Enterocloster bolteae drives hyperglycemia-associated colorectal cancer cell proliferation via Rac/Rho-MSH6-Elongin C signaling pathway

Table 1 Clinical data, n (%)/mean ± SD

Characteristic	DM group (n = 8)	DM-CRC group (n = 11)	DM vs DM-CRC, P value	CRC group (n = 10)	Control group (n = 11)	P value
Age (years)	55.7 ± 10.4	56.3 ± 11.5	0.7812	59.3 ± 11.8	53.5 ± 10.6	0.151
Male gender	4 (50)	5 (45.5)	0.9175	6 (60)	6 (54.5)	0.053
Smoking	2 (25)	3 (27.3)	0.8550	3 (30)	4 (36.4)	0.051
Alcohol drinking	2 (25)	3 (27.3)	0.9175	3 (30)	3 (27.3)	0.193
Family history of CRC	2 (25)	2 (18.2)	0.9175	2 (20)	2 (18.2)	0.524
Family history of colorectal polyps	3 (37.5)	2 (27.3)	0.6580	4 (40)	2 (18.2)	0.030
Regular antidiabetic drugs use	7 (87.5)	7 (63.6)	0.2681	0	0	< 0.001
BMI (kg/m2)	24.4 ± 3.9	22.2 ± 3.6	0.2498	22.5 ± 3.7	23.5 ± 3.5	0.025
FPG (mmol/L)	9.8 ± 1.2	10.1 ± 1.3	0.7211	4.1 ± 1.2	5.3 ± 1.1	< 0.001
TG (mmol/L)	3.0 ± 0.9	1.5 ± 0.9	0.0036	1.8 ± 0.9	2.1 ± 0.9	0.030
TCH (mmol/L)	5.1 ± 1.3	4.7 ± 1.3	0.4478	4.8 ± 1.3	4.9 ± 1.3	0.627
LDL (mmol/L)	3.01 ± 0.9	3.01 ± 1.0	0.6217	3.01 ± 1.0	3.01 ± 1.0	0.766
HDL (mmol/L)	0.9 ± 0.4	0.8 ± 0.3	0.6307	1.0 ± 0.3	2.0 ± 0.3	< 0.001

DM: Diabetes mellitus; CRC: Colorectal cancer; BMI: Body mass index; FPG: Fasting plasma glucose; TG: Triglyceride; TCH: Total cholesterol; LDL: Low-density lipoprotein cholesterol; HDL: High-density lipoprotein cholesterol.

Table 2 Primer sequences used in this study, sourced from Primer Bank

Name		Sequence
Bacteria	7F1540R	5’-CAGAGTTTGATCCTGGCT-3’
	16S rDNA	5’-AGGAGGTGATCCAGCCGCA-3’
Bacteria	27F1492R	5’-AGTTTGATCMTGGCTCAG-3’
	16S rDNA	5’-GGTTACCTTGTTACGACTT-3’
Fungus	ITS1	5’-TCCGTAGGTGAACCTGCGG-3’
	ITS4	5’-TCCTCCGCTTATTGATATGC-3’
Fungus	NS1	5’-GTAGTCATATGCTTGTCTC-3’
	NS6	5’-GCATCACAGACCTGTTATTGCCTC-3’
PCNA		Forward: 5’-CCTGCTGGGATATTAGCTCCA-3’
		Reverse: 5’-CAGCGGTAGGTGTCGAAGC-3’
Rac		Forward: 5’-AAGGAGCCCCACGAGAAAAAT-3’
		Reverse: 5’-ACCGAACTTGCATTGATTCCAG-3’
Rho		Forward: 5’-CTCGCTCAGTGCGAAGACAA-3’
		Downstream: 5’-CATTCTCTGACGACATTTTCCCT-3’
Elongin C		Forward: 5’-AACCCGGACCCTAAGAAGCTA-3’
		Reverse: 5’-TCTCCGCAAGAATGTCTACTGTA-3’
MSH6		Forward: 5’-CCAAGGCGAAGAACCTCAAC-3’
		Reverse: 5’-ACCAGGGGTAACCCTCCATC-3’
TRK		Forward: 5’-ACCCCGATTCCCCTAATAGAGG-3’
		Reverse: 5’-ACCAGAACTCGGTAATCTGACT-3’
GAPDH		Forward: 5’-TCGTGGATCTGACGTGCCGCCTG-3’
		Reverse: 5’-CACCACCCTGTTGCTGTAGCCGTAT-3’

