Isolation And Identification Of The Regulatory Mechanism Of Liver Tumor Stem Cells

Background: Hepatocellular Carcinoma(HCC) is one of the most common malignant tumor in the world, especially in our country. In china, there are about 100000 people die for primary carcinoma of liver every year, which comprise 45% of death numble coused by liver cancer in the world. Recurrence and metastasis are the key and fatal steps in the progress of malignant cancer development, especially HCC. The research on the mechanism of cancer recurrence and metastasis has been going along for more than 100 years, with the advancement of the research about tumor growth, invasion and tumor micro-environment, the diagnosis and treatment also have made great progress too. However, current clinical treatment still cannot overcome the recurrence and metastasis of cancer, and the survival rate of cancer patients has not been significantly improved.Recent studies showed that not all tumor cells have the ability to form a new tumor, more and more evidence showed that may be the fundamental reason about cancer recurrence and metastasis is a small subgroup of tumor cells, these cells have similar features like stem-cells, just like self-renewal, unlimited proliferation ability, multiple differentiation ability, and also chemo-therapy resistance ability, we called these cells cancer stem cells or tumor-initiating cells. Scientists believe that the arising of cancer stem cells including the following two possibilities, one is the mutation of normal stem cells, the other one is the dedifferentiation of differentiated cells which caused by unknown reasons. We have reasons to believe that maybe the real reasons about tumor development, metastasis and recurrence are existence of cancer stem cells. If we can isolate CSCs in vitro and find out the their regulatory mechanism of development, maintenance, it would be helpful to not only reveal the reason of tumor development, but also offer effective targets for the prevention and treatment of tumor.Today we usually use specific cell surface molecular to isolate CSCs, just like the CD34+CD38- cells in AML, the CD44+CD24- cells in mammary cancer, the CD133+ cells in colon cancer, et al. About the investigation of hepatocellular cancer stem cells, scientists use the marker as the following shows: CD44, CD90, EPCAM, CD133, et al.Recent studies showed that epigenetic regulations play important roles in the arise and maintenance of not only normal stem cells but also CSCs, No matter whatever mechanism or passways involved in cell programming and reprogramming, the regulation of epigenetic would definitely play important roles. Epigenetic regulation refers to those variations which can be inherited from generation to generation without the DNA sequence changed, it includes DNA methylation, histone modification, chromosome remodeling and non-coding RNA regulation. Recently, more and more evidence revealed that microRNAs, which belong to non-coding RNA, have close relationship to cancer development. microRNAs are those 21–25 nucleotide (nt) non-coding RNAs that bind to the 3'untranslated region (3'UTR) of target mRNAs through an imperfect match to repress their translation and stability. microRNAs are reported to involved a series of important progress of life, just like early development, cell proliferation, apoptosis, cell death, fat metabolism ,cell differentiation and so on. The research of Erwei Song showed that infecting BT-IC with let-7-lentivirus reduced proliferation, mammosphere formation, and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice[1], but the regulatory mechanism, especially the epigenetic regulation of the occurrence and maintenance about liver cancer stem cells have been reported littlely.Aim:This study use molecular marker CD90 to isolate hepatocellular cancer stem cells, and then identified the biological characteristics of CD90+ cells. After CD90+ cells have been identified as liver CSCs, we used microRNA chip to analyze the different expression between CD90+ cells and CD90+ cells, and selected the most obviously differential expressed microRNA from the result of microRNA chip. After the construction of the microRNA vector, we observe its effect on liver cancer cells. Our hope is to selecte one microRNA or more to explore the possible molecular mechanism, it could be meaningful to the diagnosis and therapy of liver cancer in the future.Methods:1. The isolation and identification of liver cancer stem cells First, we use flow cytometry and immunofluorescence to detect the expression of CD90+ cells in liver cancer cell lines and liver cancer tissues. And then we use immunomagnetic beads to isolate CD90+ cells from MHCC97H, chemo-resistance assay and tumorigenicity in nude mice assay were used to identify the different between CD90+ cells and CD90+ cells.2. The screening of specific expressed microRNA in liver cancer stem cells We use microRNA chip to analyze the different expression between CD90+ cells and CD90- cells, real-time PCR and TaqMan qRT-PCR assays have been used to verify the result of microRNA chip partly.3. The investigation about the biological effect of the microRNA which has been screenedAfter the analysis and verification about the result of microRNA chip, we screened out microRNA-146a, in order to learn its mechanism, we constructed the over-expression vector of microRNA-146a and established a liver cancer cell line which could over-express microRNA-146a stabilized. Proliferation assay and tumorigenicity in nude mice assay are designed to observe the biological effect of microRNA-146a.Conclusion:1. Flow cytometry and Immunofluorescence detected the CD90+ cells in liver cancer cell lines and liver cancer tissues, the ratio are as the following show: MHCC97H 8.23%,MHCC97L 2.23%,PLC 5.5%,HepG2 0.23%,SNU-182 0.9%,SNU-449 0.29%,SMMC-7721 0.53%,HBXF-344 0.28%, and the ratio of CD90+ cells in liver cancer tissues are 1.2%,11.07%,29.59%. And then we use immunomagnetic beads to isolate CD90+ cells from MHCC97H, the ratio of CD90+ cells enriched by immunomagnetic beads is 71.53%. The result of chemo-resistance assay showed that CD90+ cells have strong chemo-resistance possibility in four densities of oxaliplatin,Cisplatin,5-FU and oxaliplatin combined with 5-FU; The results of twice tumorigenicity in nude mice assays showed that CD90+ cells have powerful possibility of tumorigenicity. We could make a conclusion from all of these assays that CD90+ cells have feathers of liver cancer stem cells.2. The result of microRNA chip showed that 92 microRNAs were differently expressed between CD90+ cells and CD90- cells, and about 52 microRNAs are over-expressed in CD90+ cells, and 40 microRNAs are over-expressed in CD90- cells. We choose those significantly expressed microRNAs whose differentially time are more than 1 and the p-value < 0.01 . Since then we screened 31 microRNAs from the result of microRNA chip, and we use Real-Time PCR to verify these 31 microRNAs in three liver cancer cell lines, MHCC97H,MHCC97L,PLC. And finally we screened 7 microRNAs , microRNA-146a,microRNA-19b,microRNA-7,microRNA-17,microRNA-106a,microRNA- 513-5på'ŒmicroRNA-20b, their differential expression are all significant and stable. Then we use TaqMan qRT-PCR assays to verify the 7 microRNAs.3. Through the verification above, we finally screened out microRNA-146a, since its significantly and stably differential expression. we constructed the over- expression vector of microRNA-146a and established a liver cancer cell line which could stably over-express microRNA-146a about 9 times. The cck-8 proliferation assay, cells migaration assay and tumorigenicity in nude mice assay all indicated that those liver cancer cells which have been over-expressed microRNA-146a have increased the proliferate and migrate abilities, and the tumorigenicity also have been significantly enhanced.