Primary Study On The Liver Cancer Stem Cells And The Effects Of MiR-1301 On Liver Cancer Cells

Background Hepatocellular carcinoma(HCC) is a common malignant tumor in China. Researchers have long been searching for the cellular origin and causes of HCC to try to find effective preventive and treatment measures under the hints of the pathogenesis of HCC. Recent researches had discovered that there were some stem-like subpopulations in tumor tissues which had the potential to proliferate infinitely and played much crucial role in initiating carcinogenesis and development, while the other cells would die after transitory differentiation. It was thought that the tumor growth resulted from the proliferation of some tumor stem cells with special surface markers. For this reason, the tumor treatment must aim directly at the tumor stem cells but not at the bulk of tumor cells with little ability to proliferate and differentiate. This theory might bring about some revolutionary in tumor therapeutics.The key point of the research on tumor stem cells was how to establish the special surface markers of them. However, it was hope to achieve an efficient sorting scheme of tumor stem cells by detecting the surface markers with some directing antigens, which would save a lots of time and research funds. But the following findings published in recent years are exciting:the marker molecule ABCG2 of liver SP cells, which have the biological characteristics of liver cancer stem cells, acts as a marker of liver cancer stem cells; CD 133 is also considered as a marker of liver cancer stem cells; and CD90 is another specific marker of liver cancer stem cells.MicroRNAs(miRNAs), a new class of endogenous, noncoding and single-stranded RNAs, were recently discovered in both animals and plants. They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3'-untranslated regions of target mRNAs. Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation, differentiation, and aberrant expression. Metabolism of miRNAs are associated with human diseases including malignancy. Alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity, influencing cell transformation, tumor progression or metastasis. Accumulating evidences suggest that miRNAs could potentially be widely used in the diagnosis, prognosis and therapy of human cancer.Objective In this study, we screened differentially miRNAs expressed between hepatocellular carcinoma tissue and adjacent non-tumor tissue through realtime PCR technology, and hope to find that miRNAs changes in tumor tissue. Than we transfected these miRNAS into cells. We colledted the total RNA. Realtime PCR were used to examine the different expression of the potential target mRNA in test groups and the control groups. We hope our study could provide convincing evidences on miRNAs association with hepatocellular carcinoma tumorigenesis, and facilitate the search of suitable candidates for cancer targeting therapy.Using CD90 as specific marker of liver cancer stem cells,we isolate, culture and identify the stem-like subpopulations from HepG2 cells in vitro, to investigate the basic characteristics of stem-like liver cancer cells.MethodsTissue samples were obtained from 20 patients undergoing hepatectomy for hepatocellular carcinoma. Tumor samples were resected at surgery. Non-tumor samples from the macroscopic tumor margin were isolated at the same patients and used as the matched adjacent non-neoplastic tissue. Real-time PCR technology was applied to screen differentially expressed miR-155, miR-198, miR-373, miR-520b, miR-548c, miR-1289, miR-1301 between tumor tissue and adjacent non-tumor tissue.We transfected miR-1301 mimics into HepG2 cells. Real-time PCR technique was applied to screen p65,β-catenin, p53, Tg737, bcl-2, bcl-XL, caspase-3, caspase-8 mRNA between transfected groups and contral groups. Western Blot technique was applied to detect their protein. Transwell cabin was used to observe cellular invasiveness. The cellular growth activity was assayed by MTT assay.Using CD90 as specific marker of liver cancer stem cells, we isolate, culture and identify the stem-like subpopulations from HepG2 cells in vitro. Real-time PCR technique was applied to screen miR-155, miR-198, miR-373, miR-568c-5p, miR-1289, miR-145, miR-375, miR-874, miR-1183, and miR-1324 between CD90 positive cells and negative cells. P65,β-catenin, P53, Tg737, caspase-3, caspase-8,bcl-2, bcl-XL mRNA between CD90 positive cells and negative cells were also detectd by Real-time PCR method. miR-568c-5p mimics was transfected into CD90 positive cells, and change of P65,β-catenin, P53, Tg737, caspase-3, caspase-8,bcl-2, bcl-XL mRNA were detected. Western Blot technique was applied to detect their protein. Transwell cabin was used to observe cellular invasion capacity. Results 1. Compared with adjacent non-tumor tissues, miR-155, miR-198, miR-373, miR-520b, miR-1301 were down-regulated while miR-548c,miR-1289 were up-regulated in tumor tissue.2. p53 mRNA and protein were up-regulated in miR-1301 transfected groups. bcl-2, bcl-XL mRNA and protein were down-regulated in miR-1301 transfected groups. Cellular invasive capacity was depressed. Cellular inhibition ratio were high in transfected groups.3. In CD90 positive cells, miR-155, miR-198, miR-1289 were up-regulated while miR-548c-5p,miR-145, miR-375, miR-874were down-regulated.Caspase-3,bcl-2 mRNA were down-regulated in CD90 positive cells while Caspase-8 mRNA were up-regulated. There were no changes in p65,β-catenin, p53, Tg737, bcl-XL mRNA.β-catenin, Tg737, bcl-2, bcl-XL, caspase-3 were down-regulated in miR-548c-5p transfected groups in CD90 positive cells. bcl-2, bcl-XL, caspase-3 protein were down-regulated in miR-548c-5p transfected groups. Cellular invasive capacity was depressed.ConclusionsmiR-1301 may inhibitor tumor process through p53, bcl-2, and bcl-XL. miR-548c-5p may affect the level of caspase-3, bcl-2, and bcl-XL mRNAs and their proteins.Expression level of miRNAs were alterd in hepatocellular carcinoma and in liver cancer stem-like cells. p65,β-catenin, p53, Tg737, bcl-2, bcl-XL, caspase-3, caspase-8 mRNA were up-regulated or down-regulated in experiment groups. We may suppose that miRNAs may have great effects on hepatocellular carcinoma and liver cancer stem-like cells. Disorder of apotosis mechanism may plays important role in liver cancer stem cells.