Expression of miR-720 is correlated with DNMT3 in Oral squamous cell carcinomas

Background: Many factors are involved with cellular differentiation and pluripotency, such as long non-coding and microRNAs (miRNA). miR-720 has been demonstrated to affect development, differentiation and pluripotency in dental pulp stem cells but has also been linked to many cancers (renal, prostate, skin, colorectal, breast, cervical and esophageal) through shared pathways including DNA methyltransferase family members (DNMT)3a and DNMT3b, which are highly upregulated in many oral cancers. However, little is known about the expression (or function, if expressed) of miR-720 in oral cancers. Based upon this lack of information, the primary objective of this project was to evaluate the expression of miR-720 among oral squamous cell carcinomas. Methods: RNA was extracted from commercially available oral cancer cell lines (SCC4, SCC9, SCC15, SCC25 and CAL27). This was subsequently screened for DNMT1, DNMT-3a, DNMT-3b, and miR-720 expression using highspecificity primers and polymerase chain reaction (RT-PCR). Proliferation assays were performed to determine the proliferation rate of each cell line. Results: Variable expression of miR-720 was observed among the oral cancers. More specifically, expression was low or absent among SCC4 and SCC25 that correlated with relatively low expression of DNMT3a and DNMT3b mRNA expression. High expression was observed among CAL27, SCC15, and SCC9 cells that correlated with relatively higher levels of DNMT3a and DNMT3b. Pearson’s correlation found an association between proliferation rate of each cell line and expression of miR-720 assessed by RT-PCR; R = 0.8488. Conclusions: Although much is known about miR-720 – no studies to date have examined expression among oral cancers and tumor cell lines. This study may be the first to examine miR-720 expression in normal and cancerous oral tissues. These results demonstrate that miR-720 can be found in the most rapidly proliferating cell lines and may also correlate with expression of DNMT3a and DNMT3b expression in these same cell lines.


Background
Many factors are involved with cellular differentiation and pluripotency, including recently discovered modulators such as long non-coding and microRNAs (miRNA) [1][2][3]. MicroRNA expression is known to regulate protein and mRNA expression through a variety of mechanisms, including direct and indirect modulation of mRNA transcription through DNA-binding complexes and mediation of mRNA translation through complex inhibitory sequence interactions [4][5][6]. Although much remains to be discovered, several important microRNAs have been identified that may be key regulators of gene expression relating specifically to human health and disease [7,8].
One such microRNA, miR-720, has been linked to many cancers, including colorectal, breast, prostate, and cervical cancers [9][10][11][12]. Recently it was reported that miR-720 may influence dental pulp stem cell proliferation and differentiation through shared pathways including DNA methyltransferase family members DNMT3a and DNMT3b, which are also highly upregulated in many oral cancers [13][14][15]. The family of DNMT enzymes influence gene activity through methylation and epigenetic modifications and are known to play a significant role in the development and pathophysiology of many cancers [16][17][18].
However, little is known about the expression (or function, if expressed) of miR-720 in oral cancers. Based upon this lack of information, the primary objective of this project was to evaluate the expression of miR-720 among oral squamous cell carcinomas.

RNA isolation
RNA was extracted from T25 flasks of each cell line at approximately 70% confluence using the TRIzol RNA isolation reagent (Fisher Scientific) and repeated in triplicate. In brief, supernatant was aspirated from each cell culture and 1 mL of TRIzol reagent was added to homogenize the cell monolayer in each flask for approximately 5 minutes at room temperature. Each sample was then transferred to a microcentrifuge tube and 0.2 mL of chloroform was added and mixed thoroughly prior to incubation for an additional 5 minutes. Each sample was then centrifuged at 12,000 x g or relative centrifugal force (RCF) for 15 min at 4°C.
Following centrifugation, the aqueous upper phase was transferred to a sterile microcentrifuge tube and mixed with an equal volume of isopropyl alcohol prior to incubation at room temperature. Samples were then centrifuged at 12,000 RCF to precipitate the nucleic acids. Supernatant was removed and the RNA pellet was washed with 75% ethanol and centrifuged again. Supernatant was removed and 100 uL of RNA rehydration solution was added to each microcentrifuge tube and allowed to rehydrate.

RNA analysis
Quality and quantity of RNA from each sample (n = 3 per sample) was determined using a NanoDrop 2000 spectrophotometer (Fisher Scientific). Purity of RNA was measured using the absorbance readings at A260 nm and A280 nm. RNA with an A260:A280 ratio higher than 1.65 is considered to be acceptable purity for polymerase chain reaction (PCR) screening, without significant protein contamination. Quantitative analysis was determined automatically by the NanoDrop 2000 using the measured absorbance value at A260 nm and Beer-Lambert's equation, which measures the attenuation of incident light reaching the detector after passing through the sample.

PCR screening
To quantify the expression of specific mRNA, RT-PCR was performed on total RNA using the.
ABgene Reverse-iT One-Step RT-PCR Kit (ReadyMix Version) and a Mastercycler gradient. thermocycler (Eppendorf: Hamburg, Germany) using the following primers for:

Proliferation assays
Cells were plated in 96-well tissue culture plates at a density of 1.2 × 10 5 cells/mL and allowed to grow for 24, 48 and 72 h. Cells were subsequently fixed with 10% formalin and stained using Gentian violet. Absorbance readings for each plate were measured using a BioTek ELx808 microplate reader at 595 nm to approximate cell number and confluence.
Absorbance (optical density) measurements of cell growth using a microplate reader are among the most commonly techniques used to quantify and characterize proliferation of cells in culture. Previous studies have demonstrated that the absorbance reading of cells stained with Gentian violet at 595 nm are proportional to cell number (concentration of cells in a 96-well plate) [19][20][21].

