Study On MiR-1Inducing Apoptosis In Hepatoma Carcinoma Cell Hep3B

ObjectivePrimary hepatocellular carcinoma(HCC) is called “the worst cancer” with highmorbidity and mortality. The pathogenesis has not been discovered yet,but according to therecent research, abnormal cells apoptosis is related with liver cancer closely. Liver cancerhas low sensitivity to chemo-therapy and radiotherapy, It infers that there is a high effectiveand complex anti-apoptosis system in liver cancer cell.miRNAs is single-chain non-codingmolecules with18-24nucleotides which make genes silence by combing the non-codingarea in mRNA of target gene. There are60%of the genes expression have been regulatedby miRNAs in human. miRNAs play a very important role in bio-behavior of tumor. miR-1is an anti-cancer gene which has been widely proved. It was found reduced expression ofmiR-1in various tumors. Functional experiments showed miR-1can reduce proliferation,induce apoptosis and diminish metastasis. It was reported that miR-1has anti-cancer affecton lots of solid tumors by effecting oncogene such as c-met、CCND、FoxP1ect. As far ashepatocellular carcinoma was concerned, miR-1can inhibit proliferation of liver cancer cellby reducing the expression of ET-1. It was reported that expression of miR-1can effectETS1-a high expressing transcription factors in HepG2. ETS1can lead degradation ofextracellular matrix in order to enhance tumor metastasis. miR-1also can convert livercancer cell’s phenotype into a normal one. But there is no widely research reported aboutthe affect on apoptosis for miR-1as well as their mechanism. we intend to observe theinfluence of miR-1’s over-expression in liver cancer cell Hep3B on cells’early apoptosis.Furthermore we would demonstrate the possible way that how miR-1may affect Hep3B’sapoptosis.Methods：Part I:1.We transfected the miR-1over-expressing plasmids into liver cancer cell Hep3Bwith the assistance of attractene transfection reagent and set a negative control group which transfected the negative plasmids and a blank control. Then we observed the reporter geneGFP’s expression and performed RT-qPCR to detect the miR-1’s expression aftertransfection.2.In48h after transfection, we detected the cell apoptosis of miR-1group、nc groupand blank group by using annexin V/PI staining and flow cytometry scan. The analysisindex was early apoptosis rate.Part II:1.We predicted miR-1’s potential downstream targets and chose the the one which isinvolved closely in apoptosis by using bioinformatics software such as targetscan,miRTarBase and PicTar.2. We colleted16pairs of primary hepatocellular carcinoma tissues and PNLtissues, then detected the expression of protein of the downstream target we chose withWestern Blot.Part III:1.We detected the change of the downstream target’s mRNA expression by RT-qPCRafter extracting total RNA of cells from miR-1group，nc group as well as blank group.2. We detected the protein levels of the downstream target by western bolt afterextracting total protein of cells from miR-1group，nc group as well as blank group.Results：Part I:1.We observed plenty of GFP’s expression in48h after transfection both in miR-1andnc groups which indicated that we transfected the plasmids into cells successfully. TheRT-qPCR results also showed that our aim gene miR-1’expression was significantly muchhigher than it in nc and blank group(P＜0.01）.which indicated we managed to perform theover-expression of miR-1in Hep3B.2.The early apoptosis rate of miR-1group was（30.27±2.62）%, that in nc group was（4.13±0.92）%and in the blank group was（1.91±0.36）%which showed a significantstatistic difference（P<0.01）。 Part II:All the miRNA prediction software indicated that BAG4may be a potentialdownstream target molecule of miR-1.We detected the protein level of BAG4in HCC tissues and PNL tissues,the resultsshowed that almost all the HCC tissues had a higher expression compared with PNLtissues.The analysis of gray density scanning was with statistic significance（P<0.01）.Part III:1.The RT-qPCR showed a statistic difference（P<0.01) between miR-1group and ncgroup after we detected the mRNA expression of BAG4.2.Western blot results indicated the protein level of BAG4in miR-1group is reducedthan nc group.Conclusion：1.miR-1could induce apoptosis of Hep3B significantly.2.The prediction software indicted that BAG4might be a potential downstream targetof miR-1and the protein level of BAG4in HCC tissues was much higher than it in PNLtissues.3.miR-1could reduce BAG4’s expression both in mRNA and protein levels. miR-1might induce apoptosis of Hep3B by targeting BAG4.