Identification Ofdifferentially Expressed Micrornas in Human Hepatocellular Adenoma Associated

Identification ofdifferentially expressed microRNAs in human hepatocellular adenoma associated with type I glycogen storage disease: a potential utility as biomarkers

Li-Ya Chiua,Priya S. Kishnanib, Tzu-Po Chuangc,d, Cheng-Yang Tanga, Cheng-Yuan Liue, Deeksha Balib,Dwight Koeberlb,Stephanie Austinb, Keri Boyetteb,David A. Weinsteinf, Elaine Murphyg, Adam Yaoa, h, Yuan-Tsong Chenb, h, Ling-Hui Lia,h, *

Supplementary Information

Supplementary Methods

The output of the sequencing reaction consisted of two files: csfasta and qual (QV); the former contained di-base encoded sequencing reads while the latter the quality of each di-base signal.Theoutput data were analyzed in two phases: primary analysis, which focused on the mapping of reads to reference sequencesof miRNA precursors,was performed using (Applied Biosystems);secondary analysis was employed to estimate the expression level of known mature miRNAs.

Primary Analysis

The following workflow implemented by RNA2MAP(RNA Pipeline v.5.0) was adopted to process the raw sequencing outputs. The primary SOLiD sequencing reads were encoded by color space, and the entire workflow was carried out using the color space for the sake of higher accuracy and error correction.

The filtering step removed tRNAs, rRNAs, repeated sequences, and adaptor sequences included in the reference file (downloaded from with the following conditions for alignment:

Numberofbasesto be used =25

Numberoferrors (mismatches) to be tolerant=2

The alignment method used for mapping the remaining reads to miRNA precursors from miRBase v.14 (ftp://mirbase.org/pub/mirbase/14/genomes/hsa.gff)contained twosteps:

Step1: global alignment

Match “beginning part of the read” or “seed sequences”to the reference sequencesof miRNA precursors using Mapreads, which is the core ofSOLiD 3 System color mapping tool, with the following parameters:

Numberofbasesto be used =18

Numberoferrors (mismatches) to be tolerant= 3

Step2: local alignment for adaptor identificationand adaptor sequence trimming

The locationof an adaptor within each read was determined by using the reference location obtained at the first step.Every possible adaptor start point was tested by comparing each full-length read against a hypothetical read obtained by concatenating the appropriate number of bases from the reference and adaptor sequences. The location that generates the least number of mismatches determined the size of the small RNA and adaptor sequences that needed to be trimmed.

The following parameters were applied for local alignment:

Number of bases of reference sequences plus adaptor sequences =35

Maximal numberoferrors (mismatches) to be tolerant=6

Secondary Analysis

The reads mapped to miRNA precursors in the primary analysis phase were furtherinvestigated to determine the expression level and sequence variations of mature miRNAs

By default, a read aligned to either the positive or negative strands of a given precursor miRNA is considered “mapped” by RNA2MAP. Owing tothe unidirectional transcription nature of genes, all mapped reads with the oppositeorientation to the reference miRNA precursors were thus considered spurious and excluded from further analysis. The remaining reads were then organized along the precursors usingthe alignment information obtained in the primary analysis to form multiplealignment views, as illustrated below.

Afterwards, the following steps were used to determine the normalized expression levels of mature miRNAs:

(1) The regions of mature miRNAs within the precursors were defined according to miRBase version 14 (ftp://mirbase.org/pub/mirbase/14/database_files/)(bold letters in the illustration);

(2) Each nucleotide position within the mature miRNA regions covered by one read was considered expressed once; two sequencing reads, twice, and soforth. The sum of counts at individual nucleotide position was then defined as the positional expression level of the mature miRNA.

(3) It is well-documented thatvariation in mature miRNA length due tovariability in Dicer1 or Drosha cleavage positions and single-nucleotide 3’ extensionsare frequently observed in next-generation miRNA profiling(1). To better describe the expression level of a mature miRNA,we decided to use the median of positional expression levels of a mature miRNA as the raw expression level of that mature miRNA.

(4) The raw expression levels were then normalized with the total reads mapped to miRNA precursors with the correct orientation(Supplementary Table S1). The normalized expression levels were expressed ascounts per million reads (CPMR) by multiplying the factor of 1,000,000.

