| BackgroundMicro RNAs, are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level. miRNA dysfunction plays a important role in cancer progression. In this study, mi R-208-3p was highly expressed and directly repressed ARID2 expression. As a result, ARID2 expression was downregulated in hepatocellular carcinoma(HCC). In vitro, miR-208-3p down-regulation and ARID2 over-expression elicited similar inhibitory effects on HCC cell proliferation and invasion. In vivo test results revealed that mi R-208-3p down-regulation inhibited HCC tumorigenesis in Hep3 B cells. Moreover, ARID2 was possibly a downstream factor of TGFβ1/mi R-208-3p/ARID2 signal pathway. These findings suggested that mi R-208-3p up-regulation is associated with HCC cell progression and may provide a new target for liver cancer treatment.IntroductionWith a high death rate attributed to high mortality and unsatisfactory treatment options, hepatocellular carcinoma(HCC) is one of the most common malignant carcinomas worldwide [1-3]. So effective therapeutic tactic, such as gene therapy, should be developed for HCC treatment. For instance, micro RNAs(miRNAs), are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level in way of directly binding to complementary sequences in m RNAs [4, 5]. The dysregulation of mi RNAs play critical parts in tumor initiation and development. One of the well-known mi RNAs is mi R-21, which is highly expressed and plays important role in various types of cancers, including colon cancer, prostate cancer, colorectal cancer, and HCC [6-9]. In contrast, mi R-34 a, mi R-15-/16 cluster, and let-7 mi RNA are usually expressed at low levels in different malignant cancers[10-13]. However, few studies have focused on cancer dysregulation of mi R-208, which promotes cell proliferation of human esophageal squamous cell carcinoma, and contributes to cell metastasis and invasion by inducing epithelial-to-mesenchymal transition(EMT) in pancreatic cancer cells [14, 15].ARID2 is located in chromosome 12 q and is composed of 21 exons; this domain is a subunit of a polybromo-associated BRG1-associated factor(PBAF) chromatin-remodeling complex, a switch/sucrose non-fermenting(SWI/SNF) chromatin-remodeling complex involved in ligand-dependent transcriptional activation of nuclear receptors [16]. Human ARID superfamily includes 15 members, which are subunits of SWI/SNF complexes. Human ARID2 protein contains a conservative N-terminal ARID, an RFX-type winged helix, a proline- and glutamine-rich region, and two conservative C-terminal C2H2 Zn-finger motifs [17].Proteins containing ARIDs are involved in lots of biological processes, including embryonic development, cell lineage gene regulation, and cell cycle control. ARID2 is also as described in previous studies implicated in tumor progression. ARID2 have been found to be mutated and functions as a tumor suppressor gene in HCC [18, 19]. In non-small cell lung cancer and colorectal cancer, loss-of-function mutations of ARID2 have also frequently occurred [20, 21]. Although the crucial roles of ARID2 in cancer have been extensively investigated, the relationship between miR-208-3p and ARID2 in HCC remains unclear.In this study, miR-208-3p in HCC, which is regulated by TGFβ1 at transcriptional level, was expressed highly and correlated with ARID2 expression inversely. In addition, mi R-208-3p down-regulation inhibited HCC cell proliferation and invasion. This down-regulation of mi R-208-3p also suppressed tumor growth in nude mice. Morever, ARID2 was confirmed to be regulated by mi R-208-3p directly, and the depetion of ARID2 could reverse the effect of mi R-208-3p down-regulation in HCC cells. These results indicated the important role of miR-208-3p/ARID2 in HCC andmay also provide a basis to develop new and potent therapeutic agents for HCC treatment.Materials and methodsCell culture and clinical specimens collectionHepG2 and Hep3 B HCC cell lines were bought from the American Type Culture Collection(ATCC, Rockville, MD, USA). Hep G2 was maintained at 37 °C in 5% CO2 in Dulbecco’s modified eagle’s medium(GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum(FBS). Hep3 B was grown in minimum essential medium(GIBCO, Grand Island, NY, USA) containing 10% FBS at 37 °C in 5% CO2. Human HCC specimens(n = 20) and paired adjacent control tissues(n = 20), with documented informed consent from each case, were obtained from the Southwest Hospital, Third Military Medical University. The speciments information was listed in Table S1.Small interfering RNA(siRNA), mimics, and DNA constructionmi RNA inhibitor was purchased from Genewiz(Suzhou, China). mi RNA mimics and siRNA used in this study were purchased from Gene Pharma Co., Ltd.