| Background and ObjectivesHepatocellular carcinoma (HCC) is one of the common malignant tumors in the world. The Global Cancer Report in 2014 published by WHO indicated the deaths caused by HCC in China accounted for 51% of global deaths. Currently, the best treatment for HCC is still surgery, but the difficulties of the early diagnosis resulted in only 15% HCC patients fitting in with surgery. Because of the poor sensitivity to chemotherapy and strong metastasis, the recurrence rate is high and the 5-year mortality is more than 90% for HCC patients. So searching the novel and better treatments becomes extremely important. In recent years, the relationship between miRNA and tumor has arrested the attention. Some investigations showed that the miRNAs may participate in the regulation of the cell differentiation, proliferation, apoptosis, tumorigenesis, hormone secretion and many other biological processes, which may become the novel targets for HCC therapy. However, in order to realize the clinical applications of miRNA, a safety and efficient carrier system is also needed, exosome is a kind of natural lipid nanoparticles, which plays a role in transmitting the nucleic acids and proteins in the intercellular transport. In recent, exosome is used as carrier for drugs delivering which has more potential advantages than the traditional viral vector. In this study, the anti-tumor or sensitive chemotherapy miRNAs were screened, through the analyzing dysregulation miRNA in the HCC clinical samples or the different expression miRNA in the HCC cell lines which were treated with anti-tumor drugs. Subsequently, for verifying whether or not these miRNAs have the function of sensitizing the chemotherapy in Xenograft mouse models, exosome that were secreted by human adipose-derived mesenchymal stem cells (AMSC) were used, which were already transfected with these miRNAs, as a carrier to deliver these miRNAs to the tumor site.Methods and ResultsPart one The screening the miRNAs and studying the mechanisms of miRNA which has anti-hepatoma or chemo-sensibilization effects.The miRNA with anti-hepatoma effects was screened mainly through two aspects. The first aspect is by analyzing differently expressed miRNAs from HCC tissue and their matching para-carcinoma tissue samples to acquire miRNAs associated with the development of HCC, and to study their potential roles and mechanisms in chemotherapy and chemo-sensibilization effects on HCC. The second aspect is by analyzing the mechanisms of some anti-cancer drugs to find the miRNAs which are closely related to anti-hepatoma effect, then their effects in terms of anti-hepatoma or chemo-sensibilization were studied. Experimental results indicated the different expressions could be obtained through the analysis of clinical samples. Among all kinds of miRNAs, miR-122 had the inhibition effects on the proliferation of HCC cells and cycle arrest through targeted inhibition on the expression of ADAM 10, CCNG1 and IGF1R, promoting the apoptosis of HCC cells, and improving the antitumor effects of Sorafenib (the first-line chemotherapy drug for HCC). It was found that miR-199* has the anti-tumor effects by targeted inhibition on the expression of mTOR and c-Met, also could inhibit indirectly the expression of Yapl to increase the chemo-sensibilization effects of HCC to DNA damage drugs. In the screened and studied miRNAs for mechanisms of antitumor drugs, it was found that miR-34a plays an important role in the treatment of HCC by Quercetin through targeted inhibition on SIRT1 (further affecting the acetylation of p53), so that p53 is more stable and promotes the apoptosis. In the study on the anti-tumor mechanisms of Amonafide (a MEAN derivative), it was found that the upregulated expressed miR-141 could target-inhibit SPAG9 and play a role in anti-cancer and anti-metastasis effects through JNK pathway. Finally, the chemo-sensibilization effects of four miRNAs on 5-FU in Huh7 cell lines were evaluated. The results showed that four miRNAs had the chemo-sensibilization effects on 5-FU, but the chemo-sensibilization effect of miR-199* was the best and the chemo-sensibilization effect of miR-122 was better than other two miRNAs.Part two The application of therapeutic miRNA-carrying AMSC-derived exosome in the treatment of HCC.AMSCs isolated from the cultured adult adipose tissues were transfected with therapeutic miRNAs (miRNA-122 or miR-199*) and cultured for 48h, then the exosome (miRNA-Exo) with typical phenotypic traits (CD9, CD63 and CD81) was isolated from the culture supernatant. qPCR analysis showed that the exosome contained rich above miRNAs. When the HCC cell line was treated with only miRNA-Exo or miRNA-Exo plus chemotherapy drugs, it was found that the HCC cell proliferation inhibition and apoptosis were significantly increased in miRNA-Exo treatment group, and the sensitivity of HCC cells to chemotherapy drugs was much higher. At the same time, the expression levels of miRNA-122 and miR-199* significantly increased, while the expression levels of corresponding miRNAs target genes (such as the of ADAM10, CCNG1, IGF1R, HGF, mTOR, Yapl) significantly reduced in HCC line, indicating the exosome with specific therapeutic miRNAs can realize the effective transport of miRNAs towards target cells and play a role in anti-hepatoma and chemo-sensibilization effects. Also the feasibility that the exosome was used as the transport carrier of therapeutic miRNA in HCC bearing model was analyzed. In ectopic HCC bearing models, the miRNA-Exo infusion by the means of orthotopic injection or the combination with intraperitoneal injection of low dosage Sorafenib or 5-FU was performed. After 4 weeks of drug treatment, the size and weight of tumors in the miRNA-Exo infusion groups were significantly lower than those in control group. The expression of miRNA target genes in tumor tissues was significantly lower than that in control group, suggesting that AMSC-derived exosome is an ideal transport carrier of therapeutic miRNA, which can play a role in anti-hepatoma and chemo-sensibilization effects.ConclusionThe present study has further complemented the role and regulation mechanism of miRNA in HCC proliferation and metastasis. Also, it was determined that AMSC-derived exosome can be used as an effective carrier of miRNA, realize the specific transport of miRNA towards HCC cells and play role in anti-hepatoma and chemo-sensibilization effects through regulating the expression of tumor-related target genes. Our study provides a new method for the infusion of miRNA as well as a new idea and a brand-new measure for the treatment and chemo-of sensibilization effects of HCC. |
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