Calcium Lactate Promotes Hepatocellular Carcinoma Cell Proliferation And Metastasis Via Calpains-Dicer Axis

Objective: To detect the content of calcium lactate salt in HCC tissue and explore its effect and mechanism on HCC cell proliferation and metastasis.Methods:Part one: DEN was used for 14 weeks to construct HCC in situ model and HE stain was used to distinguish normal or tumor;the content of calcium lactate in HCC tissues and normal tissues were detected using corresponding kit;Part two: Different concentration(0mM,0.25 mM.1mM and 2mM)of Calcium lactate(CaLa)was used to mimic high calcium lactate salt cumulate-microenvironment;CCK8 was performed to detect the effect of CaLa on HCC cell proliferation;Clonal assay was used to detect the ability of HCC cell to form colon;Flow cytometry was used to detect the change of cell-cycle and apoptosis in HCC cell while treatment with different concentration of Cala;Wound healing assays were used to detect the effect of CaLa on HCC cell migration;Transwell assays were used to detect the effect of CaLa on HCC cell migration and invasion;Part three: Immumohistochemical staining was performed to detect the expression of Calpains and lactate metabolism associated protein,while the relationship was also detected by Pearson analysis;the mRNA level,protein level and protein activity were also detected in HCC after treatment with CaLa while the concentration of intracellular calcium was also detected using Fluo-4 am;The expression of CalpainS1,Calpain2 and Dicer in HCC tissues was detected using immumohistochemical staining and western blot,while their expression in HCC cell after treatment with CaLa was also detected;Furthermore,we construct CalpainS1 lowly-expressed cell line,Calpain2 lowly expressed cell line and Dicer highly expressed cell line to explore whether alter the expression of CalpainS1,Calpain2 and Dicer can reverse the effect of CaLa.Results: HCC in situ model was constructed successfully and we found high concentration of calcium lactate salt accumulated in HCC tissues;CaLa promoted HCC cell HEPG2 and HEP3 B proliferation,colon formation,migration and invasion signally,while its effect on cell-cycle and apoptosis was also prominent(p<0.05);A member of Calpains named CalpainS1 was co-expressed with key proteins which regulated lactate metabolism pathway;The expression of CalpainS1 was increased in HCC cell after treatment with CaLa,while the activity was also increased;High expression of CalpainS1 was associated with low expression of Dicer in HCC tissues;The expression of Dicer was decreased after treatment with CaLa;CalpainS1 and Calpain2 inhibition can reserved the effect of CaLa on the proteolysis of Dicer and cell proliferation,as well as cell migration;Recovered the expression of Dicer also revered the effect of CaLa in cell proliferation and migration.Conclusion: High calcium lactate accumulation is a feature of microenvironment in HCC and it increases the mRNA level of CalpainS1 and the expression of Calpain2,induces the proteolysis of Dicer,therefore promotes HCC cell proliferation and metastasis.