(Shanghai, China). The DNA fragments of full-length ARID2(5,508 bp) were synthesized and cloned in pcDNA3.1 vector by using Not I andXba I to construct miR-208-3p and ARID2 expression plasmids, respectively. Wild-type 3-untranslated region(UTR; 156 bp) of ARID2 and mutated 3-UTR(156 bp) of ARID2 were also synthesized by Genewiz(Suzhou, China) and then separately inserted in pmir GLO dual-luciferase reporter vector(Promega, Madison, WI, USA) by using Nhe I and SalI, respectively.Luciferase reporter assaysHepG2 and Hep3 B cells were maintained in a 96-well plate at a final concentration of 1 × 103 cells / well and kept at 37 °C in 5% CO2. After 24 h, luciferase reporter plasmids were co-transfected with mi RNA expression plasmid or its control vector. The cells were harvested exactly 48 h after transfection; firefly and Renillaluciferase activities were detected with a Dual-Glo® luciferase assay system(Promega, Madison, WI, USA). Firefly luciferase was normalized according to Renillaluciferase. Transfections were performed three times.RNA extraction and q RT-PCRTotal RNAs were extracted from tissues and cell lines, and HCC cells were isolated with Trizol(Sigma, St. Louis, MO, USA) strictly according to the manufacturer’s instructions. RNAs were used as samples in reverse transcription(RT) by using oligo(dT) or miR-208-3p-specific RT primer. cDNAs were then prepared to examine ARID2 and mi R-208-3p expressions. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and U6 small nuclear RNA(snRNA) were used as controls. Expression levels were quantified using the 2-ΔΔCt method. Each experiment was performed thrice. All of the primers could be seen in Table 1.Cell proliferation assayMTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and colony formation assays were carried out to detect HCC cell proliferation. In MTT assay, cells were seeded in a 96-well plate at final concentrations of 2 × 103 cells/ well. At 24, 48, and 72 h after transfection, the cells were transfected with MTT(0.5 mg/mL) for 4 h at 37 °C. Absorbance value was determined at 490 nm. In colony formation assay, cells were seeded in a six-well plate at final concentrations 2 × 103 cells/well. At 7 d(for Hep3 B cells) or 10 d(for Hep G2 cells) after transfection, the clones were fixed with methanol and stained using 2% Giemsa solution(Merck, Germany).5-Bromo-2-deoxyuridine(Brd U) labeling and flow cytometry analysisCells were seeded in a six-well plate at a final concentration of 2 × 105 cells / well. Transfected cells were incubated with BrdU at a final concentration of 10 μM for 40 min, stained with Brdu-fluorescein isothiocyanate antibodies and 7-aminoactinomycin D by using Brd U flow kits from BD Pharmingen(San Diego, CA), and analyzed by flow cytometry.Cell invasion assayTransfected cells were seeded in Matrigel-coated transwell cell-culture inserts(Invitrogen Carlsbad, CA, USA) with DMEM/MEM containing 2% FBS. The bottom chamber was filled with DMEM in 10% FBS. The cells were allowed to move for 8 h. Afterward, the inserts were gently washed and stained with crystal violet. The passed cells were photographed under a microscope.Western blot analysisTotal proteins(20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidenedifluoride membranes(Millipore, MA, USA). The membranes were incubated and blocked with a rabbit polyclonal antibody for ARID2(Abcam, Cambridge, UK), TGFβ1(Abcam, Cambridge, UK), and GAPDH(Abcam, Cambridge, UK). The membranes were then incubated with goat horseradish peroxidase-labeled anti-rabbit IgG secondary antibody(CST Inc., Danvers, MA, USA). Proteins were detected by chemiluminescence with chemiluminescent and Western blot detection reagents(Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK); the target proteins were then exposed to a chemiluminescent film.Tumorigenesis assay in nude miceTumorigenic potency of the cells was examined using five-week-old to six-week-old BALB/c nude mice obtained from the Animal Center of the Chinese Academy of Science(Shanghai, China). The mice were randomly assigned to four groups(8 mice/ group) and maintained under pathogen-free