PCNA: Proliferating cell nuclear antigen; MSH6: Melanocyte-stulating hormone-6; TRK: Tropomyosin receptor kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Table 3 siRNA primer sequences sourced from Primer Bank

Name	Sequence
Rac-si	Forward: 5’-GCUACUUCCUCCUCAAGAAdTdT-3’
	Reverse: 5’-CAGCGGTAGGTGTCGAAGC-3’
Rho-si	Forward: 5’-CCUACAUGUUUCUGCUGAUTT-3’
	Reverse: 5’-AUCAGCAGAAACAUGUAGGTT-3’
hMSH6-si	Forward: 5’-GGCCUUGUCUGGUUUACAATT-3’
	Reverse: 5’-UUGUAAACCAGACAAGGCCTT-3’
Elongin C-si	Forward: 5’-GCACCUGAAAUUGCACUGGAAdTdT-3’
	Reverse: 5’-UUCCAGUGCAAUUUCAGGUGCdTdT-3’
GAPDH	Forward: 5’-TCGTGGATCTGACGTGCCGCCTG-3’
	Reverse: 5’-CACCACCCTGTTGCTGTAGCCGTAT-3’

hMSH6: Melanocyte-stulating hormone-6; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Table 4 Spearman correlation analysis between the diabetes mellitus-colorectal cancer group and the diabetes mellitus group

Variable	r	95%CI	P value
Age	0.06827	-0.4104 to 0.5175	0.7812
Male	0.02548	-0.4454 to 0.4854	0.9175
Smoking	-0.04495	-0.5001 to 0.4297	0.8550
Drinking	0.02548	-0.4454 to 0.4854	0.9175
CRC history	0.02548	-0.4454 to 0.4854	0.9175
Hypertension	-0.2955	-0.6690 to 0.1973	0.2194
C polyps history	-0.1086	-0.5466 to 0.3760	0.6580
Antidiabetic drugs	-0.2676	-0.6520 to 0.2263	0.2681
BMI	-0.2776	-0.6582 to 0.2159	0.2498
FPG	0.08770	-0.3940 to 0.5316	0.7211
TG	-0.6334	-0.8487 to -0.2379	0.0036
TCH	-0.1852	-0.5992 to 0.3069	0.4478
HDL	-0.1179	-0.5532 to 0.3679	0.6307
LDL	-0.1951	-0.6057 to 0.2976	0.4236
Abundance of E. bolteae	0.8563	0.6498-0.9451	< 0.0001

CI: Confidence interval; CRC: Colorectal cancer; BMI: Body mass index; FPG: Fasting plasma glucose; TG: Triglyceride; TCH: Total cholesterol; HDL: High-density lipoprotein cholesterol; LDL: Low-density lipoprotein cholesterol.

Table 5 Spearman correlation analysis between the diabetes mellitus group and the diabetes mellitus-colorectal cancer group after stratification based on median triglyceride level

Characteristic		r	95%CI	P value
TG ≥ 2.6 mmol/L	TG	-0.3242	-0.8132 to 0.4332	0.3946
	Abundance of Bolteae	0.9376	0.7248-0.9871	0.0002
TG < 2.6 mmol/L	TG	-0.2871	-0.7764 to 0.4181	0.4213
	Abundance of Bolteae	0.9133	0.6676-0.9796	0.0002

TG: Triglyceride; CI: Confidence interval.