Study On Molecular Mechanisms Of MiR-21in Regulating Proliferation And Metastasis Of Hepatocellular Carcinoma

microRNAs (miRNAs) are a family of small highly conserved endogenousnon-coding RNAs which inhibit translation of target genes by base pairing to the3、-UTRof mRNAs，finally inhibit expression of target genes. miRNAs play an important role inmany kinds of physiological and pathological process, including cancer and tumormetastasis process. As a novel regulatory factor, miRNAs can act as oncogenes or tumorsuppressors. As a typical oncogene, miR-21is known to play a noticeable role incarcinogenesis and development of cancers including hepatocellular carcinoma (HCC).Aberrant expression of miR-21can contribute to HCC growth and migration throughregulating PTEN expression. As a typical target of miR-21, inactivation of PTEN in cancercells leads to an activation of downstream signaling including PI3-kinase/AKT (PI3K/AKT)and/or mitogen-activated protein kinases (Raf/MEK/ERK) inside the cells, and thenenhance cancer cell proliferation and movement ability. However, in PTEN-silenced cancercells, manipulation of miR-21expression also results in the changes of AKT and ERK1/2pathway activity, demonstrating that there is another mechanism involved in the regulationof AKT and ERK1/2signaling besides PTEN. hSulf-1is a heparin-degrading endosulfataseoutside the cells, which can desulfate heparan sulfate proteoglycans (HSPG) on cellularsurface and inhibit the heparin binding growth factor-mediated signaling transduction intocells. In this study, by manipulating miR-21expression in HCC cell lines, we found thatmiR-21can suppress the expression of human sulfatase-1(hSulf-1), as well as PTEN, inHCC cells. Therefore, the aim of this study is to explore the effect of miR-21on hSulf-1and its downstream pathway, then further investigate the effect of miR-21on proliferation,migration and invasion of HCC, to provides a new molecular target for clinical genetherapy of HCC. Part Ⅰ Expression of miR-21in HCC cell lines【Objective】To explore the expression of miR-21in different metastatic HCC celllines and confirm the relation between miR-21and the metastatic potential of HCC.【Methods】The expression of miR-21in different HCC and normal cell lines weredetected by real-time quantitative reverse transcription-polymerase chain reaction. Thehepatocellular cancer xenografts in BALB/c (nu/nu) mice were established. To comparethe metastatic potential of different HCC, mice were dissected under body visionmicroscope and the cancer nodules were monitored and counted after two weeks. Finally,confirming the relation between miR-21and the metastatic potential of HCC.【Results】Real time qPCR analysis showed that the relative expression of miR-21was significantly up-regulated in high-metastatic cancer cell lines (6.73±1.01inMHCC-97H; p=0.001versus normal cell lines) compared with MHCC-97L (4.35±0.53;p=0.001versus normal cell lines) and SMMC-7721cells (1.10±0.13; p=0.582versusnormal cell lines). BALB/c (nu/nu) mice tumorigenicity assay presented that MHCC-97Hcells formed more cancer nodules with larger size than SMCC-7721cells.【Conclusion】The relative expression of miR-21was significantly up-regulated inhigh-metastatic cancer cell lines, there may be a positive relation between high expressionof miR-21and metastatic potential of HCC.Part Ⅱ Effect of miR-21on motility, proliferation and neoplasticcapacity of HCC cells【Objective】To investigate the influence of miR-21on invasion, migration,proliferation, and neoplastic capacity of HCC cells.【Methods】 The effect of miR-21on motility of HCC cells were measured bywound healing, transwell migration and invasion assays after transfected miR-21expression vector or treated with miR-21inhibitor. The viability and malignancy potentialof HCC cells were measured by MTT assay and anchorage-independent growth assay aftertransfected miR-21expression vector or treated with miR-21inhibitor.【Results】Wound healing, transwell migration and invasion assays showed that thetransfection of miR-21expression vector significantly enhanced the capacity of motility in SMMC-7721cells, as compared with the parental control cells without transfection. Thedown-expression of miR-21significantly attenuated the motility of MHCC-97H cells, ascompared with the parental control cells without treatment. MTT assays demonstrated thatthe cell viability of SMMC-7721cells transfected with miR-21expression vector wassignificantly increased compared with the parental cell and negative control groups, thecell viability of MHCC-97H cells transfected with miR-21inhibitor was significantlylower than that of the control groups. The anchorage-independent growth assay suggestedthat SMMC-7721cells displayed more colonies when transfected with over-expressionvector of miR-21, compared with the control groups, and MHCC-97H that transfected withinhibitor of miR-21displayed less colonies, compared with the control groups.【Conclusion】 miR-21can influence the motility, proliferation and neoplasticcapacity of HCC cells.Part Ⅲ Possible molecular mechanism of miR-21in regulatingproliferation and motility of HCC cells【Objective】To study the possible molecular mechanism of miR-21in regulatingproliferation and motility of HCC cells【Methods】The expression vector of GFP-tagged miR-21was transfected intoSMMC-7721cells，the inhibitor of miR-21was transfected into MHCC-97H. Theexpression of miR-21were measured by real time qRT-PCR in SMMC-7721andMHCC-97H after treated by expression vector or inhibitor of miR-21. The relativeexpression levels of EMT biomarkers，PTEN, hSulf-1and downstream signal regulatoryfactors were measured by Western blotting analysis in SMMC-7721and MHCC-97H.【Results】Real time qRT-PCR analysis demonstrated the expression of miR-21wasincreased to more than18-fold after treated with miR-21vector, as compared with theparental cells without transfection in SMMC-7721. The expression of miR-21wasdecreased to more than6-fold after treated with miR-21inhibitor, as compared to theparental cell control in MHCC-97H. Western blotting analysis showed that over-expressionof miR-21in SMMC-7721decreased the the expression of E-cadherin and increased theexpression levels of β-catenin and vimentin. Meanwhile, the relative expression of p-AKTand p-ERK1/2was increased accompany by an decrease of PTEN and hSulf-1. Conversely, the inhibition of miR-21in MHCC-97H increased the protein expression of E-cadherin anddecreased the expression of β-catenin and vimentin. Meanwhile, the relative expression ofp-AKT and p-ERK1/2was decreased accompany by an increase of PTEN and hSulf-1.【Conclusion】miR-21promotes cell proliferation and movement by inducingepithelial-mesenchymal transition through both PTEN-and hSulf-1-mediatedAKT/ERK1/2pathway in hepatocellular carcinoma.