Involvement of methylation-associated silencing of formin 2 in colorectal carcinogenesis

Table 1 Clinical materials used in this article

No.	Sex	Age	Lymph nodes	TNM	Pathology	Differentiation	MSI	Organ
1	M	67	5 (14)	T4aN2aM0	Adenocarcinoma	Moderate	No	Rectum
2	M	53	0 (22)	T2N0M0	Adenocarcinoma	Moderate	-	Rectum
3	M	72	4 (20)	T4aN2aM0	Adenocarcinoma	Poor	-	Right colon
4	M	52	0 (16)	T3N0M0	Adenocarcinoma	Moderate	-	Rectum
5	M	52	3 (25)	T3N1bM0	Adenocarcinoma	Moderate	-	Sigmoid colon
6	F	57	0 (15)	T4aN0M0	Adenocarcinoma	Moderate	-	Left colon
7	F	51	0 (14)	T4aN0M0	Adenocarcinoma	Moderate	No	Rectum
8	F	66	19 (27)	T3N2bM0	Adenocarcinoma	Poor	No	Rectum
9	F	71	0 (16)	T4bN0M1b	Adenocarcinoma	Moderate	No	Rectum

Table 2 Details of the four CpG regions in the CpG islands of formin 2

Target	TSS	Start	End	Length	Target strand	Distance2TSS
FMN2_1	240255184	240255353	240255531	179	+	169
FMN2_2	240255184	240255809	240256007	199	+	625
FMN2_3	240255184	240256241	240256483	243	+	1057
FMN2_4	240255184	240256757	240257017	261	+	1573

Start: The starting position of the product on the reference genome; TSS: The mRNA transcription initiation site; End: The end position of the product on the reference genome; Length: the product length; Target strand: The product orientation; Distance2TSS: The distance from the product to the TSS.

Table 3 Primers used for the MethylTarget™ assays

Primer name	Primer
FMN2_1_F	GAGGGTYGGGATGGTTTGAG
FMN2_1_R	CCCCCRCTCCCCTTCTTT
FMN2_2F	GAGTGTGYGGATTTTTTTGAGGT
FMN2_2R	AAATATCTAAAAACAAATCCTCTTACTCC
FMN2_3F	GATTTGTTYGAGAGTTTGGTTGT
FMN2_3R	AAACRCATCCTCAAAAACATCCT
FMN2_4F	TTTTTGAGTYGAGGGTTTAGAATTG
FMN2_4R	CACRTTCTAAAAACCATCCRCAAC