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Supplementary Table S1: Clinical, pathological, and genetic mutation information of GSDIa patients.

Case # / Gender / Contraceptive Use (Age) / Mutations in G6Pase / Age (years) at Adenoma Diagnosis / Adenoma Management / Age at Procedure/ Years Post Transplant / Diameter of the main lesion (cm)
1 / F / at age 23 years for 5 months / c.1039C>T(p.Q347X) /c.1039C>T (p.Q347X) / 20 / Transplant; 38 years;
7 years post / 4.5
3 / M / N/A / c.79delC (p.35X) / c.508C>T (p.R170X) / None / None / None
4 / F / None / c.247C>T (p.R83C) / c.247C>T (p.R83C) / 13 / Liver resection; 16 years;
11 years post; Liver ablation; 26 years; 2 weeks post / 7.3 (resection)
3.2 (ablated lesion)
6 / F / NI / c.150-151delGT(p.Trp40CysfsTer10)/ c.150-151delGT(p.Trp40CysfsTer10) / 27 / Liver resection; 30 years, 3.5 years post / 2.3
7 / F / from age 13 years to age 30 years / c.79delC (p.35X) / c.1039C>T (p.Q347X) / 15 / Liver Transplant; 32 years; 8 years post / 6.5
8 / M / N/A / c.563-3c>g / c.1039C>T (p.Q347X) / 15 / Liver Resection; 45 years; Deceased / 12.6
9 / M / N/A / c.247C>T (p.R83C) / c.247C>T (p.R83C) / 21 / Liver Resection; 34 years;
7 years post / 11
10 / M / N/A / c.979delTT(p.delF327) / c.979delTT (p.delF327) / 16 / Liver Transplant; 31 years; 8 years post / 9
13 / F / Progesterone for heavy periods / c.1039C>T (p.Q347X)/ c.1039C>T (p.Q347X) / 33 / None / 0.9
29 / M / N/A / c.1039C>T (p.Q347X)/ c.1039C>T (p.Q347X) / None / None / None
30 / M / N/A / c.508C>T (p.R170X) / c.1039C>T (p.Q347X) / None / None / None
35 / M / N/A / c.724C>T (p.Q242X)/ c.1039C>T (p.Q347X) / 20 / Liver Resection; 22 years; 6 years post / 7.5
36 / F / None / c.247C>T (p.R83C)/ c.247C>T (p.R83C) / 11 / Liver Resection; 29 years;
9 years post / 6.5
37 / M / N/A / c.247C>T(p.R83C)/ c.1039C>T (p.Q347X) / NI / NI / NI
38 / M / N/A / c.247C>T (p.R83C)/ c.562G>C (p.G188R) / Between age 30 and 35 years / Liver transplant X 3;
35 years; deceased / 1.8
47 / M / N/A / c.247C>T (p.R83C)/ c.1039C>T (p.Q347X) / 14 / None / 2.5

The information of case #s 1, 4, 6, 7, 8, 9, 10, 36, 37 and 38 is taken from the previous work published in Hum Mol Genet. 2009;18:4781-90.

