Progress risk assessment of oral premalignant lesions with saliva miRNA analysis

From 2006 to 2012, 45 eligible patients treated in the department of Oral & Maxillofacial-Head & Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University (Shanghai, China) were enrolled in the present study. The patients had been diagnosed with LGD leukoplakias and were given 13-cis-retinoic acid (13cRA) treatment and close follow-ups before any event (defined as the diagnosis of HGD or CIS or OSCC). The diagnosis of leukoplakias, OSCC, and LGD were made according to World Health Organization criteria [17]. A total of 45 frozen samples were collected at baseline after enrollment. Among these 45 LGD leukoplakias patients, 10 patients progressed to carcinoma in situ or OSCC after an average time of 30.1 months. 2 were excluded because of poor RNA quality. Among the 35 patients who did not develop OSCC, 5 were excluded because of poor RNA quality, and another 12 were excluded because of new lesion occurrence. As these progressing LGD samples are very rare and difficult to obtain, we included all the 8 samples from patients who developed oral cancer (N = 8) for gene expression profiling. 7 patients who remained at a LGD stage within 3–5 years of follow-up were selected, in order to identify the differences in miRNA expression between these precancerous lesions with different clinical outcomes. Informed consent was obtained from all patients. The project was approved by the Scientific and Ethics Committee of Shanghai Jiao Tong University. Patient information and representative histological pictures is shown in Table 1 and Figure 1, respectively.

Saliva samples were collected from these patients whenever tissue biopsies were collected. But only samples collected at first visit were used for analysis in order to investigate miRNA expression differences in these premalignant lesions at LGD stage. Saliva samples from 7 healthy volunteers were also collected to determine baseline miRNA expression in oral saliva from normal control. Briefly, immediately after mouthwash, ~2 ml saliva was collected. Upon collection, saliva samples were mixed with 5 ml RNA Protect Saliva reagent (Qiagen, Copenhagen), kept at room temperature for 24 hours, and then stored at −20°C until RNA or protein isolation.

The TaqMan^® low density array (TLDA) qRT-PCR system (Applied Biosystems, Foster City CA) were used for global miR expression analysis. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen).The qualities of RNA extracts were found to be good. Total RNA was converted into specific cDNA derived from mature miRs and quantified using Multiplex RT and TaqMan Low Density Array (TLDA) Human MicroRNA Panel v3.0 and 7900 real-time RT PCR System (Applied Biosystems). A total of 800 ng RNAs were used for each array. The two-card set of TaqMan^® Array MicroRNA Cards (Cards A and B) contained 754 lyophilized human TaqMan miR sequences. And each card contains four control assays—three carefully selected candidate endogenous control assays and one negative control assay.

Real-time qPCR analysis was performed on saliva samples from these patients and 7 healthy volunteers using the TaqMan MicroRNA Assay. miRNA expression analysis was performed for 8 selected deregulated miRNAs (miR-10b, miR-145, miR-99b, miR-708, miR-181c, miR-30e, miR-660 and miR-197) with greatest fold changes in progressing LGD leukoplakias compared with non-progressing LGD leukoplakias.

Total RNA was extracted from saliva samples using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. In brief, the saliva sample is first centrifuged, and Buffer RLT is then added to the resulting pellet. After addition of ethanol, the sample is applied to an RNeasy MinElute spin column, where RNA binds to the membrane after centrifugation. Traces of genomic DNA are removed by DNase digestion, and contaminants are washed away in several wash steps. Highly pure RNA is then eluted using RNase-free water in a volume of just 14 μl. The purity of isolated RNA was determined by OD260/280 using a Nanodrop ND-1000 (Thermo Scientific, Worcester, MA). Each RNA sample was polyadenylated and reversely transcribed to cDNA using the TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA Single-stranded cDNA was synthesized from 10 ng of total RNA(normalization by amount) or from 1 μl of the RNA volume (normalization by volume) using specific miRNA primers (TaqMan MicroRNA Assay, PN 4427975, Applied Biosystems).

All assays were performed in duplicate. Expression data were normalized against RNU6; pooled saliva samples from healthy volunteers were used as the reference for analysis.

Samples were grouped as normal, non-progressive LGD leukoplakia and progressive LGD leukoplakia. Data were quantified and analyzed using Sequence Detection System (version 2.3) (Applied Biosystems). The expression level of each miRNA was quantified by its normalized threshold cycle number ΔCt, where ΔCt = [Ct (miRNA)]-[Ct (U48)], and the relative expression level was calculated as 2^-(ΔCt) which is commonly used in genom-wide profiling studies of miRNAs. In order to select differentially expressed miRNAs for further classification, the random variance t-test (RVM t-test) was used to compare the control and experiment, as the RVM t-test can raise degrees of freedom effectively in the cases of small samples. The multiple testing was corrected using the Benjamini–Hochberg false discovery rate (FDR) method. Having a differential fold change > ±2 at raw P value < 0.05 were considered as significant candidates.

In order to evaluate miRNA deregulations of progressive oral premalignant lesions, we used a systematic miRNA expression profiling analysis of 754 mammalian miRNAs on 8 progressing LGD leukoplakias and 7 non-progressing LGD leukoplakias. A filter procedure reduced the number of miRNAs to a total of 617 hsa-miRNAs, eliminating genes with low expression variation across the experiments (SD < 0.3). We performed a differential expression analysis to detect significant differences in miRNA expression between different types of LGD leukoplakias.

We identified 25 miRNAs differentially expressed between progressive and non-progressive LGD leukoplakias. Compared to non-progressive LGD leukoplakias, 13 miRNAs were down-regulated and 12 miRNAs were up-regulated in progressive LGD leukoplakias. According to previous studies on oral squamous cell carcinomas, most of these deregulated miRNAs in progressive LGD leukoplakias were changed with same changing patterns as that in OSCC, such as down-regulation of miR-99, miR-let-7, miR-145 and up-regulation of miR-708, miR-10b, miR-26a and miR-30e. Interestingly, in our study both miR-181c and miR-181b were found to be under-expressed in progressing LGD leukoplakias, which was contrary to the result of a previous study on miRNA expression changes during progression of oral premalignancy. The miRNAs that were differentially expressed are listed in Table 2 and Figure 2.

To explore the possibility of using salivary microRNA markers to distinguish OPL patients with different malignant transform potential, we tested the expression of 8 miRNAs (miR-10b, miR-145, miR-99b, miR-708, miR-181c, miR-30e, miR-660 and miR-197) in saliva samples from these patients using qRT-PCR. According to the TLDA findings, miR-10b, miR-660, miR-708 and miR-30e demonstrated significant over-expression in progressive LGD leukoplakias; miR-145, miR-99b, miR-181c and miR-197 were under-expressed in this group of leukoplakias.

Expressions of miR-10b, miR-145, miR-99b, miR-708 and miR-181c were significantly different in saliva of progressive LGD leukoplakia patients, compared to that of non-progressive LGD leukoplakia patients (Figure 3). Trend (P < 0.10) were observed for miR-197 and miR-30e, the p-values were 0.057 and 0.089 respectively. Expression of miR-660 was under-expressed in non-progressing LGD leukoplakias (p = 0.038) but over-expression of this miRNA in progressing leukoplakias did not reach statistical significance (P = 0.098), which was inconsistent with our initial TLDA findings of this gene in progressing leukoplakias.