Table 4 Methylation level of each CG site in the CpG island regions

Target	Position	Type	P value (t-test)	OR (L95-U95) (logistic)	C mean	N mean
FMN2_1	25	CG	0.007774578	2.1731 (0.9323-5.0653)	0.17	0.04
FMN2_1	27	CG	0.01061292	1.5266 (0.8980-2.5953)	0.16	0.03
FMN2_1	30	CG	0.1359111	1.3535 (0.7970-2.2986)	0.15	0.03
FMN2_1	32	CG	0.01875771	1.7416 (0.8662-3.5016)	0.16	0.04
FMN2_1	35	CG	0.03998355	1.2722 (0.8959-1.8068)	0.18	0.05
FMN2_1	38	CG	0.01419169	1.6993 (0.8462-3.4125)	0.16	0.03
FMN2_1	46	CG	0.003990128	2.2454 (0.9140-5.5161)	0.15	0.02
FMN2_1	90	CG	0.1134924	1.3103 (0.6401-2.6825)	0.16	0.03
FMN2_1	108	CG	0.06252571	1.2532 (0.7873-1.9948)	0.17	0.04
FMN2_1	114	CG	0.1134924	1.1858 (0.8641-1.6273)	0.18	0.05
FMN2_1	117	CG	0.05030852	1.3499 (0.8867-2.0550)	0.19	0.04
FMN2_1	123	CG	0.01061292	2.2247 (0.9162-5.4017)	0.16	0.03
FMN2_1	126	CG	0.09391197	1.2852 (0.7471-2.2110)	0.16	0.03
FMN2_1	134	CG	0.01875771	2.4645 (0.6195-9.8050)	0.16	0.02
FMN2_2	27	CG	0.000493624	1.3442 (1.0433-1.7321)	0.28	0.07
FMN2_2	51	CG	0.01419169	1.2762 (0.9670-1.6843)	0.17	0.04
FMN2_2	63	CG	0.007774578	1.4062 (0.9872-2.0030)	0.37	0.25
FMN2_2	70	CG	0.000781571	1.3753 (1.0378-1.8226)	0.24	0.05
FMN2_2	74	CG	0.000493624	1.3079 (1.0283-1.6636)	0.31	0.07
FMN2_2	78	CG	0.000287947	1.3676 (1.0603-1.7639)	0.25	0.05
FMN2_2	81	CG	0.00185109	1.3630 (0.9664-1.9224)	0.21	0.05
FMN2_2	107	CG	0.000493624	1.3025 (0.9942-1.7063)	0.24	0.06
FMN2_2	131	CG	0.007774578	1.2706 (0.9662-1.6708)	0.15	0.03
FMN2_2	137	CG	0.002756067	1.3002 (0.9986-1.6928)	0.21	0.05
FMN2_2	143	CG	0.00123406	1.3743 (1.0297-1.8341)	0.20	0.04
FMN2_2	164	CG	0.000287947	1.3527 (1.0108-1.8101)	0.29	0.07
FMN2_2	170	CG	0.00123406	1.2431 (1.0019-1.5424)	0.28	0.08
FMN2_3	24	CG	0.005635541	1.3854 (0.9994-1.9207)	0.20	0.05
FMN2_3	31	CG	0.005635541	1.4241 (0.9731-2.0841)	0.23	0.04
FMN2_3	47	CG	0.002756067	1.3776 (0.9695-1.9574)	0.24	0.06
FMN2_3	50	CG	0.003990128	1.3787 (1.0219-1.8599)	0.22	0.05
FMN2_3	57	CG	0.00123406	1.3592 (1.0212-1.8091)	0.25	0.06
FMN2_3	60	CG	0.003990128	1.5630 (0.9433-2.5897)	0.19	0.04
FMN2_3	68	CG	0.00185109	1.3474 (1.0273-1.7673)	0.23	0.06
FMN2_3	75	CG	0.00123406	1.3787 (0.9962-1.9082)	0.26	0.06
FMN2_3	80	CG	0.00123406	1.3582 (0.9518-1.9380)	0.24	0.05
FMN2_3	92	CG	0.00185109	1.3665 (0.9746-1.9159)	0.23	0.05
FMN2_3	98	CG	0.00123406	1.3200 (0.9414-1.8509)	0.27	0.06
FMN2_3	105	CG	0.000781571	1.4801 (0.9296-2.3566)	0.22	0.04
FMN2_3	107	CG	0.007774578	1.2402 (1.0141-1.5168)	0.21	0.05
FMN2_3	119	CG	0.00123406	1.4326 (0.9810-2.0921)	0.19	0.04
FMN2_3	131	CG	0.000493624	1.7790 (0.9589-3.3006)	0.22	0.04
FMN2_3	147	CG	0.007774578	1.3343 (0.9443-1.8856)	0.20	0.04
FMN2_3	153	CG	0.000493624	1.5405 (0.9677-2.4523)	0.22	0.05
FMN2_3	158	CG	8.23E-05	1.9417 (0.8981-4.1979)	0.31	0.08
FMN2_3	170	CG	4.11E-05	1714240.0000 (0.0000-Inf)	0.32	0.08
FMN2_3	174	CG	4.11E-05	11577516733.0000 (0.0000-Inf)	0.34	0.08
FMN2_3	183	CG	0.000287947	1.6836 (0.9224-3.0731)	0.28	0.06
FMN2_3	187	CG	0.000287947	1.4526 (0.9861-2.1399)	0.27	0.06
FMN2_3	203	CG	0.000781571	1.4980 (0.9815-2.2861)	0.25	0.05
FMN2_4	36	CG	0.003990128	1.1409 (1.0248-1.2702)	0.41	0.14
FMN2_4	42	CG	0.002756067	1.2068 (1.0353-1.4068)	0.38	0.12
FMN2_4	47	CG	0.000781571	1.2264 (1.0309-1.4589)	0.37	0.10
FMN2_4	61	CG	0.005386639	1.1552 (1.0206-1.3075)	0.38	0.10
FMN2_4	66	CG	0.0003108	243.9222 (0.0000-Inf)	0.40	0.09
FMN2_4	69	CG	8.23E-05	1.7294 (0.6520-4.5874)	0.42	0.09
FMN2_4	72	CG	0.003990128	1.2209 (1.0296-1.4477)	0.4	0.13
FMN2_4	80	CG	4.11E-05	2515571.0000 (0.0000-Inf)	0.48	0.18
FMN2_4	84	CG	0.00185109	1.2366 (1.0241-1.4931)	0.39	0.12
FMN2_4	94	CG	0.002756067	1.2121 (1.0363-1.4178)	0.38	0.12
FMN2_4	98	CG	0.003990128	1.1618 (1.0236-1.3186)	0.36	0.11
FMN2_4	110	CG	0.00185109	1.1712 (1.0253-1.3378)	0.44	0.19
FMN2_4	123	CG	0.000781571	1.2218 (1.0321-1.4463)	0.40	0.10
FMN2_4	127	CG	0.00185109	1.2220 (1.0324-1.4464)	0.39	0.11
FMN2_4	131	CG	8.23E-05	1.3179 (1.0259-1.6931)	0.38	0.08
FMN2_4	136	CG	0.003990128	1.1979 (1.0209-1.4054)	0.34	0.09
FMN2_4	150	CG	4.11E-05	1217702.0000 (0.0000-Inf)	0.54	0.22
FMN2_4	152	CG	0.003990128	1.1644 (1.0196-1.3297)	0.39	0.12
FMN2_4	166	CG	0.000164541	1.3446 (0.9896-1.8269)	0.45	0.16
FMN2_4	168	CG	0.001710676	1.2557 (1.0089-1.5628)	0.44	0.16
FMN2_4	176	CG	4.11E-05	26059.6300 (0.0000-Inf)	0.52	0.21
FMN2_4	186	CG	0.000781571	1.1735 (1.0224-1.3469)	0.54	0.2
FMN2_4	189	CG	0.003990128	1.1348 (1.0221-1.2600)	0.49	0.18
FMN2_4	213	CG	0.00185109	1.1572 (1.0241-1.3076)	0.47	0.14
FMN2_4	221	CG	0.002756067	1.1534 (1.0276-1.2947)	0.45	0.13
FMN2_4	234	CG	0.00185109	1.1981 (1.0181-1.4099)	0.32	0.08
FMN2_4	236	CG	0.000493624	1.2631 (1.0149-1.5720)	0.36	0.08

