Association of expression of inflammatory response genes and DNA repair genes in colorectal carcinoma

Abstract

Inflammation is an important etiological factor of colorectal carcinoma and may be related to colorectal carcinoma growth and proliferation. This study aimed to verify whether the presence of chronic inflammation represented by tumor necrosis factor-α, interleukin-2, interleukin-6, and interleukin-10 gene expression is related to hMLH1, hMSH2, hMSH6, and PMS2 gene expression and the corresponding protein levels of these genes from the DNA repair system. A total of 83 patients were operated on for curative or palliative colorectal carcinoma. Expression of the inflammatory response genes tumor necrosis factor-α, interleukin-2, interleukin-6, and interleukin-10 as well as expression of the hMLH1, hMSH2, hMSH6, and PMS2 genes of the DNA repair system (mismatch repair) and the expression levels of the corresponding mismatch repair proteins were measured in neoplastic tissue by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Associations were observed between hMSH6 mRNA expression and interleukin-2 mRNA expression (p = 0.026) as well as between hMLH1 and hMSH2 gene expression and tumor necrosis factor-α gene expression (p = 0.042). Higher tissue levels of interleukin-2 and tumor necrosis factor-α gene expression were associated with lower hMSH6, hMLH1, and hMSH2 gene expression.

Keywords

Colorectal neoplasms inflammation tumor necrosis factor alpha interleukin DNA mismatch repair

Introduction

Colorectal carcinoma (CRC) is the third most frequent malignant neoplasm worldwide, affecting an estimated 1.4 million people annually and accounting for 700,000 deaths per year.^1,2

The chronic inflammation that occurs in obesity and inflammatory bowel diseases³ is considered an important factor in the development of intestinal neoplasia, which in turn may be directly related to the duration and intensity of the chronic inflammatory state.^4,5 A previous study showed that inflammation alters immunity and normal cell proliferation, which can result in the loss of cellular growth control capacity, favoring genomic destabilization and consequent development of neoplasms.⁶

Malignant neoplasms, such as colorectal adenocarcinoma, arise in association with chronic inflammation, which induces irreversible and persistent somatic changes in DNA in normal colonic tissue.^2,4,7 This state remains until a second stimulation occurs after colon cells are exposed to chronic inflammation, which then recruits inflammatory cells, induces cell proliferation, and produces reactive oxygen species.

These events lead to oxidative DNA damage by transcription of the dimeric transcription factor, NF-kB, outside of the cell cytoplasm, and when this inflammatory pathway is activated, DNA recognition and repair mechanisms become damaged, thus promoting the development of neoplasia.^7–12

At the onset of inflammatory responses, macrophages and T lymphocytes produce tumor necrosis factor alpha (TNF-α), a cytokine related to proliferation, differentiation, and tumor angiogenesis.^13,14 TNF-α stimulates the proliferation of macrophages that favor chronic inflammation. Macrophages in the presence of neoplasms may facilitate breaking down the extracellular matrix, increase tumor progression and invasion in the surrounding tissues, and cause metastases to form.¹⁵

T lymphocytes in chronic inflammation also release macrophage migration inhibitory factor (MIF), a cytokine that concentrates macrophages in inflammatory sites and suppresses the transcription of p53 protein, causing the loss of the regulatory function of this protein in tissues with inflammation.¹⁶ Suppression of p53 protein activity creates a deficiency in DNA repair mechanisms, increasing the number of potential oncogenic mutations. In addition, p53 suppression retards apoptosis of damaged cells, which favors the onset and spread of neoplasias, such as CRC.^17–19

Several of these cytokines, especially TNF-α and interleukin (IL)-6, have been identified as tumor growth factors and influence the survival of patients. These substances act on premalignant cells, stimulate angiogenesis and favor inflammation, a situation that can promote carcinogenic conditions.¹⁴ TNF-α and IL-6 stimulate macrophage proliferation and differentiation, and these factors facilitate tissue invasion by neoplastic cells in the presence of neoplasias.^20,21

