微型RNA miR-134於口腔癌化之探讨

AbstractBeing the sixth most common cancer worldwide, oral squamous cell carcinoma (OSCC) also ranks the fourth among Taiwanese male cause of cancer death. MicroRNAs (miRNA; miR) are regulatory RNAs involved in post-transcriptional gene regulation, which can affect homeostasis, metabolism, and carcinogenesis. Aberrant expression of miRNAs can be seen in various cancers. Our results indicate that miR-134is markedly up-regulated in cancerous tissues than in non-cancerous matched tissues in clinical OSCC specimens after qRT-PCR analysis. Higher miR-134 expression is also associated with more extensive tumor size and vascular invasion. According to the result of in vitro study, over-expression of miR-134enhances oncogenic potentials of OSCC cells by increasing proliferation, migration, and colony formation abilities; conversely, when miR-134 expression is down-regulated, the oncogenic potentials of OSCC cells are inhibited. Besides, increased radio-resistant ability is noted in miR-134over-expressed OSCC cells. In vivo study also shows more and larger tumor formation in nude mice injected by miR-134 over-expressed OSCC cells. In addition, transient induction of NFκB, AKT, and HIF signals increases miR-134 expression, and down-regulation of NFκB by co-transfecting IκB reduces expression of miR-134. miR-134may be related to the development of OSCC. A thorough comprehending towards regulation of miR-134expression and miR-134target gene will be beneficial to OSCCdiagnosis and therapeutic intervention.Keywords: carcinoma, microRNA, oral cavity, miR-134IntroductionOral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide.[1] Taiwan also has one of the highest incidences of OSCC in the world.[2] In Taiwan, OSCC median age of cause of cancer death is only 55 years old; OSCC standardized mortality rate in male increased 36.4% in past 10 years[3]. Although diagnosis and treatment of OSCC have improved, the survival rate has not increased substantially in recent years. Altered microRNA (miRNA) expression profiles have been observed in numerous malignancies, including OSCC.[4] miRNAs are short RNAs that direct messenger RNA degradation or disrupt mRNA translation in a sequence-dependent manner and thereby significantly influence cell development, homeostasis, metabolism, neuronal and immunological regulation, even carcinogenesis[5]. In recent studies of Professor Chang’s team show that miR-31 contributes to the development of OSCC by impeding FIH to activate HIF under normoxic conditions[6], and up-regulation of miR-221/222 is associated with OSCC development by enhancing cell proliferation abilities.Among all kinds of miRNAs, miR-134is found to be a differential marker of stem cells[7] and is considered to be involved in the regulation of memory as well[8]. Recently, miR-134is reported to be involved in drug-resistant small cell lung cancers[9]. It is also reported that miR-134 is elevated in tissues of tongue OSCC in Honk Kong people[10]. Furthermore, the level of miR-134is high in the saliva ofnormal individuals, which means miR-134may be useful to be a biomarker for detecting and monitoring various physiopathological conditions of oral cavity[11]. In our preliminary data, we have found that the level of miR-134is elevated in the plasma of OSCC patients (n=10), which indicates that miR-134 is associated with OSCC development.This study hypothesizes that miR-134to be an oncogenic miRNA. Our short-term aims are as below: first, to validate the elevation of miR-134in the plasma of OSCC patients by using more samples. Second is to verify whether the level of miR-134 is elevated in saliva of OSCC patients, and to see if the level of miR-134in saliva is valuable for diagnosis of OSCC. Our long-term aims are as following: first, to confirm the in vitro function of miR-134through transient transfection; second, to see the induction of in vivo tumorigenesis driven by miR-134 by using stable cell lines and perform animal studies. By using stable cell lines, our final aim is to find out the target of miR-134 for pathogenesis.Materials and MethodsOSCC tissueTissue specimens were taken from 42 patients with primary OSCC, together with matched paired non-cancerous oral mucosa (NOM) samples. The epithelial cells were retrieved from frozen-sectioned NOM or OSCC tissues using laser capture micro-dissection (LCM) for total RNA isolation; the blood samples were available in 34 patients prior to surgery and 20 control subjects without oral lesion. The study was approved by an Institutional Review Board. All samples were collected only after receiving informed consent from the patients.qRT-PCRA TaqMan MicroRNA Assays kit and TaqMan microRNA Assays supplies were used to quantify the expression of miR-134according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Quantitative RT-PCR (qRT-PCR) was carried out on a TECHNE QUANTICATM thermocycler (TECHEN, Milford, MA) using RNU6B and/or Let-7a as the internal controls. Ct was the number of cycle at which the fluorescence signal passed threshold. △Ct was the difference in Ct values between miR-134 in relation to the controls. -△△Ct was the difference in △Ct values between the paired samples or the different experimental settings. 2Ct represents the fold change in miR-134 expression.Cell cultureThe OECM-1 OSCC cell line was grown in RPMI medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel). The SAS OSCC cell line was grown in DMEM medium (Invitrogen) supplemented with 10% FBS. Both cells were cultured at 37℃in a humidified atmosphere of 5% CO2. SAS is a tumorigenic cell line, while OECM-1 is a non-tumorigenic cell line. Precursor miR-134 and antisense miR-134 together with control scrambles (Scr) were purchased from Ambion (Austin, TX). The doses of precursor miR-134 and antisense miR-134 were determined as 30 nM and 60 nM by pilot studies, respectively.Cell proliferationA total of 5 x 103 cells were seeded into 24-well culture plates. The viability of cells was measured with the trypan blue exclusion assay.Cell migrationA total of 1 x 105 cells were grown in a Transwell (Corning, Acton, MA) having a pore diameter of 8 μm. The cell growth was arrested by the addition of hydroxyurea (Sigma-Aldrich) to a final concentration of 1 μM. The bottom chamber and lower side of the membrane was coated with 20 μg/ml fibronectin (Sigma-Aldrich) to induce migration. The bottom chamber and lower side of the membrane was soaked in 10% FBS supplemented medium, with upper side soaked in FBS-freemedium to induce cell migration.Anchorage-independent colony formation assayIn a 6-well plate, a lower agar layer was fabricated using a 1:1 mixture of 1.8% agarose and 60% FBS. An upper agar layer was formed with 1:1 mixture of methylcellulose (Sigma-Aldrich, St Louise, MO) and 60% FBS after lower layer had set. A total of 1 x 105 cells for SAS OSCC cell lines and 8 x 104 for OECM-1 OSCC cell lines were seeded into 6-well culture plates. Colonies with a diameter larger that 50 and 100 μM were counted by crystal violet staining.Establishment of SAS-miR-134 cellsStable cell line of SAS OSCC cells were established by infecting lentivirus containing premature miR-134with its negative control (BioSettia). Transduced cells were selected using DMEM medium (containing 10% FBS) with puromycin (25 mg/ml) for 7 days. The concentration of puromycin was determined by pilot studies. The vector contained RFP to monitor the infection rate by fluorescence microscopy. The expression of ectopic miR-134 was assayed by qRT-PCR.In vivo tumorigenesis assay105 SAS OSCC cells per nude mice were injected subcutaneously to total 10 nude mice. The xenografts grown afterwards were collected and analyzed by qRT-PCR to quantify miR-134 expressions.Radio-resistance assay300 SAS-miR-134-RFP and SAS-RFP were seeded to 3-cm plate and incubated for 14 days. Cisplatin were then treated. Different X-ray exposures of 0 Grey, 2 Grey and 8 Grey were then given.Reporter assayCo-transfection of reporter plasmids with pCMV-Luc into lentivirus-transduced cells was performed with TransFectin Lipid Reagent (BIO-RAD, Hercules, CA, USA). Over-expressed miR-134 targeted to their complementary sequence and decreased β-galactosidase activity. Normalization was done by luciferase activity.Statistical analysisMann-Whitney and ANOVA analysis were used for statistical analysis and p < 0.05 was considered statistically significant.ResultsmiR-134 expression in OSCCqRT-PCR analysis from LCM-retrieved NOM and OSCC showed higher expression of miR-134in tumors than in normal tissues (Fig. 1 A). In larger and invasive tumors, the miR-134 expression was even higher (Fig. 1 B). As in 34 pairs of patient blood samples, miR-134 expression was slightly