conditions. Cells(1 × 107 cells/100 μL PBS) were injected subcutaneously to the nude mice. The nude mice were monitored at an interval of 3 d for the appearance of tumors. The mice were sacrificed on 28 th day after injection, and tumor sizes were measured. Tumor size was calculated using the following equation: tumor size = width2 × length × 0.5.Statistical analysisSPSS® was carried out forstatistical analyses(SPSS Inc., Chicago, IL, USA). All data were recorded as mean ± standard deviation. Student’s t-test or one-way ANOVA test was performed to determine significant differences. A P-value < 0.05 was considered statistically significant.ResultsHigh mi R-208-3p expression directly decreased ARID2 expression in HCCTo determine the miRNA that regulates ARID2 expression, we selected four mi RNAs, mi R-203, miR-208-3p, mi R-132, and mi R-155, which contain ARID2 binding sites. Western blot analysis results in HCC cells showed that ARID2 protein level was lower in Hep G2 and Hep3 B than that in the three other cell lines(Fig. 1A). qRT-PCR results showed that two cell lines, HepG2 and Hep3 b, expressing high level of mi R-208-3p. In addition, mi R-208-3p expression was higher than those of the three other miRNAs in Hep G2 and Hep3 B cell lines(Fig. 1B and C). To further confirm the relationship between mi R-208-3p and ARID2, mi R-208-3p inhibitor was transfected to HepG2 and Hep3 B cell lines. q RT-PCR and WB results showed that ARID2 protein levels ascended after miR-208-3p was inhibited(Fig. 1D and E). Luciferase reporter assay was then performed to confirm that mi R-208-3p directly bound to the 3-UTR of ARID2(Fig. 1F).Furthermore, we found that ARID2 m RNA levels were lower in HCC specimens and inversely correlated miR-208-3p expression(Fig. 1G and H). These observations demonstrated that miR-208-3p could directly bind to the 3-UTR of ARID2 and repress its expression.mi R-208-3p down-regulation inhibited HCC cell proliferation and invasionTo investigate the function of mi R-208-3p dysregulation on HCC, we transfected mi R-208-3p inhibitor in Hep G2 and Hep3 B cell lines. Then MTT, colony formation, transwell, and flow cytometry assays were performed to analyze the proliferation and the invasion ability of HCC cells. MTT, colony formation assay and flow cytometry analysis results suggested that miR-208-3p down-regulation could depress HCC cell proliferation(Fig. 2A, B and C). Transwell analysis results showed that mi R-208-3p inhibitor inhibited invasion of HCC cells(Fig. 1D).ARID2 over-expression exhibited similar effect to that of mi R-208-3p downregulation in HCC cellsThe above results indicated that mi R-208-3p directly decreased ARID2 expression and promoted HCC cell progression. Considering that miRNAs are regulatory genes, we speculated that this mi R-208-3p effect on HCC cells was possibly exhibited by ARID2.To confirm this hypothesis, we transfected ARID2 expression plasmids in Hep G2 and Hep3 B cells. Results of western blot analysis showed that ARID2 and IFITM1 were up-regulated after the transfection of ARID2expression plasmids(Fig. 3A). Colony formation and flow cytometry analysis results showed that ARID2 over-expression inhibited HCC cell proliferation(Fig. 3B and C). HCC cell Invasive potential was also inhibited in over-expressed ARID2 groups compared with those in control groups(Fig. 3D).ARID2 restoration partly reversed the effect of mi R-208 down-regulation on HCC cellsTo further verify the causal role of ARID2 in miR-208-3p regulation signal pathway, we negatively regulated ARID2 expression in mi R-208-3p inhibitor pretreated HepG2 and Hep3 B cells by using si RNA. The transfection of ARID2-specific si RNA decreased ARID2 protein levels compared with that of the control group in mi R-208-pretreated HCC cells(Fig. 4A). Furthermore, we recorded the effects of ARID2 restoration on theproliferation and invasion. The results showed that ARID2 silence could rescue cell proliferation(Fig. 4B and C) and invasion ability(Fig.4D) of HCC cells that inhibited by miR-208-3p inhibitor. These data suggested that the effects of mi R-208-3p on HCC cells were achieved by targeting ARID2 to a certain degree.MiR-208 inhibited HCC tumorigenesis potential in vivoTo investigate the role of mi R-208-3p in vivo, we injected control or mi R-208-3p-inhibitor transfected HepG2 and Hep3 B cells subcutaneously into BALB/c nude mice. No solid tumors were detected in