| Background and ObjectiveHepatic carcinoma is one of the most common malignant tumors in clinic. It contains primary hepatic carcinoma and metastatic hepatic carcinoma. At present in the global, hepatocellular carcinoma (HCC) accounts for 70%-85% of primary liver cancers and ranks as the second leading cause of male cancer death. Especially in Asia and Africa, HCC becomes a major cause of tumor death. HCC is one of common diseases characterized by high malignant degree and quickly development. However, the pathogenesis of HCC remains unknown. Accumulating evidences suggest that to explore the mechanism of HCC will provide an important theoretic evidences for the the establishment of antitumor therapy for HCC.It usually develops in an affected liver with cirrhosis due to viral infection (hepatitis B virus, HBV; hepatitis C virus, HCV), alcohol abuse, metabolic disorders, or a carcinogenic agent. HCC has heterogeneous etiologic and molecular profiles and varied response to therapeutics. This tumor diversity arises from multistep hepatocarcinogenic processes requiring sequential genetic and epigenetic alterations including gene mutations and/or chromosome instability. Therefore, to further study the molecular biological features of HCC will provide significant novel insights into the diagnosis and finding new therapeutic targets of liver cancers.PcG (Polycomb group protein), a kind of transcription factor, plays an important role in regulating target genes by epigenetic modification at the chromatin level. PRC (Polycomb repressive complex) is the common form of PcG existence. In the present study, it has become apparent that PRC 1 (Polycomb repressive complex1) and PRC2(Polycomb repressive complex2) are function as the inhibitors of target genes transcription, which lead to transcriptional silencing. PHF19 (PHD finger protein 19), as a component of PRC2, encodes a member of the polycomb group of proteins that function by maintaining repressive transcriptional states of many developmental regulatory genes. The previous study proved that PHF19 is frequently up regulated in several types of cancer. Ghislin.et al proved that PHF19 silencing reduces the cell proliferation rate and increases the transendothelial migration capacities of melanoma cell lines. Li.et al showed that the expressions of and PHF19 increased in glioblastoma multiforme samples and correlated positively with the astrocytoma grades. These observations suggest that PHF19 is strongly linked to aggressive behavior of the tumors and are in agreement with its increased expression in various human tumor types.MicroRNAs are a class of conserved, small (about 17-25 nucleotides), single-stranded non-coding RNAs. MiRNAs are small noncoding RNAs that control gene expression by modulating stability and/or translation of messenger RNA (mRNA) through interactions with specific sequences located in either the coding or the untranslated regions (UTR). Many studies have shown that miRNAs are implicated in many cancers, and altered miRNA levels can result in the aberrant expression of gene products that may contribute to cancer biology. Increasing evidence demonstrates that miRNA as a novel modulator implicated in some common tumors. The expression of miRNA can lead to the functional change involvement of gene in tumor. Recently, miR-195 was found to be down-regulated in a variety of cancers, including liver cancer, gastric cancer, bladder cancer, breast cancer, and adrenocortical cancer. Introduction of miR-195 markedly suppressed colony formation in vitro and tumor development in nude mice. Several in vitro data suggest that miR-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. However, the association between miR-195 and PHF19 contributes to hepatocellular carcinoma is currently undetermined.Therefore, to further uncover the role of mechanism of PHF19 and miR-195 in HCC, we next choose PHF19 and miR-195 as research objects to investigate. In this study, we sought to identify the molecular biology characteristics of PHF19 and miR-195 and the functional role of them in the liver cancer cells and tissues.Taken together, our studies aim to reveal and