elevated in pre-operative OSCC patients than in normal controls. After operation, miR-134expression reduced in plasma samples (Fig. 1 C).After qRT-PCR analysis, OC3, SAS, and OECM-1 OSCC cells all showed higher miR-134 expression than NHOK cells (Fig. 1 D). OECM-1 and SAS OSCC cells were chosen due to their slightly elevated endogenous miR-134 expression; OC3 cells were discarded because of their higher endogenous miR-134 expression might interfere with experiments.Modulation of miR-134 expressionUp-regulated and down-regulated miR-134OSCC cells after transfecting precursor and antisense miR-134 were established as transient models to study oncogenic phenotypes. OECM-1- and SAS-precursor-miR-134cells showed an increase in miR-134 expression for 30000 folds and 10000 folds, respectively (Fig.2 A). OECM-1- and SAS-antisense-miR-134 cells showed a decrease in miR-134 expression for 0.6 folds and 0.5 folds, respectively (Fig. 2 B).Stable OSCC cell line (SAS-miR-134-RFP cells)showed bright red fluorescence relative to negative control (Fig. 2 C) with an increase in miR-134 expression for 19.6 folds (Fig. 2 D).Association between miR-134 expression and oncogenic phenotypes.Both OECM-1- and SAS-precursor-miR-134 cells had a higher growth rate than controls (Fig. 3 A). OECM-1- and SAS-precursor-miR-134 showed higher migration abilities relative to controls (Fig. 3 C). The anchorage-independent colony formation assays also revealed that OECM-1- and SAS-precursor-miR-134formed more colonies (Fig. 3 D). Conversely, OECM-1- and SAS-antisense-miR-134 showed slower growth rate (Fig. 3 B), lower migration abilities (Fig. 3 C), and smaller and less colony formation (Fig. 3 D) than their controls. Both OECM-1- and SAS-mutant-miR-134showed no statistical significant differences in proliferation assay, migration assay, and anchorage-independent colony formation assay compared to controls (Fig. 4 A, B , C, D); OECM-1 and SAS-precusor-miR-134 were set as positive controls, which showed higher growth rate, more competent migration ability, and fortified colony formation ability.Stable clones of SAS OSCC cells were also studied. SAS-miR-134–RFP showed higher growth rate, strengthened migration ability, and reinforced colony formation ability compared to SAS-RFP (Fig. 5 A, B, C). Xenografts retrieved from nude mice also showed more and larger tumor formed by SAS-miR-134. TheqRT-PCR analysis of xenografts showed elevated miR-134expression in tissue samples retrieved from SAS-miR-134-RFP injected nude mice compared to SAS-RFP injected nude mice (Fig. 5 D). Radio-resistance assay showed more colonies of SAS-miR-134-RFP survived after exposure of 2 Grey and 8 Grey radiation compared to SAS-RFP (Fig. 6).By transfecting NFκB, AKT, and HIF into OECM-1 and SAS OSCC cells, respectively, increases of miR-134 expression were detected by qRT-PCR analysis (Fig. 7 A). After co-transfecting IκB to inhibit NFκB expression, expression of miR-134 decreased (Fig. 7 B). The result showed that miR-134 might be regulated through the oncogenic NFκB, AKT, or HIF signaling pathways.The result of reporter assay of PDCD-7 (programmed cell death protein 7) showed no significant statistical differences between SAS-miR-134-RFP and control.DiscussionOur data had shown elevated miR-134expression in both tissue specimens and plasma samples from OSCC patients, which was consistent with recent studies showing that increased miR-134expression might be related to carcinogenesis. Elevated expression of miR-134was also related with more advanced cancer stages, such as larger tumor formation and invasion; miR-134plasma levels reduced after surgical removal of cancer tissues. These results indicated that the up-regulation of miR-134 was associated with OSCC development.Our results of transient OSCC cell lines revealed that in both OECM-1- and SAS-precursor-miR-134 cells, the oncogenic potentials were enhanced; conversely, the oncogenic potentials were inhibited in OECM-1- and SAS-antisense-miR-134 cells. To further confirm the specificity of miR-134 sequence used in our study, we used mutant-miR-134. The results for phenotype studies of OECM-1- and SAS-mutant-miR-134 showed no significant difference from controls, which implied the specificity of miR-134sequence used in our study was reliable despite that transfecting antisense-miR-134did not significantly reduce the expression