| Objective:hepatocellular carcinoma cells are the most common malignant tumor in prime liver cancer, occult onset, and high degree of malignancy. Multidrug resistance, MDR phenomenon often led to failure of hepatocellular carcinoma chemotherapy. Tumor cells’ MDR to chemotherapeutic mostly belong to acquired drug resistance; it is the result of chemotherapy-drug induced and closely related with the genetic variation of epigenetic level. The non-coding RNA could regulate the express activity of its’ related genes. A lot of studies found that the abnormal expression of miRNA, in the HCC organization, including the let-7 family. Let-7c, an importment member of let-7 family, is closely related to many biological characteristics of hepatocellular carcinoma cell, like proliferation, apoptosis, invasion, and metastasis et al. But the regulation mode between let-7c and c-Myc in HCC is not clear. We assume that through the regulation of let-7c and c-Myc may reverse multidrug resistance of hepatocellular carcinoma cells and clarify the mechanism. In this study, firstly we use the c-Myc antisense phosphorothioate oligodeoxynucleotide technology regulate c-Myc activity in HepG2 cell, change biological characteristics of HepG2 cell and the sensitivity to 5-Fu; secondly, let-7c mimic and let-7c mimic NC were transfect into HepG2 cells, observe the targeted regulation mode between let-7c and c-Myc expression. We also analyzed the effects of let-7c changing on the expression of c-Myc and the booactivity of HepG2 and HepG2/5-Fu cells and explored possible mechanism of reversing the multidrug resistance.Methods:the first part, use liposome mediated c-Myc antisense phosphorothioate oligodeoxynucleotides combined with 5 fluorouracil regulate the expression of c-Myc in hepatocellular carcinoma cells, and the influence on the biological activity of HepG2 cells; the second part, let-7c mimic and let-7c mimic NC were designed and synthesized, transiently transfected into HepG2 cells, real-time qPCR andwestern blot were used to detect let-7c expression levels and the effect on the expression of c-Myc; and the changes of HepG2 cells’biological characteristics, like proliferation, apoptosis and metastasis, et al. Luciferase reporter assay was performed to detect whether the c-Myc was regulated by let-7c. The third part, detecting drug resistance of HepG2 cells and drug resistant cell strain HepG2/5- to DDP, VCR and 5-Fu; through let-7c mimic and let-7c inhibitor were transiently transfected into HepG2 cells and resistant strains of HepG2/5- Fu cells, the related regulation of target genes:such as c-Myc gene expression, than changing the sensitivity of HepG2 and HepG2/5- Fu cells to different chemotherapeutic drugs.Results:(1) c-Myc antisense oligonucleotide transfection combined with 5-Fu, down-regulation of c-Myc gene activity and protein expression in HepG2 cells, inhibit the proliferation and invasion of HepG2 cells, promote the apoptosis; and increase the sensitivity of HepG2 cells to 5-Fu. (2) The luciferase reporter assay confirmed that c-Myc is one of the target genes of let-7c; let-7c mimic can regulate the expression of c-Myc transcription and translation in two opposite directions; let-7c mimic transfection can up regulate the expression of let-7c, down regulate c-Myc mRNA translation, inhibit the expression of c-Myc, promote apoptosis, inhibit the proliferation of HepG2 cells; (3) drug resistant cells HepG2/5-Fu cells resistant 5-Fu, and other chemotherapy drug, like VCR, DDP; in let-7c mimic group, the expression of let-7c increased in the intracellular, inhibit the expression of c-Myc, inhibit HepG2/5-Fu cells’ proliferation, induce apoptosis, and improving the sensitivity of HepG2/5-Fu to different chemotherapeutic drugs; and after let-7c inhibitor transfection, the results showed reversed.Conclusion:liposome mediated c-Myc antisense oligonucleotide combined with 5-Fu can inhibit the expression of c-Myc gene, inhibits the proliferation of HepG2 cell invasion and promote its apoptosis; c-Myc antisense oligonucleotide can inhibit HepG2 cell activity, improve the sensitivity of HepG2 cells to 5-Fu and reduce the dose of 5-Fu. C-Myc is one of the target genes of let-7c, let-7c combined with the target site of c-Myc mRNA 3’UTR, play a role, directly regulation of the c-Myc expression, which regulate the proliferation and apoptosis of tumor cells. Let-7c mimic increase let-7c high expression in HepG2/5- Fu cells, inhibit c-Myc expression, inhibit the proliferation of HepG2/5- Fu cells, and improve the sensitivity of HepG2/5- Fu to different chemotherapy drugs, like DDP, VCR and 5-Fu. Let-7c is an important tumor suppressor gene in HCC; let-7c could reverse multidrug resistance of hepatocellular carcinoma cells by downregulation of c-Myc. Let-7c is likely to become a useful molecular targeted therapeutic index of malignant liver cancer in the future. |
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