verify miR-195 as a novel regulator of PHF19 and effects of miR-195 on the molecular biology mechanism of HCC progression. Our research also provides a significant novel view into the crucial importance of miR-195 served as therapeutic target to treat HCC.Methods1. PHF19 gene expressions in HCC tumor tissues and adjacent tissues1.1 Paired HCC tissue and adjacent non-tumor liver tissues were obtained from 30 HCC patients undergoing HCC resection. HCC tissue and adjacent non-tumor liver tissues were treated astreatment group and negative control group. The tissuses were measured by Real-Time quantitative PCR by extract RNAs from these tissuses.2. Effects of PHF19 on invasion, migration, proliferation and apoptosis of HepG2 cells.2.1 Lentiviral vector carrying PHF19 gene has been successfully constructed and transfected into with HepG2 cells.Western blot analysis of PHF19 protein expression in HepG2 cells treated with LV-PHF19.2.2 HepG2 cells were treated as indicated and then transwell assays were performed to assess migration and invasion by analyzing the cell numbers on the transwell membrane.2.3 HepG2 cells were treated as indicated. Cells were seeded in 96-well plates and then the CCK-8 cell proliferation assay was performed. The effects of proliferation of PHF19 on HepG2 cell were measured.2.4 The LV-PHF19 HepG2 cell and LV-Mock HepG2 cell were collected, fixed and stained, then examined on flow cytometry. The cell cycle and proportion of apoptotic cells were measured.3. Effects of miR-195-5p on PHF19 expression.3.1 We identified a putative miRNA, designated miR-195-5p, which targets human PHF19, utilizing a combination of TargetScan, miRanda, RNA22, miRDB, miR-Gen, PITA, EMBL-EBI, starBase, PicTar, RNAhybrid and miRBase. To establish the effect of miR-195-5p on the 3’UTR of human PHF19, the dual luciferase assay was performed.3.2 The protein level of PHF19 was analyzed by western blotting after treatment with has-miR-195-5p mimics or negative control in HepG2 cells.3.3 Paired HCC tissue and adjacent non-tumor liver tissues were obtained from 30 HCC patients undergoing HCC resection. HCC tissue and adjacent non-tumor liver tissues were treated as treatment group and negative control group. The tissuses were measured by real-time quantitative PCR.4. Effects of miR-195-5p on invasion, migration, proliferation and apoptosis of HepG2 cells4.1 Lentiviral vector carrying miR-195-5p gene has been successfully constructed and transfected into with HepG2 cells. HepG2 cells were treated as indicated and then transwell assays were performed to assess effects of miR-195-5p on invasion, migration, proliferation and apoptosis of HepG2 cells by analyzing the cell numbers on the transwell membrane.4.2 HepG2 cells were treated as indicated. Cells were seeded in 96-well plates and then the CCK-8 cell proliferation assay was performed. The effects of proliferation of miR-195-5p on HepG2 cell were measured.4.3 The LV-miR-195-5p HepG2 cell and LV-Mock HepG2 cell were collected, fixed and stained, then examined on flow cytometry. To observe the cell cycle and proportion of apoptotic cells regulated by miR-195-5p, the cells were measured.5. Effects of PHF19 and miR-195-5p on xenograft tumors.5.1 HepG2 cells infected with LV-PHF19, LV-miR-195-5p or LV-Mock were subcutaneously configured to cell suspension and then injected into the nude mice. The body weights of all of nude mice and metastatic tumors were measured every 3 days. All mice were sacrificed and the formed tumors were removed after two weeks.6. Statistical AnalysisData are expressed as mean±standard deviation (S.D.). The results of qPCR detection PHF19 in HCC tissue and adjacent to carcinoma tissues and Nude mice tumor experimental results were analyzed by paired t-test. The effect on cell proliferation ability of the experimental and cell cycle were also analyzed by factorial design analysis of variance.The result of phfl9 protein expression were analyzed by single factor analysis of variance. The other results between two way were compared with two independent samples t-test.The Student’s t-test using SPSS v13.0 statistical software (SPSS, Inc., Chicago, IL, USA). P