HepG2-injected mice. After 28 d, Hep3B-injected mice were sacrificed, and the tumors were resected and photographed. Tumor volumes in mice with low mi R-208-3p levels were smaller than those of control group(Figs. 5A and 5B). qRT-PCR and WB were conducted to evaluate the expression of mi R-208-3p and ARID2 in 6 pair solid tumors. The results indicated that miR-208-3p expressions were lower in miR-208-3p inhibitor-transfected groups(Fig. 5C), in which, ARID2 was upregulated(Fig. 5D and E). These results suggested that down-regulation of mi R-208-3pcould suppress HCC tumorigenesis in vivo.mi R-208-3p mediated the repression of ARID2 by TGFβ1 in HCC cellsTGFβ1-specific si RNA was transfected into Hep3 B cells separately or co-transfected with mi R-208-3p mimics or control mimics to confirm whether ARID2 is a downstream response element of TGFβ1/mi R-208-3p regulatory network. Western blot analysis was performed to analyze TGFβ1, ARID2 and IFITM1 protein levels, and q RT-PCR was carried out to determine mi R-208-3p expression. The transfection of TGFβ1-specific si RNA could repress TGFβ1 expression, repress mi R-208-3p expression, and upregulate the protein level of ARID2 and IFITM1, This effect of TGFβ1 si RNA on miR-208-3p, ARID2 and IFITM1 expressions could be reversed by miR-208-3p mimics(Fig. 6A and B). The results of transwell and colony formation assay indicated that depeted TGFβ1 inhibited HCC cell invasion and colony formation ability, but the over-expression of miR-208-3p could reverse the effect of TGFβ1 down-regulation on HCC cells(Fig. 6C and D). These data provided a new signal pathway, in which ARID2 is regulated by TGFβ1 via mi R-208-3p(Fig. 6E).DiscussionIn all these years, even more researchers focused on the potent therapeutic miRNAs for cancer treatment. MiR-122 a is minimally expressed and is inversely correlated with cyclin G1 expression in HCC [22]. In addition, mi R-124 and miR-203 are down-regulated and cell growth is inhibited by regulating multiple targets during hepatocarcinogenesis [23]. By contrast, miR-181 b, a TGFβ-activated mi RNA is up-regulated and can promote HCC cell invasion and proliferation by targeting TIMP3 [24]. Moreover, miR-21 and miR-17-92 were upregulated in HCC, and cell proliferation is significantly reduced when these two mi RNAs were knocked down [9]. Previous studies showed the role of miR-208 in myocardial injury, EMT, and cancer biological regulation [14, 15, 25]. However, limited information is available regarding the role of mi R-208-3p in HCC. In this study, upregulated expression levels of mi R-208-3p were detected in HCC speciments compared to that in paired normal hepatic tissues. In HepG2 and Hep3 B cells, repression of mi R-208-3p inhibited HCC cell proliferation and invasion. In addition, silencing mi R-208-3p inhibited HCC tumorigenesis in nude mice. Furthermore, miR-208-3p expression level was inversely correlated with level of ARID2 protein in HCC specimens.To further understand the potential association of mi R-208-3p and ARID2, luciferase reporter and Western blot assays wer performed. The results showed that miR-208-3p repressed ARID2 expression and directly bound to its 3-UTR. ARID2 mutated and lost the function as a tumor suppressor gene inboth small cell lung carcinoma and HCC [19, 20]. In this study, ARID2 expression was down-regulated, and ARID2 over-expression inhibited HCC cell proliferation and invasion. In addition, ARID2 expression could partly reverse the effect of miR-208-3p down-regulation on HCC cell progression. Early study has shown that mi R-208 was regulated by TGFβ1, which was involved in the TGFβ1-SMAD4-ARID2 pathway [26, 27]. As a subunit of PBAF, ARID2 was also found to regulate the expression of IFITM1[16], which could enhance the transcriptional activity of p53 and negatively regulated HCC cell cell proliferation and tumorigenesis[28]. Here we found that depletion of TGFβ1 by si RNA repressed mi R-208-3p, upregulated ARID2 and IFITM1 expression. This up-regulation of ARID2 and IFITM1 were down-regulated by transfection of mi R-208-3p mimics in TGFβ1 si RNA and mi R-208-3p mimics co-transfection group. Furthermore, the inhibition of HCC cell invasion and colony formation ability caused by TGFβ1 si RNA were partly reversed by mi R-208-3p mimics. These results may provide clearer understandings about ARID2 signal regulatory pathways in cancer. |
PDF Full Text Request