Characterization of DNA hydroxymethylation profile in cervical cancer

Figures & data

Figure 1. The overall levels of 5mC/5hmC in CSCC. The dot plot (A) and IHC staining (C) with the corresponding statistical analysis (B, D) of 5mC in CSCC. The dot plot (E) and IHC staining (G) with the corresponding statistical analysis (F, H) of 5hmC in CSCC. Experiments were performed in triplicates. “*” represents the comparison between I-IIa group of CSCC and cervicitis with p values less than 0.05. “#” represents the comparison between IIb and IV group of cervicitis and control with p values less than .05.

Figure 2. The genomic distribution of 5mC/5hmC in CSCC. The MA plot of IIb–IV group of CSCC and cervicitis contrast of 5mC (A) and 5hmC (B) peaks normalized with tag density. The X axis indicates the normalized mean and the Y axis indicates the log2-fold change. The red pixels mean the differential peaks with FDR less than 0.05. The blue pixels mean the peaks with no significant difference. The genome-wide mapping of 5mC (C) and 5hmC (D) in cervicitis and different stages of CSCC. Normalized DMRs and DHMRs tag density distribution across the gene body. Each gene body was normalized to 0–100%. Normalized Tag density is plotted from 20% of upstream of transcription start sites (TSS) and 20% of downstream of transcription terminal sites (TTS). -3 kb to TSS and +3 kb to TTS is shown.

Table 1. The summary of MeDIP and hMeDIP-seq data.

Sample	Total raw reads	Total raw bases	Total clean reads	Total clean bases	Mapped reads	Mapped ratio (%)	Unique mapped reads	Unique mappedratio (%)
Cervicitis_1	4658933	3.5E + 08	4527698	3.4E + 08	4267998	94.26	3077184	67.96
Cervicitis_2	4866549	3.7E + 08	4729759	3.6E + 08	4457715	94.25	3215524	67.98
I–IIa_1	4789317	3.6E + 08	4654346	3.5E + 08	4387511	94.27	3163059	67.96
5hmC
I–IIa_2	4977856	3.8E + 08	4837617	3.7E + 08	4559156	94.24	3285198	67.91
IIb–IV_1	5107214	3.9E + 08	4963388	3.8E + 08	4679174	94.27	3374284	67.98
IIb–IV_2	5166223	3.9E + 08	5020926	3.8E + 08	4733070	94.27	3413340	67.98
Cervicitis_1	4601442	3.5E + 08	4590342	3.5E + 08	4483939	97.68	2973858	64.79
Cervicitis_2	4658933	3.5E + 08	4647606	3.5E + 08	4540046	97.69	3011401	64.79
I–IIa_1	4789317	3.6E + 08	4777779	3.6E + 08	4667329	97.69	3097092	64.82
5mC
I–IIa_2	4866549	3.7E + 08	4854865	3.7E + 08	4742431	97.68	3147006	64.82
IIb–IV_1	4877129	3.7E + 08	4865326	3.7E + 08	4752950	97.69	3151706	64.78
IIb–IV_2	4977856	3.8E + 08	4965935	3.7E + 08	4851746	97.70	3218583	64.81
Cervicitis_1	4658933	3.5E + 08	4637594	3.5E + 08	4311095	92.96	2923315	63.04
Cervicitis_2	4977856	3.8E + 08	4955132	3.7E + 08	4604933	92.93	3123040	63.03
I–IIa_1	5021006	3.8E + 08	4998307	3.8E + 08	4646036	92.95	3149488	63.01
Input
I–IIa_2	4789317	3.6E + 08	4767682	3.6E + 08	4432209	92.96	3007027	63.07
IIb–IV_1	4866549	3.7E + 08	4844124	3.6E + 08	4502630	92.95	3052616	63.02
IIb–IV_2	4089748	3.1E + 08	4071043	3.1E + 08	3784021	92.95	2566918	63.05

Figure 3. The associated functions of DMR/DHMRs in CSCC. The count of selected DMRs/DHMRs with the similar or opposite variation trend of 5mC/5hmC compared among cervicitis, I–IIa and IIb–IV of CSCC (A). Upper and down arrow mean the up- and down-regulated 5mC compared among cervicitis, I–IIa and IIb–IV of CSCC. Left and right arrow mean the up- and down-regulated 5hmC compared among cervicitis, I–IIa and IIb–IV of CSCC. “-” means no variation of 5mC/5hmC among these three groups. The distribution of the selected DMRs/DHMRs in genomic regions (B). GO (C) and KEGG (D) analysis of the genes associated with DMRs/DHMRs.

