| Background&Objectives:Intrahepatic cholangiocarcinoma(ICC) is a malignant tumor which originally arises from the secondly ductular epithelium of the biliary tree. It is also called peripheral cholangiocarcinoma. According to some epidemiological surveys in the United States, Western Europe and Japan, the incidence of intrahepatic cholangiocarcinoma has increased rapidly over the past decades. Nowadays it accounts for10%to15%of primary hepatic malignancies and it is the second most common primary liver cancers after hepatocellular carcinoma. Patients with intrahepatic cholangiocarcinoma often present at late stages of the disease owing to its nonspecific symptoms, growth pattern and lack of early detection biomarks. Although researchers spent so much efforts on the studies and explores in intrahepatic cholangiocarcinoma, the treatment outcomes and survival for patients with this cancer have improved little over the past decades. Therefor it is of importance to improve the research on molecular biological characteristics of cholangiocarcinoma cell and explore some new treatment modalities.MicroRNAs are a goup of noncoding RNAs18-25nucleotides in length which exists in almost all living species. miRNAs are highly conservative in the evolution and play a vital role in cell proliferation, differentiation and apoptosis via regulating protein expression through sequence-specific interaction with messenger RNA. With the contrast between the normal gene expressions and aberrant miRNA expression profiles associated with different diseases, we can draw the conclusion that miRNAs have been linked closely to the genesis and development of diseases. Nowadays the research on miRNAs has been a new hotpot around the world. Through the findings, miRNAs have dual function in malignant tumor, which means it can work as oncogene or antioncogene. The mutation, deletion or abnormal expression of miRNAs can led to the initiation and the progression of human cancer. Lethal-7(let-7) was discovered as the second miRNA in the world and the whole let-7family consists of9different miRNAs. According to previous research reports on several disease including lung cancer, breast cancer, colon cancer, etc, let-7may act as a tumor suppressor and has identified as potential early tumor biomarker or prognostic marker.Up to now, the relationship between let-7a and ICC has reported less worldwide, and it remains unknown that how the role of let-7effects on the genesis and development of ICC. In our study we aimed to detect the expression of let-7a in intrahepatic cholangiocarcinoma and normal bile duct tissues by means of Real-time RT-PCR. Then in corder to up-regulation or down-regulation the expression of let-7a in ICC cell line HuCCT-1, we introduced miRNA mimics or miRNA inhibitor respectively into cell and examine their effects on cell biological processes trying to provide an experimental and theoretical basis for potential brand new treatment.Methods:1. Real-time reverse transcription polymerase chain reaction(Real-time PCR)for mature let-7a were performed to detect the expression of it in16intrahepatic cholangiocarcinoma tissues and normal bile duct tissues collected from patients at the peoples hospital of Hunan Province from March to October in2012. 2. HuCCT-1cell line was cultured in DMEM media supplemented with10%FBS. The cell line was transfected with RNA oligonucleotide respectively (let-7a mimics, let-7a inhibitor or corresponding control RNA) using lipofectamin2000and the nontransfected cell was set as blank control group. We used real-time PCR to analyze the expression of let-7a in different cell groups and evaluate the transfection effect.3. Cell proliferation assay, colony formation assay and apoptosis assay were used to measure the biological behavior changes in ICC cell by over expression and low expression of let-7a.Results:1. Expression of let-7a is down-regulated remarkably in intrahepatic choangiocarcinoma compared to normal bile duct (P=0.00). It is showed that the down-regulation of let-7a expression was associated with lymph node metastasis (P=0.001) and histological grade (P=0.00), but there were no significant correlations between expression of let-7a and sex, age, potral vein invasion and tumor size (P>0.05).2. let-7a mimics, let-7a inhibitor and both negative control RNA were transfected into HuCCT-1cell line successfully and its transfection efficiency reached to80%. qRT-PCR verified that let-7a expression levels increased significantly in HuCCT-1cells after transfected with mimics whereas let-7a was down-regulated after transfected with inhibitor. Remarkably, there was no distinct differences in blank group, let-7a negative control group and anti-let7a negative control group.3. In vitro assays indicated that let-7a overexpression significantly inhibited HuCCT-1cell proliferation and decreased cell colonies. Conversely, let-7a inhibitor notably cell proliferation and promoted cell proliferation and stimulated HuCCT-1cells to grow more and larger colonies. Moreover, let-7a promoted apoptosis while repression of let-7a levels provided protection from apoptosis. Of all the in vitro assays, there were no significant differences among blank group and two negative control groups.Conelus ions:1. Let-7a is aberrantly expression in intrahepatic cholangiocarcinoma which indicates its down expression may associated with the genesis and development of ICC.2. The lower expression of let-7a may indicates higher lymph node metastasis and lower histological grade.3. Significant increase of let-7a in cell was observed after let-7a mimics transfection and conversely down-regulated let-7a leads to totally opposite results.4. Let-7a might act as tumor suppressor gene in intrahepatic cholangiocarcinoma. |
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