BL/6 mouse's liver,and the expression of ML-1 in liver injury tissues was significantly decreased?P<0.001?compared with the control group.Moreover,the average expression of ML-1 in the liver tissue of Kunming mouse was significantly higher in the central vein than in the portal vein?P<0.001 or P<0.05?,and the expression of ML-1 in the liver tissue at the 5th week after stimulation was observed.There were significantly more fat areas than non-fat areas?P<0.001?;similar patterns also existed in the livers of C57 BL/6 mouse.Next,we initially explored the mechanism of ML-1 affecting liver injury and liver cancer at the cellular level.The results of MTT assay showed that after HepG2 cells were treated with ML-1 activator ML-SA1,the survival rate of HepG2 cells maintained at about 1.0?P>0.05,no significant difference?.1.ML-1 protein was present in HepG2 cells.When the concentrations of ML-SA1 were 6?M12?M,ML-1 expression was significantly increased with increasing concentrations?P<0.001?,while the phosphorylation of ERK1/2 gradually decreased with the increase of ML-SA1.Comparison with a stimulation concentration at 6?M,12?M the phosphorylation of ERK1/2 was also significantly reduced?P<0.01?.2.The expression of ML-1 gradually decreased with the prolongation of time when the ML-SA1 stimulation concentrations were 6?M and 12?M;the expression of ML-1 was significantly lower than that of the control group at 8?P<0.01?and/or 4 h?P<0.001?.However,the phosphorylation of ERK1/2 increased with the time prolonged with the stimulation of ML-SA1,at first increased and then decreased.The stimulation of ML-SA1with P-ERK1/2 expression and ERK1/2 phosphorylation reached the maximum at 1 h or/and 2 h,and were significantly higher than that of the control group?P<0.001?.3.ML-SA1 and PKA inhibitor Rp-cAMPs stimulated HepG2 cells for 4 h.And the optimal concentration of Rp-cAMPs stimulation was 50?M.Compared with no ML-SA1,the expression of ML-1 was significantly up-regulated after co-stimulation?P<0.001?,and the phosphorylation of ERK1/2 was also significantly up-regulated?P<0.05?.4.ML-SA1 and Epac inhibitor ESI-09 alone or jointly stimulated HepG2 cells for 4 h.The ML-1 expression was significantly down-regulated?P<0.05?when ESI-09 was added 5 min prior to stimulation compared with ML-SA1 alone.Compared with adding them at the same time and adding ML-SA1 5 min earlier,the phosphorylation of ERK1/2 was significantly increased?P<0.001?when ESI-09 was added 5 min before.5.HepG2 cells were stimulated by ML-SA1 and ESI-09 individually or together for 1 h.Compared with adding them at the same time and adding ML-SA1 5 min earlier,the expression of ML-1 was significantly down-regulated?P<0.01?when ESI-09 was added 5min earlier.Compared with the control group,the expression of P-ERK1/2 in each group was significantly decreased?P<0.001?.Compared with adding ML-SA1 5 min earlier,the phosphorylation of ERK1/2 was significantly down-regulated when ESI-09 was added 5 min earlier?P<0.01?.6.HepG2 cells were stimulated with 1?M and 10?M intracellular calcium chelator BAPTA-am for 0.1 min,1 min,3 min,5 min.The expression of ML-1 was down-regulated more significantly when stimulated with 10?M BAPTA-am?P<0.001?for 3 min,5 min than that 1?M BAPTA-am?P<0.01?.The phosphorylation of ERK1/2 was significantly up-regulated?P<0.001?in HepG2 cells after stimulated with BAPTA-am for 0.1 min,reaching the maximum at 1 min?P<0.001?;and then the level of it was rapidly decreased to the level of control group?P<0.001?,after stimulated for 5 min.ML-SA1,extracellular calcium chelator EGTA and Intracellular calcium chelator BAPTA-am stimulated HepG2 cells separately or co-stimulated for 1 min,5 min,10 min and 30 min.At 5 min stimulation alone,ML-1 was significantly up-regulated in BAPTA-am group?P<0.001?.At 30 min stimulation alone,ML-1 in EGTA group?P<0.001?was significantly up-regulated.The