Table 2. The annotation of RefSeq genes associated with DMRs/DHMRs.

chr	Start	End	Width	Annotation	Genechr	GeneStart	Geneend	Genelength	Genestrand	Distanceto TSS	Ensemble	Symbol	Gene name	5hmC（cancer vs.cervici tis）	5mC（cance r vs.cerviciti s）
chr10	1.04E + 08	1.04E + 08	2182	Promoter (< =1kb)	10	1.04E + 08	1.04E + 08	54717	2	0	ENSG000 00065618	COL17A 1	Collagen type XVII alpha 1 chain	No	Down
chr10	1.15E + 08	1.15E + 08	1131	Intron (uc001lcc. 4/57700, intron 16of 16)	10	1.15E + 08	1.15E + 08	3124	1	14472	ENSG000 00151553	FAM160 B1	Family with sequence similarity 160 member B1	Up	Up
chr10	1209185	1209837	652	Intron (uc009xh q.4/105, intron 7 of 9)	10	1183151	1200385	17235	2	−8801	ENSG000 00185736	ADARB2	Adenosine deaminase, RNA specific B2(inactive)	DOWN	DOWN
chr10	1.32E + 08	1.32E + 08	761	Intron (uc001lkx. 5/282973, intron 15 of 23)	10	1.32E + 08	1.32E + 08	41094	1	6889	ENSG000 00188385	JAKMIP 3	Janus kinase and microtubule interacting protein 3	Up	Up
chr10	17226768	17228949	2181	Promoter (< =1kb)	10	17214239	17226898	12660	2	0	ENSG000 00229124	VIM- AS1	VIM antisense RNA 1	No	Up
chr10	41882611	41884809	2198	Distal intergenic	10	42475543	42495336	19794	1	#####	ENSG000 00185904	LINC008 39	Long intergenic non-protein coding RNA 839	Up	Up
chr10	42104070	42104409	339	Distal intergenic	10	42475543	42495336	19794	1	#####	ENSG000 00185904	LINC008 39	Long intergenic non- protein coding RNA 839	Up	Up
chr10	43408670	43410867	2197	Promoter (< =1kb)	10	43385617	43408882	23266	2	0	ENSG000 00169813	HNRNP F	Heterogeneous nuclear ribonucleoprotein F	Up	Down
chr10	79067549	79069735	2186	Promoter (< =1kb)	10	78943328	79067895	124568	2	0	ENSG000 00224596	ZMIZ1- AS1	ZMIZ1 antisense RNA 1	Up	Up
chr11	1.16E + 08	1.16E + 08	2170	Promoter (< =1 kb)	11	1.15E + 08	1.16E + 08	335288	2	0	ENSG000 00182985	CADM1	Cell adhesion molecule 1	Up	Up
chr11	31817033	31820872	3839	Promoter (< =1 kb)	11	31784792	31817961	33170	2	0	ENSG00000007372	PAX6	Paired box 6	Up	Down
chr11	65558439	65560872	2433	Promoter (< =1 kb)	11	65539411	65558930	19520	2	0	ENSG000 00168056	LTBP3	Latent transforming growth factor beta binding protein 3	Up	Down
chr1	2146821	2147888	1067	Promoter (1–2 kb)	1	2145387	2185116	39730	1	1435	ENSG000 00067606	PRKCZ	Protein kinase C zeta	Down	Down
chr12	91719517	91720391	874	Distal intergenic	12	91986626	91993403	6778	2	273012	ENSG000 00257242	LINC016 19	Long intergenic non-protein coding RNA 1619	Up	Up
chr13	77975030	77977208	2178	Intron (uc001vks.3/100874 222, intron 1 of 2)	13	77989372	77990646	1275	2	13438	NA	LINC010 69	long intergenic non-protein coding RNA 1069	Up	Up
chr14	1.01E + 08	1.01E + 08	2518	Promoter (< =1kb)	14	1.01E + 08	1.01E + 08	49776	1	0	ENSG000 00214548	MEG3	Maternally expressed 3 (non-protein coding)	Up	Up
