| BackgroundAdvanced hepatocellular carcinoma(HCC)has a poor prognosis due to the loss of surgical opportunities.Currently,the targeted drug apatinib plays an important role in the treatment of advanced HCC.However,a phenomenon of decreased sensitivity to apatinib has been observed in clinical treatment of HCC,and the relevant mechanisms need to be clarified.Stanniocalcin2(STC2)is upregulated in HCC and its overexpression reduces the sensitivity of HCC to certain anti-tumor drugs,but it is currently unclear whether STC2 can affect the sensitivity of HCC to apatinib.Hypoxia-inducible factor 1α(HIF-1α)can mediate the decreased sensitivity of HCC to certain anti-tumor drugs,and in other tumors,it has been found that HIF-1α can upregulate the expression of STC2 to promote cell proliferation and migration.However,whether HIF-1αcan affect the sensitivity of HCC to apatinib by promoting the expression of STC2 has not been reported.ObjectiveThis study aims to investigate the changes in the expression level of STC2 in HCC cells with decreased sensitivity to apatinib and to explore the regulatory relationship between HIF1α and STC2 in HCC.We also aim to explore the impact of the upregulation of HIF-1α and STC2 expression in HCC on the treatment efficacy of apatinib.This research provides new ideas and theoretical support for overcoming the reduced efficacy of apatinib in the treatment of HCC.Methods1.Huh-7 cells of liver cancer were cultured with different concentrations of apatinib,and the absorbance of Huh-7 cells under different concentrations was detected using the CCK-8 assay,and the half-maximal inhibitory concentration(IC50)was calculated.2.Real-time quantitative PCR(qPCR)and Western blot(WB)assays were used to detect the expression levels of STC2 mRNA and protein in cells treated with different concentrations of apatinib,respectively.3.Bioinformatics analysis was performed to investigate the correlation between STC2 and HIF-1α in HCC.4.Hypoxia was induced in Huh-7 cells treated with apatinib,and HIF-1α was knocked down in the cells.The expression of HIF-1α and STC2 mRNA in cells under different treatment conditions was detected using qPCR.5.Chromatin immuneprecipitation combined with qPCR(ChIP-qPCR)was used to investigate whether HIF-1α binds to the STC2 promoter in Huh-7 cells.6.The CCK-8 assay was used to detect changes in cell proliferation activity after knocking down HIF-1α in Huh-7 cells treated with apatinib.7.The Transwell assay was used to detect changes in cell migration ability after knocking down HIF1α in Huh-7 cells treated with apatinib.Results1.With the increase of apatinib culture concentration,the IC50 of Huh-7 cells significantly increased.Compared with the sensitive control group,the expression of STC2 in the apatinibinsensitive group was higher.2.Bioinformatics analysis showed a significant positive correlation between STC2 and HIF-1α in HCC.3.Real-time quantitative PCR results showed that the mRNA expression level of STC2 decreased after knocking down HIF-1α in Huh-7 cells,while the mRNA expression level of STC2 increased as HIF-1α levels increased under hypoxia.4.ChIP-qPCR results suggested that HIF-la could bind to the promoter of the STC2 gene.5.Using the CCK-8 kit to detect cell proliferation activity,the results showed that the proliferation activity of Huh-7 cells decreased after knocking down HIF-1α on the basis of apatinib culture.6.Transwell experiments were used to detect cell migration ability,and the results showed that the migration level of Huh-7 cells decreased after knocking down HIF-1α on the basis of apatinib culture.ConclusionThis study suggests that the increase of STC2 may be related to the decreased sensitivity of HCC to apatinib.HIF-1α can bind to the promoter region of STC2,and changes in HIF-1αcan affect the expression of STC2.Moreover,high expression levels of HIF-1 a and STC2 can weaken the inhibitory effect of apatinib on HCC proliferation and migration,and thus reduce the sensitivity of HCC to apatinib. |
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