Abbreviations: N/A, not applied; NI, no information

Supplementary Table S2

Sample IDa / Total reads / Mapped to Filter / Mapped to miRBase / Mapped to Genome / # of mappable reads / % of mappable reads / % of mappable reads* (miRBase and genome) / % of miRNAs (compared to total reads) / % of miRNAs (compared to mappable reads) / Mapped to miR precusor at the same direction
GSD-01C / 15,897,860 / 8,540,828 / 1,221,168 / 1,306,390 / 11,068,386 / 69.6219 / 15.8987 / 7.6813 / 11.0329 / 1,200,293
GSD-01D / 15,486,091 / 6,257,406 / 1,302,694 / 1,649,640 / 9,209,740 / 59.4710 / 19.0644 / 8.4120 / 14.1447 / 1,283,222
GSD-04C / 15,972,056 / 4,306,700 / 2,355,658 / 2,219,316 / 8,881,674 / 55.6076 / 28.6436 / 14.7486 / 26.5227 / 2,320,623
GSD-04D / 20,469,562 / 6,182,991 / 3,053,949 / 3,094,862 / 12,331,802 / 60.2446 / 30.0388 / 14.9195 / 24.7648 / 3,012,170
GSD-08C / 19,598,022 / 8,790,424 / 1,954,988 / 2,072,714 / 12,818,126 / 65.4052 / 20.5516 / 9.9754 / 15.2517 / 1,923,948
GSD-08D / 18,148,034 / 4,506,028 / 3,296,927 / 2,619,348 / 10,422,303 / 57.4294 / 32.6001 / 18.1669 / 31.6334 / 3,256,934
GSD-09C / 17,801,082 / 6,447,696 / 1,701,323 / 2,136,342 / 10,285,361 / 57.7794 / 21.5586 / 9.5574 / 16.5412 / 1,660,081
GSD-09D / 18,141,355 / 7,848,340 / 2,565,488 / 2,376,134 / 12,789,962 / 70.5017 / 27.2395 / 14.1417 / 20.0586 / 2,525,500
GSD-10C / 17,516,645 / 5,501,100 / 2,324,005 / 2,482,822 / 10,307,927 / 58.8465 / 27.4415 / 13.2674 / 22.5458 / 2,282,072
GSD-10D / 16,230,248 / 7,841,419 / 1,723,215 / 2,101,821 / 11,666,455 / 71.8809 / 23.5673 / 10.6173 / 14.7707 / 1,691,559
GSD-36C / 13,834,273 / 4,644,660 / 1,529,431 / 1,983,261 / 8,157,352 / 58.9648 / 25.3912 / 11.0554 / 18.7491 / 1,494,556
GSD-36D / 20,432,895 / 6,465,002 / 3,436,053 / 2,111,262 / 12,012,317 / 58.7891 / 27.1489 / 16.8163 / 28.6044 / 3,420,583
GSD-37C / 10,309,672 / 3,893,838 / 1,107,739 / 919,426 / 5,921,003 / 57.4315 / 19.6627 / 10.7447 / 18.7086 / 1,102,219
GSD-37D / 22,319,530 / 6,370,578 / 4,419,550 / 2,298,435 / 13,088,563 / 58.6418 / 30.0991 / 19.8013 / 33.7665 / 4,405,560
Sum / 242,157,325 / 87,597,010 / 31,992,188 / 29,371,773 / 148,960,971 / 860.6154 / 348.9062 / 179.9051 / 297.0953 / 31,579,320.00
Average / 17,296,952 / 6,256,929 / 2,285,156 / 2,097,984 / 10,640,069 / 61.4725 / 24.9219 / 12.8504 / 21.2211 / 2,255,665.71
SD / 2,941,121 / 1,521,278 / 941,138 / 519,481 / 1,985,624 / 5.2533 / 4.9484 / 3.5877 / 6.7057 / 940,509.05

aThe samples include seven GSD Ia HCA and seven paired non-neoplastic liver tissues. C denotes tumor tissue and D denotes corresponding non-neoplastic tissue.

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Supplementary Table S3. List of the miRNAs with differential expression in GSD Ia HCA.