chr1	4653242	4655419	2177	Promoter (< =1 kb)	1	4654732	4777794	123063	1	0	ENSG000 00196581	AJAP1	Adherens junctions associate d protein 1	Up	Up
chr16	4425172	4427411	2239	Promoter (< =1 kb)	16	4425805	4456772	30968	1	0	ENSG000 00103423	DNAJA3	DnaJ heat shock protein family (Hsp40)member A3	Up	Up
chr16	48931526	48932225	699	Distal intergenic	16	48546097	48620148	74052	2	#####	ENSG000 00102921	N4BP1	NEDD4 binding protein 1	Up	Up
chr16	90031782	90032741	959	Promoter (2–3 kb)	16	90029541	90044961	15421	1	2242	ENSG000 00141013	GAS8	Growth arrest specific 8	UP	UP
chr17	181132	183414	2282	Promoter (< =1 kb)	17	142789	181636	38848	2	0	ENSG000 00272636	DOC2B	DoubleC2 domain beta	No	Up
chr17	31390129	31392485	2356	Promoter (< =1 kb)	17	31391624	31538217	146594	1	0	ENSG000 00131242	RAB11FI P4	RAB11 family interacting protein 4	Up	Up
chr17	59876159	59877817	1658	Promoter (< =1kb)	17	59874539	59878762	4224	2	945	ENSG00000108423	TUBD1	tubulindelta 1	Up	Up
chr17	62625907	62632834	6927	Promoter (< =1 kb)	17	62627401	62693597	66197	1	0	ENSG000 00011028	MRC2	Mannose receptor C type 2	Up	Up
chr17	81512564	81514760	2196	Promoter (< =1 kb)	17	81509971	81512874	2904	2	0	ENSG00000184009	ACTG1	actingamma 1	Up	Up
chr17	81523347	81525549	2202	Promoter (< =1 kb)	17	81511346	81523847	12502	2	0	ENSG00000184009	ACTG1	Actin gamma 1	Up	Down
chr17	82092446	82094674	2228	Exon(uc002kd u.5/2194, exon 4 of 43)	17	82078340	82098187	19848	2	3513	ENSG000 00169710	FASN	fatty acid synthase	Up	Up
chr18	78978515	78981707	3192	Promoter (< =1 kb)	18	78980275	78997397	17123	1	0	ENSG000 00256463	SALL3	Spalt like transcripti on factor 3	Up	Up
chr20	21704172	21707170	2998	Promoter (< =1 kb)	20	21705659	21715982	10324	1	0	ENSG00000125813	PAX1	Paired box 1	Up	Up
chr20	57192385	57197334	4949	Intron(uc010gip.2/655, intron 3 of 6)	20	57177696	57187156	9461	2	−5230	ENSG000 00101144	BMP7	Bone morphogenetic protein 7	Up	Up
chr20	57215678	57220266	4588	Promoter (< =1 kb)	20	57214872	57215866	995	1	807	NA	BMP7- AS1	BMP7 antisense RNA 1	Up	Up
chr20	59298942	59301166	2224	Promoter (< =1 kb)	20	59300427	59325992	25566	1	0	ENSG00000124205	EDN3	Endothelin 3	Up	Up
chr20	61491753	61492584	831	Intron(uc032po k.1/1002, intron 2 of 15)	20	61570135	61937218	367084	1	−77551	ENSG000 00179242	CDH4	Cadherin 4	Down	No
chr20	63101496	63102157	661	Promoter (< =1 kb)	20	63102205	63104386	2182	1	−48	NA	HAR1A	Highly accelerated region 1A (non-protein coding)	Up	Up
chr21	10676553	10677170	617	Distal intergenic	21	10521575	10605716	84142	1	154979	ENSG000 00274391	TPTE	Transmembrane phosphatase with tensin homology	Up	Up
chr21	10698147	10699885	1738	Distal intergenic	21	10521575	10605716	84142	1	176573	ENSG000 00274391	TPTE	Transmembrane phosphatase with tensin homology	Up	Down