ERK1/2 phosphorylation rate of EGTA group was significantly increased?P<0.001?at 5 min stimulation alone.At 30 min stimulation alone,EGTA group's phosphorylation of ERK1/2 was significantly decreased?P<0.001?.Conclusion:1.For the first time,TRPML1?ML-1?was demonstrated in human hepatocellular carcinoma and animal liver lesions,and the expression of ML-1 was associated with the occurrence and development of hepatocellular carcinoma.2.ML-SA1 stimulation had no significant effect on the proliferation of HepG2 cells.The ML-1 protein was found in HepG2 cells.HepG2 cells showed a response when stimulated with 6?M ML-SA1 and the ML-1 expression reached its maximum at 12?M.While the ERK1/2 phosphorylation were negatively correlated with the expression of ML-1.In subsequent experiments,6?12?M ML-SA1 stimulation was chosen to stimulate HepG2 cells and detect protein expression.ML-SA1 stimulation,with the passage of time,ML-1expression decreased,showing a negative correlation.However,the phosphorylation rate of ERK1/2/2 increased first and then decreased with the increase of ML-SA1 stimulation time and reached the maximum value.In subsequent experiments,two ML-SA1 stimulation times of 1h and 4 h were chosen to stimulate HepG2 cells and detect protein expression.3.The optimal concentration of Rp-cAMPs stimulation was found to be 50?M.The effect of Rp-cAMPs on the expression of ML-1 and the phosphorylation of ERK1/2 in ML-SA1 was not significant.That is,the expression of ML-1 stimulated and the phosphorylation rate of ERK1/2 by ML-SA1 were not achieved via the cAMP-PKA pathway.4.At 4 h stimulation,ESI-09 affected the expression of ML-1 and the phosphorylation of ERK1/2,and the effect was completed when it was added 5 min earlier.That is,ML-SA1stimulated the expression of ML-1 and the ERK1/2 phosphorylation rate was achieved via the cAMP-Epac pathway.In HepG2 cells,expression of ML-1 for ESI-09 stimulation was synergistic with ML-SA1 stimulation.The proteins change trend detected at 1 h after stimulation was almost the same as that at 4 h.It seems to indicate that there may be a response-feedback loop in HepG2 cells when ESI-09 affected ML-SA1-stimulated HepG2cell-producing proteins expression changed.First of all,ESI-09 affected the expression of ML-1 stimulated by ML-SA1,and then the expression of ML-1 affected the phosphorylation of ERK1/2.5.BAPTA-am short-term stimulation of HepG2 cells can cause both ML-1 expression and ERK1/2 phosphorylation decreased.In contrast,it was found that the expression of ML-1 was significantly down-regulated when stimulated with 10?M BAPTA-am.Therefore,we chose10?M as the experimental concentration of BAPTA-am in subsequent experiments.The effect of EGTA on the expression of both ML-1 and ERK1/2 phosphorylation lagged behind BAPTA-am,it was suggested that intracellular Ca2+mainly affects the expression of both ML-1 and ERK1/2 phosphorylation rate.That is,both ML-1 and P-ERK1/2 expression changes are mainly caused by intracellular Ca2+outflow.Combined with the previous results,we propose that ML-SA1 stimulation of HepG2 cells affect the expression of both ML-1 and P-ERK1/2,and ERK1/2 phosphorylation rate;it was a dynamic process of feedback:first,ML-SA1 affects ML-1 expression;then,the expression of the altered ML-1 caused the outflow of intracellular Ca2+and the expression of P-ERK1/2,the expression of P-ERK1/2 in turn affected Ca2+outflow and the expression of ML-1.That is,there is a ML-1-Ca2+-P-ERK1/2/2 pathway in HepG2 cells.Taken together,it is generally believed that the expression of P-ERK1/2 in cancerous tissues is up-regulated and we found that the expression of ML-1 in liver injury and liver cancer of human and animals is down-regulated.We think that ML-1-Ca2+-P-ERK1/2 pathway also exists in liver injury and liver cancer of human and animals. |
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