Table 5 Relationship between formin 2 methylation levels and clinicopathological data

Factor	No.	FMN2		P value
		Low, n (%)	High, n (%)
Age (yr)
< 60	136	66 (45.83)	70 (28.69)	0.00061
≥ 60	252	78 (54.17)	174 (71.31)
Gender
Male	210	72 (50.00)	138 (56.56)	0.2105
Female	178	72 (50.00)	106 (43.44)
Height (cm)
< 170	138	52 (48.60)	86 (47.25)	0.8250
≥ 170	151	55 (51.40)	96 (52.75)
Weight (kg)
< 80	156	56 (49.12)	100 (51.55)	0.6812
≥ 80	152	58 (50.88)	94 (48.45)
T
T1	11	5 (3.47)	6 (2.47)	0.4830
T2	54	16 (11.11)	38 (15.71)
T3	270	106 (73.61)	164 (67.77)
T4	51	17 (11.81)	34 (14.05)
N
N0	212	69 (47.92)	143 (59.34)	0.02931
N1 + N2	173	75 (52.08)	98 (40.66)
M
M0	264	98 (80.33)	166 (85.13)	0.2651
M1	53	24 (19.67)	29 (14.87)
Stage
I + II	197	62 (45.26)	135 (58.19)	0.01611
III + IV	172	75 (54.74)	97 (41.81)
Lymphovascular invasion
Yes	231	77 (60.63)	154 (72.99)	0.01801
No	107	50 (39.37)	57 (27.01)
Vascular invasion
Yes	78	31 (24.80)	47 (22.71)	0.6627
No	254	94 (75.20)	160 (77.29)
Perineural invasion
Yes	59	20 (24.39)	39 (26.53)	0.7225
No	170	62 (75.61)	108 (73.47)
Tumor status
Tumor-free	246	86 (71.07)	160 (77.29)	0.2094
Tumor-present	82	35 (28.93)	47 (22.71)
KRAS mutation
Yes	28	8 (42.11)	20 (54.05)	0.3972
No	28	11 (57.89)	17 (45.95)

¹Indicates a statistically significant result. TNM stage was defined according to the AJCC Cancer Staging Manual. Tumor status: The state or condition of an individual's neoplasm at a particular point in time.