chr21	10699915	10727771	27856	Distal intergenic	21	10521575	10605716	84142	1	178341	ENSG000 00274391	TPTE	Transmembrane phosphatase with tensin homology	Up	Up
chr2	1.91E + 08	1.91E + 08	2186	Promoter (< =1 kb)	2	1.91E + 08	1.91E + 08	30381	2	0	ENSG000 00189362	NEMP2	Nuclear envelope integral membrane protein 2	Up	Up
chr4	49102367	49104228	1861	Distal intergenic	4	49028692	49031276	2585	1	73676	ENSG000 00109182	CWH43	Cell wall biogenesis 43 C-terminal homolog	Up	Up
chr4	49110432	49111161	729	Distal intergenic	4	49028692	49031276	2585	1	81741	ENSG000 00109182	CWH43	Cell wall biogenesis 43 C-terminal homolog	Up	Down
chr5	1.51E + 08	1.51E + 08	2147	Promoter (< =1 kb)	5	1.51E + 08	1.51E + 08	8555	1	0	ENSG000 00211445	GPX3	Glutathione peroxidase 3	Down	Up
chr6	2971675	2973873	2198	Promoter (< =1 kb)	6	2948159	2971856	23698	2	0	ENSG000 00124570	SERPIN B6	Serpin family B member 6	Up	Up
chr7	38025211	38027380	2169	Promoter (< =1 kb)	7	37909659	38025695	116037	2	0	ENSG000 00106483	SFRP4	Secreted frizzled related protein 4	No	Up
chr7	72446668	72448836	2168	Promoter (< =1 kb)	7	71779491	72447061	667571	2	0	ENSG00000183166	CALN1	Calneuron 1	Down	Up
chr8	1.24E + 08	1.24E + 08	801	Intron(uc003yq y.3/157376, intron 1 of 4)	8	1.24E + 08	1.24E + 08	125450	2	18854	ENSG000 00253868	FER1L6- AS2	FER1L6 antisense RNA 2	No	Up
chr8	1.41E + 08	1.41E + 08	649	Distal intergenic	8	1.41E + 08	1.41E + 08	2297	1	5267	ENSG000 00253595	LINC013 00	Long intergenic non-protein coding RNA 1300	Up	No
chr8	1.42E + 08	1.42E + 08	2264	Intron (uc003yw k.4/203062, intron 13 of 13)	8	1.42E + 08	1.42E + 08	10648	1	23195	ENSG000 00254008	LINC000 51	Long intergenic non-protein coding RNA 51	Up	Up
chr8	41308229	41311186	2957	Promoter (< =1 kb)	8	41261958	41309497	47540	2	0	ENSG000 00104332	SFRP1	Secreted frizzled related protein 1	No	Up
chr8	54456446	54459252	2806	Promoter (< =1 kb)	8	54457935	54460888	2954	1	0	ENSG00000164736	SOX17	SRY-box17	Up	Up
chr9	1.15E + 08	1.15E + 08	2194	Promoter (< =1 kb)	9	1.14E + 08	1.15E + 08	102890	2	0	ENSG00000095397	WHRN	Whirlin	Up	Up
chr9	1.22E + 08	1.22E + 08	593	Distal intergenic	9	1.22E + 08	1.22E + 08	8187	1	46250	ENSG000 00136848	DAB2IP	DAB2 interacting protein	Up	Up
chr9	96619350	96621535	2185	Promoter (< =1 kb)	9	96490241	96619830	129590	2	0	ENSG000 00081377	CDC14B	Cell division cycle 14B	Up	Up
chrY	10671476	10674208	2732	Distal intergenic	24	9910798	9911962	1165	1	760679	ENSG000 00239225	TTTY23	Testis-specific transcript, Y-linked 23 (non-protein coding)	Up	Up

Figure 4. Vector picture for clarification of the sensitivity of variation trend of 5mC/5hmC. The area of A and C mean the associated genes with 5hmC sensitive change while area B and D mean the associated genes with 5mC sensitive change in the malignancy of CSCC. The X/Y axis indicates the normalized log2-fold change of 5mC/5hmC.