miRNA ID / P-value / t / Mean (adenoma) / Mean (normal) / MeanRatio (adenoma/normal) / FoldChange (adenoma/normal)
hsa-miR-101 / 0.000932 / -6.97488 / 3.75726 / 4.20432 / 0.357228 / -2.79934
hsa-miR-1180 / 0.033686 / 2.90268 / 1.58495 / 1.26943 / 2.06784 / 2.06784
hsa-miR-122* / 0.004302 / -4.94549 / 4.01076 / 4.35545 / 0.452178 / -2.21152
hsa-miR-130b / 0.007454 / 4.33646 / 2.87203 / 2.52023 / 2.24803 / 2.24803
hsa-miR-135b / 0.005942 / -4.58095 / 0.515721 / 1.28742 / 0.169161 / -5.91153
hsa-miR-138 / 0.002704 / -5.50528 / 0.876018 / 1.79696 / 0.119965 / -8.33574
hsa-miR-141 / 0.009537 / -4.08029 / 1.28151 / 1.63145 / 0.446742 / -2.23843
hsa-miR-182 / 0.039054 / 2.77671 / 2.19423 / 1.64682 / 3.52704 / 3.52704
hsa-miR-187 / 0.040128 / -2.75383 / 0.961991 / 1.62543 / 0.217049 / -4.60726
hsa-miR-199a-5p / 0.018731 / -3.4253 / 3.31611 / 3.74053 / 0.376337 / -2.6572
hsa-miR-199b-5p / 0.005084 / -4.75444 / 1.97834 / 2.56562 / 0.258655 / -3.86615
hsa-miR-19a / 0.003404 / -5.22207 / 3.1164 / 3.59124 / 0.335089 / -2.98429
hsa-miR-19b / 0.000962 / -6.92724 / 4.05959 / 4.41548 / 0.440669 / -2.26928
hsa-miR-200a / 0.026966 / -3.09629 / 2.10595 / 2.59112 / 0.327214 / -3.05611
hsa-miR-200b / 0.00952 / -4.0821 / 1.92885 / 2.40031 / 0.337709 / -2.96113
hsa-miR-21 / 0.00614 / 4.54516 / 5.00169 / 4.55414 / 2.80252 / 2.80252
hsa-miR-214 / 0.024798 / -3.17061 / 2.99743 / 3.33144 / 0.463437 / -2.15779
hsa-miR-219-5p / 0.004712 / -4.84064 / 0.297256 / 0.887868 / 0.256678 / -3.89594
hsa-miR-224 / 0.001749 / 6.07292 / 3.23918 / 2.22319 / 10.375 / 10.375
hsa-miR-224* / 0.007315 / 4.35636 / 2.42457 / 1.57013 / 7.15225 / 7.15225
hsa-miR-26a-2* / 0.0417 / -2.72151 / 0.676917 / 1.03354 / 0.439919 / -2.27315
hsa-miR-296-5p / 6.44E-05 / 12.2405 / 1.91232 / 1.39236 / 3.31099 / 3.31099
hsa-miR-32 / 0.010731 / -3.96112 / 1.93754 / 2.25211 / 0.484656 / -2.06332
hsa-miR-335* / 0.000174 / -9.96791 / 1.06097 / 1.45672 / 0.402027 / -2.4874
hsa-miR-33b* / 0.012854 / -3.78271 / 0.62113 / 1.08529 / 0.343433 / -2.91178
hsa-miR-34a / 0.009044 / 4.13465 / 3.59867 / 3.16439 / 2.71822 / 2.71822
hsa-miR-34a* / 0.003541 / 5.17468 / 2.50057 / 2.05844 / 2.76776 / 2.76776
hsa-miR-378 / 0.015534 / -3.6006 / 3.53967 / 3.86732 / 0.470274 / -2.12642
hsa-miR-421 / 0.005111 / 4.74867 / 2.14951 / 1.84056 / 2.03682 / 2.03682
hsa-miR-422a / 0.002748 / -5.48505 / 0.569424 / 0.973536 / 0.394356 / -2.53578
hsa-miR-424 / 0.029231 / -3.02553 / 3.03108 / 3.50789 / 0.333573 / -2.99785
hsa-miR-450a / 0.019408 / -3.39252 / 1.86796 / 2.26102 / 0.404523 / -2.47205
hsa-miR-452 / 0.005121 / 4.74642 / 2.47197 / 1.50287 / 9.31315 / 9.31315
hsa-miR-452* / 0.004566 / 4.87678 / 1.86538 / 1.02926 / 6.85672 / 6.85672
hsa-miR-455-5p / 0.012716 / -3.79329 / 2.2282 / 2.71766 / 0.323998 / -3.08644
hsa-miR-489 / 0.03277 / 2.92638 / 1.27541 / 0.896796 / 2.39117 / 2.39117
hsa-miR-499-5p / 0.001069 / -6.76981 / 0.564266 / 1.1557 / 0.256191 / -3.90333
hsa-miR-532-3p / 0.019283 / 3.39849 / 2.20266 / 1.61823 / 3.84092 / 3.84092
hsa-miR-545 / 0.017235 / -3.50269 / 1.03308 / 1.40266 / 0.427001 / -2.34192
hsa-miR-548b-3p / 0.028003 / -3.0631 / 0.342756 / 1.02101 / 0.209771 / -4.7671
hsa-miR-548h / 0.047042 / -2.62098 / 0.335857 / 0.717479 / 0.415315 / -2.40781
hsa-miR-551b / 0.000227 / 9.42221 / 2.53263 / 1.02673 / 32.0558 / 32.0558
hsa-miR-570 / 0.02968 / -3.01222 / 0.418909 / 0.780393 / 0.435026 / -2.29871
hsa-miR-582-3p / 0.011031 / -3.93358 / 0.828996 / 1.2426 / 0.385834 / -2.59179
hsa-miR-582-5p / 0.009461 / -4.08843 / 1.72373 / 2.16029 / 0.365967 / -2.73249
hsa-miR-599 / 0.0106 / -3.9734 / 1.34424 / 1.65933 / 0.484067 / -2.06583
hsa-miR-652 / 0.012159 / 3.83713 / 1.92513 / 1.42702 / 3.14853 / 3.14853
hsa-miR-671-3p / 0.009067 / 4.13208 / 1.47214 / 1.14562 / 2.12087 / 2.12087
hsa-miR-708 / 0.01542 / -3.60756 / 0.735398 / 1.05628 / 0.477655 / -2.09356
hsa-miR-891b / 0.041285 / -2.7299 / 0.471713 / 0.935944 / 0.343375 / -2.91227
hsa-miR-95 / 0.016547 / 3.54089 / 2.52782 / 1.86863 / 4.56236 / 4.56236
hsa-miR-99a* / 0.002794 / -5.46433 / 2.1733 / 2.52548 / 0.444449 / -2.24998