Statistical analysis
Differences between continuous variables (absorbance readings) were calculated using parametric statistical analysis methods, including two-tailed Student's t-tests and an alpha level of 0.05 to determine significance. Averages for normal and oral cancer cell signal band intensity (SBI) were calculated using Microsoft Excel and differences in SBI were calculated using Analysis of Variance (ANOVA) and an alpha level of 0.05 to determine statistical significance, which is appropriate for the evaluation of multiple two-way t-tests of parametric data.

Results
Cultures of each cell line were successfully established (Fig. 1). Proliferation rates varied among each of the various cell lines, with the most rapidly dividing oral cancer cell line CAL27 achieving 90-95% confluence within 48-72 h following a 1:2 cell passage. The slowest among the oral cancer cell lines was SCC4, which only achieved approximately 30-40% confluence after 48-72 h following the 1:2 cell passage. However, the normal non-cancerous oral gingival cell line, HGF-1, took approximately 10-12 days to achieve 70% confluence.
Total RNA was isolated from each cell line at 70% confluence and repeated in triplicate ( Table 1). Analysis of these data demonstrated that RNA was successfully extracted from each cell line, ranging from 639.4-887.0 ng/uL (average 745.7 ng/uL +/− 70.2). In addition, the purity as measured by A260:A280 ratio of absorbances demonstrated that each sample was of sufficient quality for processing and screening using PCR, ranging from 1.71 to 1.88 (average 1.78 +/− 0.08).
The RNA from each cell line was then used to screen for mRNA expression of differentiation markers DNMT1, DNMT3a and DNMT3b -as well as miR-720 (Fig. 2) and was repeated in triplicate (n = 3) for each cell line. These data revealed relatively robust expression of the differentiation marker DNMT1 only within the normal, non- To determine any correlation between the expression of miR-720 and growth rate of each cell, these data were plotted against one another and Pearson's correlation calculated (Fig. 3). These data demonstrate a strong linear relationship between miR-720 expression and proliferation rate of each cell line, regardless of the differentiation status, R = 0.8488. These data strongly suggest that miR-720 expression may be associated with growth rate in cell line and may therefore have an indirect or dietary effect on the pathways that influence and modulate these responses.

Discussion
Due to the lack of information regarding miR-720 in oral cancers, the primary objective of this project was to evaluate the expression among oral squamous cell carcinomas. These data revealed not only that miR-720 may be expressed widely among oral cancers but also that the levels of expression correlate with other phenotypic markers, such as proliferation and differentiation. This appears to support other studies of miR-720 that suggest this microRNA may have prognostic significance relating to proliferation rate, migration and invasion in colorectal, renal, and cervical cancers through pathways including Rab35 that may not be active in oral cancers [12,22,23].
However, these findings may conflict with other reports of miR-720 directly inhibiting other tumors, such as breast, through direct modulation of TWIST1 [24]. Expression (and overexpression) of TWIST has been documented in the development and phenotype of many oral cancers, which may suggest that other mediators of TWIST may be responsible for these observations [25,26]. In fact, TWIST1 has been shown to be associated with many facets of epithelial-mesenchymal transitions in oral cancers and may therefore be regulated by  multiple convergent pathways, including Let-7d and CCN3 that are also active in many oral cancers [27][28][29]. Despite these contradictions, many studies have demonstrated miR-720 is a downstream target of ERK activation that may, in turn, promote migratory and invasive phenotypes in other cancers [10,[30][31][32]. This pathway is central to molecular carcinogenesis and pathogenesis of oral cancers and may represent one of the most important mechanisms of interactive modulation [30,31]. This may also represent one of the first demonstrations of miR-720 activity with relation to DNMT3, although other microRNAs have recently been identified, including miR-29, miR-143 and miR-182 [33][34][35].
One potential regulatory mechanism may be through modulation of vasohibin-1 (VASH1)a recently identified novel prognostic factor for head and neck squamous cell carcinomas [36]. VASH1 is an angiogenesis inhibitor active in normal tissues, which may be suppressed (directly or indirectly) in many cancers and tumor types including head and neck cancers [37]. Previous research identified miR-31 and miR-720 as direct modulators and inhibitors of VASH1 in endothelial cells, although no direct evidence of miR-720 regulation of VASH1 in cancers has yet been established [38].
Another potential mechanism of action that should be explored in future studies may be through an indirect relationship with Sp3, a previously identified transcription factor that directly modulates DNMT3 activity in many types of cancer [39]. TWIST1 has previously been demonstrated to interact with both Sp1 and Sp3 in mesenchymal cells [40]. As previous studies have noted miR-720 induction of TWIST1, this may represent a potential downstream indirect relationship between miR-720 and TWIST1 with Sp3 and DNMT3 expressionalthough more studies will be needed to evaluate these pathways.

Conclusions
Although much is known about miR-720no studies to date have examined expression among oral cancers and tumor cell lines. This study may be the first to examine miR-720 expression in normal and cancerous oral tissues. These results demonstrate that miR-720 can be found in the most rapidly proliferating cell lines and may also correlate with expression of DNMT3a and DNMT3b expression in these same cell lines.  Correlation between miR-720 expression and proliferation rate. Pearson's correlation demonstrated a strong, positive association between miR-720 and cellular growth, R = 0.8488 using a standardized amount of total RNA (1.0 ng). This did not vary according to differentiation status (normal, cancerous) but was strongly correlated with proliferation rate only