value<0.05 was considered statistically significant.Results1. PHF19 gene expressions in HCC tumor tissues and adjacent tissues1.1 To further confirm and extend this finding, we examined the expression of PHF19 in 30 paired HCC and adjacent non-tumor tissues by real-time PCR analysis. We found that mRNA levels of PHF19 were increased in HCC tissues as compared to adjacent non-tumor tissues. These results suggest that induced PHF19 expression is a frequent event in human HCC and may be involved in hepatocarcinogenesis. The differences between the two groups have statistical significance (t=-3.909,p< 0.05).2. Effects of PHF19 on invasion, migration, proliferation and apoptosis of HepG2 cells.2.1 To explore the biological role of PHF19 in hepatoma carcinoma cells, we analyzed the effect of PHF19 on migration and invasive capacity in HepG2 cells. HepG2 cells were infected with empty LV vectors (LV-Mock) or human PHF19 overexpression LV vectors (LV-PHF19). PHF19 protein expression in HepG2 cells was assessed by western blot analyses. As shown, in comparison to the LV-Mock treated cells, PHF19 expression was markedly increased in LV-PHF19 treated cells. The differences among four groups have statistical significance (F=50.43,p< 0.05).2.2 The transwell assay was employed to investigate the effect of PHF19 on HepG2 cell migration and invasion. HepG2 cells infected with LV-PHF19 displayed significantly increased migration and invasion abilities compared to HepG2 cells infected with LV-Mock (Figure 2B). The differences between the two groups have statistical significance (invasion:t=8.376,p< 0.001;migration:t=-20.476, p<0.001).2.3 Next, we determined whether PHF19 expression influenced HepG2 cells survival or proliferation in vitro using the CCK-8 assay, which showed that PHF19 LV constructs markedly increased the proportion of living HepG2 cells. The differences between the two groups in 24 hours have statistical significance (F=13.958,P=0.001). The differences between the two groups in 48 hours have statistical significance too (F=20.582,P<0.001)2.4 We further investigated the effects of PHF19 on the cell cycle via flow cytometry. As shown, there was a decrease of the percentage of cells in the G1 phase (F=263.311,P<0.001), a increase of those in the S phase (F=249.19,P<0.001), and no change in those in the G2M phase post-infection as compared to control cells (F=0.175,P=0.679). Apoptosis in HepG2 cells expressing the LV-Mock and LV-PHF19 vectors was evaluated using co-labeling with annexin V (AV) and propidium iodide (PI) via flow cytometry. We found that, treatment with the LV-PHF19 vector significantly decreased the percentage of apoptotic cells in comparison to treatment with the LV-Mock vector in HepG2 cells. Together, these data strongly suggest important pathological roles of PHF19 in HCC. The differences between the two groups have statistical significance (t=11.724,p< 0.05).3. Identification of endogenous miRNAs to control PHF19 regulation in Liver Cancer.3.1 We identified a putative miRNA, designated miR-195-5p, which targets human PHF19, utilizing a combination of TargetScan, miRanda, RNA22, miRDB, miR-Gen, PITA, EMBL-EBI, starBase, PicTar, RNAhybrid and miRBase. The sequence and seed pairing of this miRNA are conserved across multiple species. To establish the effect of miR-195-5p on the 3’UTR of human PHF19, the dual luciferase assay was performed. The differences between PHF193’UTR-Wt group and nc group have statistical significance (t=-69.474,p<0.001). The differences between PHF19 3’UTR-Mutl group and nc group have statistical significance (t=-57.320,p<0.001).The differences between PHF193’UTR-Mut2 group and nc group have statistical significance(t=-31.338,p<0.001).3.2 Upon co-transfection of 293T cells with wild-type (pMIR-PHF19-wt) reporter vectors and hsa-miR-195-5p using Lipofectamine 2000 transfection reagent, luciferase activity was significantly decreased. Mutation of binding site 1 or 2 markedly suppressed the effect of hsa-miR-195-5p, while mutation of both binding sites completely reversed the effect of hsa-miR-195-5p. Consistent with the results of the luciferase assays, mimics