Establishment And Primary Application Of Real-time Fluorescence Quantitative PCR Detection Of HULC LncRNA And Research Of HULC LncRNA Function In Myeloid Leukemia

Objective:1. To establish a real-time fluorescent quantitative PCR method for thedetection of long non-coding RNA (highly up-regulated in liver cancer,HULC lncRNA).2. To detect the expressions of HULC lncRNA in solid tumors, WBCsof myeloid leukemia peripheral blood and tumor cell lines by establishedreal-time fluorescent quantitative PCR method and compare the differenceof the expressions in various cancers.3. To research the function of HULC lncRNA in myelogenousleukemia cell line K562by RNA interference technology.Methods:1. Specific primers for HULC lncRNA gene were designed accordingto full HULC lncRNA sequence in Genbank (AY914050.1). The T cloningvectors for the quantitative standard of HULC lncRNA and β-actin genedetection was constructed and named pMD18-T-HULC and pMD-β-actin,respectively. The SYBR Green Ⅱ dye real-time fluorescence quantitativePCR method of HULC lncRNA was established and methodologicallyevaluated. 2. The primary application of this method to detect HULC lncRNA in8types of tumor cell lines, liver cancer, other cancer tissues andparacancerous tissue, WBCs of myeloid leukemia patients was investigatedand compared for the difference of HULC lncRNA expressions.3. The recombinant interference plasmid HULC-shRNA wasconstructed and transfected into K562cells by lipofectamine mediation,then monoclonal cell line was selected by G418pressure. The expressionsof HULC lncRNA were measured by the real-time fluorescent quantitativePCR. The changes of biological behavior of malignant cells were observedby MTT and flow cytometry.Results：1. We had successfully established a real-time fluorescent quantitativePCR for detection of HULC lncRNA. The minimum detection limit ofdeveloped method was1.80×10~3copies/L； The linear range was1.80×10~3copies/L~1.80×10~13copies/L；2. The expression levels of HULC lncRNA in hepatocellularcarcinoma tissues were8.6×10~5copies/L~1.2×10~7copies/L, significantlyhigher than other cancer and paracancerous tissues (all <1.80×10~3copies/L). It was first found that HULC lncRNA was high expressed inhuman bladder cancer cell line T24[(1.41±0.54)×10~5copies/L], but wasnot detected in bladder carcinoma tissues (<1.80×10~3copies/L).Furthermore, it was also first found that the HULC lncRNA gene wasexpressed in K562cell line, the expressions were no significant differencewith HepG2cell lines (P>0.05). There were significant expressions ofHULC lncRNA gene in peripheral WBCs of acute and chronic myeloidleukemia patients, the positive expression rate was80%in acute leukemiapatients and79%in chronic leukemia patients. However, there was no expression of HULC lncRNA gene in peripheral WBCs of healthy humanperipheral WBCs.3. The recombinant interference plasmid HULC lncRNA-shRNA wasconstructed and transfected into K562cells. HULC lncRNA-shRNA-K562(interference group) and HK-K562group (negative control group) cell lineswere established. The HULC lncRNA expression levels of interferencegroups and cell proliferation were significantly decreased, and there weresignificant differences compared with those of negative control group andK562(blank group)(all P<0.05)； while those of control group and blankcontrol group were not significantly different (P>0.05). Interference group,negative control group and blank group of cells in G0/G1phase were(68.85±0.27)%(P<0.05, vs HK-K562and K562groups),(53.05±0.35)%and (53.54±0.33)%, respectively； the percentage of apoptotic cells were(9.6±0.20)%(P<0.05, vs HK-K562and K562groups),(1.79±0.23)%and(1.75±0.20)%,separately.Conclusion:1. The real-time fluorescence quantitative PCR detection of HULClncRNA is successfully established.2. The positive HULC lncRNA gene expression is first found inmyeloid leukemia patient’s peripheral blood WBCs and cell line K562, andthe assay of HULC lncRNA gene expression may be with some clinicalsignificance in the diagnosis and treatment of myeloid leukemia.3. HULC lncRNA-shRNA-K562is constructed by RNA targetinterference technology. HULC lncRNA plays some role in theproliferation, cell cycle and apoptosis of K562cell. It could provide thefoundation for further study of HULC lncRNA gene function in thepathogenesis of myeloid leukemia.