of miR-134.In SAS-miR-134 stable clones, our results also showed increased cell proliferation, migration, and colony formation abilities in miR-134 up-regulated SAS cells, which conformed to the results of transient cell lines. The radio-resistance assay also indicated SAS-miR-134-RFP possessing stronger radio-resistant ability.The result of qRT-PCR analysis also showed higher miR-134 expression in tumors retrieved from SAS-miR-134-RFP injected nude mice groups. These results further supported our hypothesis that elevation of miR-134 expression might contribute to OSCC development.Moreover, our results implied that regulation of miR-134could be possibly through oncogenic NFκB, HIF, or AKT signaling pathways. The result was consistent with our in vitro experiments which showed intensified cell proliferation, migration, and colony formation abilities, and our in vivo experiments showing more and larger tumors formed in SAS-miR-134-RFP injected nude mice.The result of reporter assay of PDCD-7 revealed that PDCD-7 might not be the target of miR-134. Other possible targets could be screened by using stable clone of over-expressed miR-134 OSCC cells.References1. Jemal, A., et al., Cancer statistics, 2009. CA Cancer J Clin, 2009. 59(4): p.225-49.2. Ferlay, J., et al., GLOBOCAN 2000: Cancer Incidence, Mortality andPrevalence Worldwide, Version 1.0. IARC CancerBase No. 5. Lyon, IARCPress, 2001. Links.3. Department of Health, E.Y., R.O.C. (Taiwan). 2008 statistics of causes ofdeath. 2009 Oct. 1, 2009 [cited 2009 Feb. 28]; Available from: .tw.4. Henson, B.J., et al., Decreased expression of miR-125b and miR-100 in oralcancer cells contributes to malignancy. Genes Chromosomes Cancer, 2009.48(7): p. 569-82.5. Cho, W.C., OncomiRs: the discovery and progress of microRNAs in cancers.Mol Cancer, 2007. 6: p. 60.6. Liu, C.J., et al., miR-31 ablates expression of the HIF regulatory factor FIH toactivate the HIF pathway in head and neck carcinoma. Cancer Res, 2010.70(4): p. 1635-44.7. Tay, Y.M., et al., MicroRNA-134 modulates the differentiation of mouseembryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells, 2008. 26(1): p. 17-29.8. Gao, J., et al., A novel pathway regulates memory and plasticity via SIRT1and miR-134. Nature, 2010. 466(7310): p. 1105-9.9. Guo, L., et al., Gene expression profiling of drug-resistant small cell lungcancer cells by combining microRNA and cDNA expression analysis. Eur J Cancer, 2010. 46(9): p. 1692-702.10. Wong, T.S., et al., Mature miR-184 as Potential Oncogenic microRNA ofSquamous Cell Carcinoma of Tongue.Clin Cancer Res, 2008. 14(9): p.2588-92.11. Weber, J.A., et al., The MicroRNA Spectrum in 12 Body Fluids. Clin Chem,2010.Figure 1. miR-134expression in OSCC. A, qRT-PCR analysis performed to detect expression of miR-134in LCM-retrieved NCMT (non-cancerous matched tissues) and OSCC tissues. C t was the number of cycle at which the fluorescence signal passed threshold. ΔC t was the difference of C t values between miR-134 and internal control, RNU6B. Note the up-regulation of miR-134 in OSCC tissues. B, Comparison across different clinical settings. N, lymph node involvement. T, tumor size. T1-3 represented smaller tumor size, and T4 represented largest tumor size. VI (-), no vascular invasion. VI (+), vascular invasion. Higher expression of miR-134 could be seen in tissues with larger tumor size and vascular invasion. Slightincrease in expression of miR-134could be found in tissues with lymph nodeinvolvement. The tissues indicated that miR-134might be involved in the carcinogenesis and progression of OSCC. C, miR-134expression in patients’ plasma before and after operation of removing OSCC. It was noted that plasma miR-134levels were found to be slightly elevated in OSCC patients relative to normal controls, and the miR-134 levels were reduced after surgery. Un-paired or paired t-test. D, endogenous miR-134expressions in OC3, OECM-1, SAS, and NHOK cells. miR-134 expressions were higher in OC3, OECM-1, and SAS OSCC cells relative to NHOK cells.Figure 2. Modulation of miR-134 expression. A, qRT-PCR analysis performed to compare the expressions of miR-134 between control and miR-134 up-regulated OSCC cells. 