of miR-195-5p was able to suppress endogenous PHF19 protein expression. The differences among four groups have statistical significance (t =6.67,P=0.001).3.3 Besides, we further evaluated the levels of miR-195-5p in 30 paired HCC and adjacent non-tumor tissues by real-time PCR analysis. Levels of miR-195-5p were decreased in HCC tissues as compared to adjacent non-tumor tissues, which was consistent with the previous report. The differences between the two groups have statistical significance (F=32.033,P=0.001)4. Effects of miR-195-5p on invasion, migration, proliferation and apoptosis of HepG2 cells4.1 HepG2 cells were infected with empty LV vectors (LV-Mock) or miR-195-5p overexpression LV vectors (LV-miR-195-5p). Transwell assay revealed that LV-miR-195-5p-infected hepG2 cells displayed a significantly decreased ability to migrate and invade in comparison with LV-Mock-infected HepG2 cells. The differences between the invasion ability of two groups have statistical significance (t=11.490,p< 0.001). The differences between the migeration ability of two groups have statistical significance (t=11.355,p< 0.001).4.2 The CCK-8 assays found that LV-miR-195-5p treatment significantly decreased the proportion of living HepG2 cells. The differences between the two groups in 24 hours have statistical significance (F=13.489,P=0.001). The differences between the two groups in 48 hours have statistical significance (F=16.988,P=0.000)4.3 LV-miR-195-5p-infected HepG2 cells showed a decrease in the percentage of cells in the G1 phase (F=37.759,P=0.000), an increase in those in the S phase(F=32.710,P=0.000), and no change in those in the G2/M phase as compared to control cells. LV-miR-195-5p significantly induced the HepG2 cell apoptosis rate. The differences between the two groups have statistical significance (t=11.728,p< 0.001).5. Effects of PHF19 and hsa-miR-195-5p on xenograft tumors.5.1 HepG2 cells infected with adenovirus expressing PHF19 (LV-PHF19), miR-195-5p (LV-miR-195-5p) or control adenovirus (LV-Mock) were subcutaneously injected into the nude mice. Four weeks after the subcutaneous injections, tumors were removed and weighed. Tumor growth was significantly promoted in mice injected with LV-PHF19-treated HepG2 cells(t=6.14,p<0.001) while reduced in mice injected with LV-miR-195-5p-treated HepG2 cells as compared to mice injected with LV-Mock-treated HepG2 cells(t=4.552,p<0.001). These results strongly suggest that PHF19 can indeed promote while miR-195-5p acts as a tumor suppressor in HCC.Conclusions1.Expression of PHF19 is frequently induced in HCC tissues.2.Overexpression of PHF19 in hepatoma carcinoma cells potently promoted cell migration, invasion and proliferation while suppressed apoptosis in vitro.3.MiR-195-5p was able to suppress endogenous PHF19 protein expression.4.Levels of miR-195-5p were decreased in HCC tissues as compared to adjacent non-tumor tissues.Induction of miR-195-5p suppressed HepG2 cell invasion, migration and proliferation while promoted apoptosis in vitro.5.PHF19 promotes tumorigenicity of HCC cells while miR-195-5p suppressed tumorigenicity of HCC cells in nude miceInnovativeThe study performed different expression of PHF19 in HepG2 cells to explore. We firstly demonstrated that overexpression of PHF19 promoted hepatoma cells migration, invasion and proliferation in vitro.This is the first study to prove that PHF19 was a potential target of hsa-miR-195-5p according to our bioinformatics analysis and luciferase reporter assay.We performed different expression of miR-195-5p in HepG2 cells to explore the expression profiles of PHF19 mRNA. On the contrary, overexpression of hsa-miR-195-5p in hepatoma cells reduced PHF19 expression, leading to suppression of hepatoma cells invasion, migration and proliferation in vitro.PHF19, which is regulated by the tumor suppressor miR-195-5p, could promote hepatocellular carcinoma by exploring the mechanisms of PHF19 on HCC in vivo and in vitro.To conclude, this work will further suggest that miR-195-5p is an molecular targets and might represent a potential therapeutic target for HCC. |
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