Curcumin Enhances Taxol-Induced Hepatocellular Carcinoma Cell Proliferation And Induces Apoptosis Via NF-κB/Lin28B/let-7 Pathway

Objective In this study,firstly,the parental cell Hep3B was used as a research subject.The paclitaxel-resistance cell line Hep3B/TAX was established by intermittent drug induction in vitro,secondly,we used MTT,molecular interference technology,flow cytometry,Western Blotting,real time fluorescence quantitative PCR to explore the relationship between Lin28B/let-7 pathway and paclitaxel resistance in HCC cells;thirdly we combined curcumin and paclitaxel to study the effects of curcumin on paclitaxel-induced NF-κB and Lin28B via MTT,Western blotting,realtime fluorescence quantitative PCR and immunofluorescence.To explore the potential of curcumin enhances taxol-induced hepatocellular carcinoma cell proliferation and induces apoptosis via NF-κB/Lin28B/let-7 pathway,and provide theoretical basis on curcumin reversing paclitaxel resistance in hepatocellular carcinoma.Methods ①Hep3B/TAX resistant cell lines were established by intermittent drug induction in vitro,and Hep3B/TAX cells were identified by microscopy,drug sensitivity experiments and flow cytometry.②Realtime fluorescence quantitative PCR and Western Blot assays were used to detect the levels of Lin28B on gene and protein expression in Hep3B and Hep3B/TAX cells;molecular interference technique was used to up-regulate or down-regulate the expression levels of Lin28B,and realtime fluorescence quantitative PCR and Western Blot assays were used to detect Lin28B on gene and protein again,MTT assay was used to determine the effect of paclitaxel on Hep3B/TAX cell proliferation,Flow cytometry was used to detect the effect of paclitaxel on Hep3B/TAX cell apoptosis,and Western Blot assay was used to detect paclitaxel related apoptosis protein in resistant cells.The expression level of let-7 family was detected by realtime fluorescence quantitative PCR in Hep3B and Hep3B/TAX cells,and the expression of related apoptotic proteins in Hep3B and Hep3B/TAX cells was detected by Western Blot assay.③MTT assay was used to detect the effects of different concentrations of curcumin and curcumin combined with paclitaxel on the proliferation of drug-resistant cells.Western blot was used to detect the effect of curcumin and paclitaxel on the expression of apoptosis-related proteins in drug-resistant cells,the effect of curcumin and paclitaxel on the expression of Lin28B in drug-resistant cells detected by realtime fluorescent quantitative PCR,and the combination of curcumin and paclitaxel detected by Western Blotting on the effect of Lin28B and NF-κB protein expression in drug-resistant cells was detected by immunofluorescence.The effect of curcumin combined with paclitaxel on the activity of NF-κB in drug-resistant cells was detected,immunohistochemical technique for detecting the expression of Lin28B and NF-κB in drug-resistant cells in combination with curcumin and paclitaxel.Results ①Hep3B/TAX resistant cell lines were successfully established and the identification of drug-resistant cell lines was completed.②Real-time quantitative PCR and Western Blot assays showed that Lin28B was highly expressed in Hep3B/TAX cells,indicating that Lin28B expression was associated with paclitaxel resistance.After silencing the Lin28B gene in Hep3B/TAX cells,the expression level of Lin28B in Hep3B/TAX cells was significantly reduced compared to the negative control group.At the same time,recovery experiments were performed to overexpress the Lin28B gene in Hep3B/TAX cells.The results showed that the expression level of Lin28B was significantly increased,and the relationship between Lin28B and paclitaxel resistance in HCC cells was verified in reverse.Fluorescent quantitative PCR results showed that compared with Hep3B cells,the expression levels of seven members of let-7 family were significantly decreased in Hep3B/TAX cells,and the expression levels of Bcl-xL,Bcl-2,caspase9,and caspase3 proteins in Hep3B/TAX cells were significantly increased.Increased;Bax expression levels decreased significantly.③Curcumin significantly inhibited the proliferation of Hep3B/TAX cells.The IC₅₀ of Hep3B/TAX cells against curcumin was 34.99±2.43 μmol/L.MTT assay detected the synergistic effect of curcumin and paclitaxel.The results showed that paclitaxel significantly reduced the IC₅₀ of Hep3B/TAX cells in a dose-dependent manner when combined with paclitaxel and curcumin.After combined use of paclitaxel and curcumin,the apoptosis rate of Hep3B/TAX cells increased significantly,the expression of apoptotic protein increased,the expression of Lin28B and NF-κB decreased,and the transcriptional activity of NF-κB decreased.Conclusion In summary,a preliminary study of the association between Lin28B/let-7 pathway and paclitaxel resistance in HCC cells found that Lin28B activation in HCC cells inhibits let-7 expression,leading to decreased apoptosis of HCC cells and promotes tumorigenesis.development of.The activation of NF-κB can induce the high expression of Lin28B,thereby blocking the maturation of let7-miRNA and inhibiting the apoptosis of tumor cells.Curcumin can inhibit the activity of NF-κB,down-regulate Lin28B expression,inhibit cell proliferation,and promote tumor cell apoptosis.Therefore,our results suggest that curcumin may increase sensitization to paclitaxel in the NF-κB/Lin28B/let-7 pathway to inhibit the proliferation of hepatoma cells and promote apoptosis.