2 ΔΔCt represented fold change in miR-134 expression. OECM-1 and SAS were both OSCC cells. OSCC cells transfected by precursor-miR-134to up-regulate miR-134. Note the over-expression of miR-134after transfecting precursor-miR-134into OECM-1 and SAS OSCC cells. B, miR-134expression between control and miR-134down-regulated OSCC cells. The expression of miR-134 slightly decreased after transfecting antisense-miR-134 into OECM-1 and SAS OSCC cells. Data shown were mean ± SE from duplicate analysis. C, Redfluorescence in stable SAS OSCC cells infected with miR-134-RFP and RFP (ascontrol). D, qRT-PCR analysis of stable SAS OSCC cells. The result showed an increase in ectopic miR-134 expression in stable cells.A B CDFigure 3. Association between miR-134expression and oncogenic phenotypes in transient models.A, Proliferation assays performed after transfecting precursor-miR-134 and scramble (as control)into OECM-1 and SAS OSCC cells. Note the significant difference between precursor-miR-134 transfected OECM-1 (p = 0.0011) and SAS (p = 0.0176) OSCC cells and control. B, Proliferation assays performed after transfecting antisense- miR-134and scramble (as control) into OSCC cells. There were decreases in growth rate in antsense-miR-134transfected OSCC cells. C, Migration assays performed after transfecting precursor- and antisense-miR-134 into OECM-1 and SAS OSCC cells. Note the up-regulated miR-134 enhanced migration abilities in both OECM-1 (p < 0.0001) and SAS (p = 0.001) cells. D, Anchorage-independent colony formation assays of OECM-1 and SAS OSCC cells after transfecting precursor- and antisense- miR-134, respectively. Precursor-miR-134transfected OSCC cells showed larger and more colonies formed; antisense-miR-134transfected OSCC cells showed smaller and less colonies formed. Data shown were mean ± SE from duplicate or triplicate analysis. *, p< 0.1, **, p < 0.01; ***, p< 0.001. Two-Way ANOVA analysis or Mann-Whitney analysis.ABCFigure 4. Comparison of mutant-miR-134expression and oncogenic phenotypes with control groups.A, Proliferation assays performed after transfecting precursor-, mutant-miR-134, and scramble (as control) into OECM-1 and SAS OSCC cells. No statistical significant difference between mutant-miR-134 and control was noted. Precursor-miR-134was set as a positive control. B, Migration assays performed after transfecting precursor-, mutant-miR-134and scramble into OECM-1 and SAS OSCC cells. There was no significant differencebetween mutant-miR-134 transfected OSCC cells and control. Precursor-miR-134 was set as a poasitive control. C, Anchorage-independent colony formation assays of OECM-1 and SAS OSCC cells after transfecting precursor-, mutant-miR-134 and scramble into OSCC cells. There was no significant difference between mutant-miR-134 and control. Data shown were mean ± SE from duplicate or triplicate analysis. *, p< 0.1, **, p < 0.01; ***, p< 0.001. Two-Way ANOVA analysis or Mann-Whitney analysis.Figure 5. Association between miR-134expression and oncogenic phenotypes in stable clones.A, Proliferation assays performed after infecting miR-134-RFP and RFP (as control) via lentivirus into SAS OSCC cells. SAS-miR-134-RFP showed higher growth rate than control (p < 0.0001). B, Migration assays performed after infecting miR-134-RFP and RFP via lentivirus into SAS OSCC cells. SAS-miR-134-RFP showed higher migration ability than control. C, Anchorage-independent colony formation assays of SAS OSCC cells after infecting miR-134-RFP and RFP. SAS-miR-134-RFP showed larger and more colonies formed than control. D, qRT-PCR analysis of xenografts retrieved from nude mice. Xenografts from SAS-miR-134-RFP injected nude mice showed highermiR-134expression compared to control. Data shown were mean ± SE from duplicate or triplicate analysis. *, p< 0.1, **, p < 0.01; ***, p< 0.001. Two-Way ANOVA analysis or Mann-Whitney analysis.Figure 6. Radio-resistance assay of stable clone. The result of radio-resistance assay of SAS OSCC cell showed more colonies survived of SAS-miR-134-RFPafter 2 Grey and 8 Grey radiation exposures compared to SAS-RFP (as control).A BFigure 7. Possible pathways influencing miR-134 expression. A, qRT-PCR analysis performed to detect expressions of miR-134after transfecting NFĸB, HIF and AKT plasmids into SAS OSCC cells. miR-134expressions were found to be elevated after transfection. B, miR-134 expressions quantified by qRT-PCR after transfecting NFĸB, IĸB, and NFĸB with IĸB.Note the reduction of miR-134 expression after co-transfecting IĸB to inhibit NFĸB action.。