The mean expression level of miRNAs was presented on the basis of Log10 transformed counts per million reads (Log10CPMR).

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Supplementary Table S4. Statistic difference of selected miRNA expression between GSD Ia HCA and general population HCA.

MiRNA / P-value
hsa-miR-21 / 1.75E-04
hsa-miR-32 / 0.536
hsa-miR-34a / 0.2523
hsa-miR-34a* / 0.2105
hsa-miR-95 / 0.0079
hsa-miR-130b / 0.0079
hsa-miR-199a-5p / 0.6806
hsa-miR-199b-5p / 0.351
hsa-miR-214 / 0.1142
hsa-miR-224 / 0.536
hsa-miR-224* / 0.6065
hsa-miR-424 / 0.536
hsa-miR-450a / 0.0115
hsa-miR-452 / 0.0712
hsa-miR-455-5p / 1
hsa-miR-551b / 0.0115
hsa-miR-582-5p / 0.351

Supplementary Table S5. Statistic difference of selected miRNA expression between GSD Ia HCA and HCC cell lines.

MiRNA / P-value
hsa-miR-21 / 0.536
hsa-miR-32 / 0.0052
hsa-miR-34a / 0.2991
hsa-miR-34a* / 0.9182
hsa-miR-95 / 0.0311
hsa-miR-130b / 1.75E-04
hsa-miR-199a-5p / 0.0164
hsa-miR-199b-5p / 0.0418
hsa-miR-214 / 0.0164
hsa-miR-224 / 0.4698
hsa-miR-224* / 0.0907
hsa-miR-424 / 0.2991
hsa-miR-450a / 1
hsa-miR-452 / 0.6806
hsa-miR-455-5p / 1
hsa-miR-551b / 0.1142
hsa-miR-582-5p / 0.0079

Supplementary Figure S1.Evaluation of the consistency of expression levels of three internal controls across samples. Ten paired samples were included for the evaluation, andC denotes tumor tissue and D denotes corresponding non-neoplastic tissue.10 ng of total RNA per sample was subjected to qRT-PCR for RNU6B, miR-140-3p and let-7f, respectively.Ct values were determined with automatic setting in analysis software SDS 2.3.

Supplementary Figure S2.Validation of miRNAs that showed differential expression in GSD Ia HCA.Relative expression level of seventeen miRNAs that showed differential expression in GSD Ia HCA by deep sequencing weredeterminedin ten paired samples using qRT-PCR.Relative expression levelsof miRNAs in tumor tissues compared to paired non-neoplastic liver tissues are presented as –ddCt with miR-140-3p as an internal control. (A)Nine miRNAs are validated to be overexpressed in tumor tissues. (B) Eight miRNAs are validated to be underexpressed in tumor tissues.

A.

B.

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