| Objective: Hepatocellular carcinoma(HCC) is one of the most common cancers worldwide with a very poor prognosis and its multiple drug resistance(MDR) emergence that still is a challenge of the distressed effective treatments. The over-expression of P-glycoprotein(P-gp) encoded by the multiple drug resistance 1(MDR1) gene is identified as one of the most important reasons for MDR formation in HCC. Although the abnormal activation of the inflammatory medium nuclear factor-kappa B(NF-?B) has been implicated and plays an important role in HCC during the development and progression. However, the molecular mechanism between MDR in HCC and NF-?B still remains to be clarified. The objectives of this study were to investigate the expression of P-gp in HCC tissues, its correlations with the clinicopathological features, and patient's survival, and the models of hepatoma resistant Hep G2/adriamycin(Hep G2/ADM) cells were used to explore the interfering NF-?B gene transcription by specific mi RNA on MDR1 gene regulation and combining with metformin to down-regulates P-gp by inhibiting NF-?B activation to observe the effect on reversing MDR of HCC.Methods: The P-gp expressions were detected in 75 HCC and their para-cancerous tissues by immunohistochemistry. Human hepatocyte(LO2), hepatoma Hep3 B, SMMC-7721, MHCC-97 H,BEL-7404, Hep G2 and adrimycin-resistant Hep G2/ ADM cells were used in this study in vitro. Four set specific p CMV-NF-?Bmi RNAs were successfully constructed for silencing NF-?B activation and were confirmed by sequencing, and one of the most effective plasmids after screening was transfected into Hep G2 and Hep G2/ADM cells. Alterations of proteins or gene transcription were analyzed by Western blotting or fluorescent quantitative PCR(FQ-PCR) technology. Cell proliferation, cell cycle or apoptosis were checked by the CCK-8 assay, flow cytometry, or Annexin-VPE/7- ADD double staining assay, respectively.Results: The positive expression of hepatic P-gp was mainly localized in cell membrane and cytoplasm. The incidence of P-gp expression(70.67%) in the HCC tissues was significantly higher(?2=27.312, P<0.001) than that in the self-control para-cancerous tissues(28.00%). The over-expression of P-gp in the HCC tissues was closely related to lymph node metastasis(P=0.037), TNM stage(P=0.048), and 5-year survival(P=0.007), and not to age, gender,AFP, portal vein invasion, hepatitis B virus infection, liver cirrhosis,differentiation degree, tumor size, and Child classification. Both of higher P-gp expressions and NF-?B phosphorylation were found after the hepatoma Hep3 B, SMMC-7721, MHCC-97 H, BEL-7404,and Hep G2 cells treated with adrimycin compared with untreated group and the Hep G2 cells were one of the most significant among them. The IC50 values of the Hep G2 or Hep G2/ADM cells to adriamycin was 0.489?M or 4.166?M at 24 h,0.221?M and 1.522 ?M at48 h, and 0.224?M or 1.380?M. The resistance index(RI) of the Hep G2/ADM cells was 8.519 at 24 h, 6.874 at 48 h, and 6.166 at 72 h,respectively. The ?ct values of mdr1(3.310±0.154) and NF-?B(2.580±0.040) expression at m RNA transcriptional levels in the Hep G2/ADM cells were significantly higher(P<0.001) than those of mdr1(0.084±0.038) and NF-?B(0.6067±0.032) in the Hep G2 cells. The p CMV-NF-?B-mi RNA1(mi RNA1) plasmids were screened as one of the most efficiency(71%) and specific plasmid among 4 sets of the successful constructed mi RNAs for cell transfection. After the Hep G2/ADM cells transfected with mi RNA1,the relative expression of the mdr1 m RNA(2-??ct=0.326±0.011) was significantly lower(t=14.262, P<0.01) in mi RNA group than those in the mi RNA-neg group(0.804±0.057) with 40.55% of inhibition,the decreasing of cystolic t-p65, karyon p-p65 and P-gp expression,the dramatic decline of the clone formation abilities(P<0.01),moreover cell cycle at G1 arrest, and increasing apoptosis rate(14.1±1.77%). After the Hep G2 or Hep G2/ADM cells transfected with mi RNA1 at 48 h, the IC50 values were 0.221?M or 1.522?M in the untreated group, 0.212?M or 1.139?M in the mi RNA-neg group,and 0.094?M or 0.254?M in the mi RNA group, respectively.Significant decline of the values was 2.26 times in the Hep G2 and4.48 times in the Hep G2/ADM cells between the mi RNA-neg group and the mi RNA-neg group, and the specific mi RNA1 could reverse notably MDR of hepatoma cells. The Hep G2/ADM cells after pretreatment with meformin were sensitized to adriacin and confirmed with the mechanism of P-gp downregulation through the NF-?B signaling pathway. The synergistic inhibition effect of both collaboration were founded in the Hep G2/ADM cell proliferation inhibition, cell cycle arrest and inducing cell apoptosis and the same molecular mechanism that inhibited the P-gp expression via NF-?B signaling pathway.Conclusions: The present data suggested that the abnormal expression of MDR1/P-gp in HCC was related to MDR information,which could be down-regulated through inhibiting activation ofNF-?B signaling pathway by specific mi RNA and increasing sensitivity to chemotherapy drugs, and mi RNA with metformin drug showing more effective reversing MDR of HCC. |
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