| Background and Objectives:As a major discovery of the life sciences in the late twentieth century, microRNAs(miRNAs) has quickly become a focus of multi-disciplinary research. Studies have shownthat the conserved non-coding, endogenous single-stranded RNA molecules, plays a role ofcancer gene (oncomiRNAs) or tumor suppressor genes (tumor suppressor miRNAs) in theprocess of tumorigenesis and metastasis through a combination of a specific sequence oftarget mRNA in regulating gene expression.China is one of the highest incidence of liver cancer in the world. There are about110,000cases of patients die from liver cancer each year. Among them, hepatocellularcarcinoma (hereinafter referred to as HCC) accounts for85-90%in primary liver cancer,which is the most common histological type. The prognosis of liver cancer is poor.Metastasis of liver cancer is the key obstacle of the curative effect and long-term survival ofpatients with HCC, and the metastatic mechanism remains unclear. Consequently, earlydiagnosis and effective treatment are still vacant. Although medical science has made greatprogress, the recurrence and metastasis are common and the5-year survival rate is still low.Therefore, the role of miRNA in HCC may further clarify the internal mechanism of thedevelopment of liver cancer, and to provide new ideas for the diagnosis and treatment ofliver cancer. In this study, starting from the disorders of miRNA expression in HCC, weintend to explore the mechanism of development of liver cancer.Methods:1. miRNA gene chip technology was applied to screen miRNAs which may beinvolved in regulation of tumor metastasis;2. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify themetastasis-related microRNAs of HCC in hepatoma cell lines with different metastaticpotential; 3. QRT-PCR was applied to verify the expression of metastasis-related microRNAs ofHCC in the plasma of patients with or without liver cancer;4. QRT-PCR was applied to demonstrate the expression of metastasis-relatedmicroRNAs of HCC in surgical specimens (hepatocellular carcinoma and adjacent non-cancerous tissues);5. The relationship between expression of miRNA and clinical pathologicalcharacteristics, clinical prognosis were analyzed;6. QRT-PCR was applied to verify the the expression of metastasis-relatedmicroRNAs of HCC in different hepatoma cell lines, hepatocellular carcinoma and adjacentnoncancerous liver tissue cells;7. The expression of miRNAs in hepatoma cells were raised by infection oflentiviruses, which were loaded with miRNA sequences;8. MTT assay, flow cytometry, colony formation assay, wound healing assay andtranswell invasion assay were applied to analyze the function of metastasis-relatedmicroRNAs of HCC, observing their impact on the biological behavior of hepatocellularcarcinoma cell line in vitro;9. Bioinformatics techniques were used to predict target genes of metastasis-relatedmicroRNAs of HCC;10. Western blot was applied to validate the role of miRNA regulation of target genes.Results:1. Results found by miRNA microarray technology are as follows:22miRNAs wereup-regulated more than2-fold or more in highly metastatic hepatocellular carcinoma celllines,2miRNAs were down-regulated more than2-fold or more in highly metastatichepatocellular carcinoma cell lines. And by using technique of qRT-PCR, miR-149-5p(formerly known as miR-149), miR-345, miR-193b*, miR-1301are found to bedifferentially expressed more than3-fold in the high and low metastatic potential ofhepatocellular carcinoma cell lines, the most obvious difference is of miR-149-5p;2. In hepatocellular carcinoma with (26cases) or without metastasis (14cases),cirrhosis (3cases), patients with metastatic liver cancer (6cases) and normal (30cases)plasma, expression of miR-149-5p were found by qRT-PCR no statistically significant (p <0.05) for liver cancer diagnosis and differential diagnosis;3. By application of qRT-PCR we detected expression of miR-149-5p in95pairs ofsurgical specimens(liver cancer and adjacent non-cancerous liver tissues), and found thatthe relative espression of miR-149-5p in cancer tissues was significantly lower thanadjacent non-cancerous liver tissues(6.982±1.166vs.12.208±2.461, p <0.05);4. Further analysis of the relationship between the relative expression ratio (C/P) ofmiR-149-5p and gender, age, hepatitis, AFP, liver cirrhosis, tumor size, number of tomors,differentiation, staging and lymph node metastasis in95cases of liver cancer and adjacenttissues, we found that the C/P ratio was significantly correlated with history of livercirrhosis (p<0.05), and it was negatively correlated with AFP levels and lymph nodemetastasis, but there is no significant difference (p>0.05), which may be related to too fewcorresponding samples. At the same time, the survival curve analysis showed that thegroup of C/P>1had survived more time than that of C/P <1, but there is still no significantdifference (p>0.05), which may be related to too short follow-up time;5. By using qRT-PCR, we detected expression of miR-149-5p in MHCC97-H,MHCC97-L, Huh-7, SMMC-7721, HepG2, PLC, LO2cell lines and hepatocellularcarcinoma, adjacent noncancerous liver tissue cells, discovering that the expression ofmiR-149-5p in cell lines and cancer tissue cells are lower than in adjacent liver tissuecells(p<0.01);6. Expression of miR-149-5p in MHCC97-H cells were up-regulated successfully bylentivirus infection. MTT assay showed that over-expression of miR-149-5p could inhibitthe MHCC97-H cell’s proliferative capacity (p<0.01). Flow cytometry showed thatover-expression of miR-149-5p could not impact on cell cycle of MHCC97-H significantly.Colony formation assay showed that over-expression of miR-149-5p can inhibit cloneforming ability of MHCC97-cell (colony formation rate:0.137±0.013vs.0.263±0.018,0.256±0.002, p <0.05). Wound healing assay showed that over-expression of miR-149-5pcan inhibit migration capacity of MHCC97-H cells in vitro (24h scratch width/the0hscratch width:0.643±0.036vs.0.454±0.037,0.469±0.013, p <0.05). And transwellinvasion assay showed that over-expression of miR-149-5p can inhibit invasion ability ofMHCC97-H cells in vitro (the cells through the membrane:(71.7±7.3)/vision vs.(133.2 ±4.5)/vision,(122.3±3.4)/vision, p <0.01);7.MiR-149-5p may regulate67target genes predicted by bioinformatics, in whichthe transcription factor, specificity protein1(SP1), is related with metastasis closely. WBtechnical validated initially that SP1may be one of the targets of miR-149-5p.Conclusions:We firstly discovered that the expression of miR-149-5p (formerly known as miR-149)in hepatocellular carcinoma was decreased, which was closely related to the prognosis ofpatients with HCC. And over-expression of miR-149-5p could significantly inhibit invasionand metastasis of liver cancer cells in vitro. Further study indicated that miR-149-5p mightinhibit invasion and metastasis of liver cancer cells through regulation of its potential targetgenes, SP1. This study has clarified initially the mechanism of liver cancer metastasis at thelevel of miRNA and provided a new target for diagnosis and treatment of liver cancer. |
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