The level of IL-6 produced by endothelial cells, fibroblasts, macrophages, and monocytes²² is related to the aggressiveness of the neoplasia and the prognosis of the patient. In general, the elevated level of IL-6 in serum samples and/or its expression in CRC tissue correlates with unfavorable prognosis.²³

T lymphocytes and inflammatory cells, such as natural killer (NK) and dendritic cells, secrete IL-2, a cytokine that regulates and differentiates T lymphocyte subtypes, and their production is regulated by another cytokine, IL-10. Decreased serum IL-2 levels may be related to the progression of the neoplasm or to the poorer prognosis of the cancer patient.²⁴ IL-10 is produced by B lymphocytes, T lymphocytes, monocytes, and macrophages, and it modulates the inflammatory process. Decreased levels of IL-10 may enhance the immune response to antigens, such as viruses or chemical agents, and cause tissue damage, such as in chronic enterocolitis.^25–27

In addition to correcting DNA replication errors, the DNA repair system (mismatch repair (MMR)) genes, hMLH1, hMSH2, hMSH6, and PMS2, also preserve the DNA of cells in lesions caused by oxidative or alkylating stimuli resulting from inflammatory processes.^28,29

Better understanding of these interactions will aid in controlling the oncogenic stimuli involved in chronic inflammation, which may contribute to identifying mechanisms that, once inhibited, may reduce the incidence of sporadic and hereditary intestinal neoplasia.^30–33

This study aimed to verify whether chronic inflammation represented by TNF-α, IL-2, IL-6, and IL-10 gene expression influences the DNA repair gene system evaluated by hMLH1, hMSH2, hMSH6, and PMS2 gene expression and their corresponding protein levels.

Materials and methods

Study subjects and samples

This study was conducted according to the ethical standards determined by the Helsinki Declaration of the World Medical Association, and it was approved by the Research Ethics Committee of the participating institutions. This study was observational, longitudinal, and retrospective, involving patients operated on between 2007 and 2009 in the Department of Surgery, State Public Servant Hospital (São Paulo, Brazil).

Adult patients of both genders with histologically confirmed CRC and who were operated on with curative or palliative intent in an elective or urgent surgery were included in this study. Patients diagnosed with familial adenomatous polyposis, colorectal neoplasia other than adenocarcinoma, or inflammatory bowel disease, as well as those submitted to neoadjuvant chemotherapy and/or radiotherapy, were excluded.

Overall, 83 patients were operated on curatively or palliatively for CRC. Of which, 22 of these patients were excluded, and 61 were studied, including 34 men (55.7%) and 27 women (44.3%). The mean age was 65.4 ± 10.7 (range = 38–88) years.

CRC tissue sample slides were analyzed for the confirmation of CRC diagnosis. Immunohistochemistry (IHC) assays were performed on the tissue samples to verify the expression of the proteins encoded by the hMLH1, hMSH2, hMSH6, and PMS2 genes of the DNA repair system (MMR) (Figure 1).

Figure 1.

Photomicrographs of colorectal carcinoma of the study patient (immunohistochemistry, 200×): (a) anti-MLH1 antibody-positive immunoexpression, (b) anti-MLH1 antibody-negative immunoexpression, (c) anti-MSH2 antibody-positive immunoexpression, (d) anti-MSH2 antibody-negative immunoexpression, (e) anti-MSH6 antibody-positive immunoexpression, (f) anti-MSH6 antibody-negative immunoexpression, (g) anti-PMS2 antibody-positive immunoexpression, and (h) anti-PMS2 antibody-negative immunoexpression. (a), (c), (e), and (g) represent positive immunoexpression for anti-MLH1, anti-MSH2, anti-PMS2, and anti-MSH6 antibodies, respectively, characterized by the presence of brownish color in the nucleus of neoplastic cells. (b), (d), (f), and (h) represent immunoexpression of anti-MLH1, anti-MSH2, anti-PMS2, and anti-MSH6 antibodies, respectively, characterized by the absence of brownish color in the nucleus of neoplastic cells.

The following clinical and pathological data were analyzed (Table 1): age, gender, tumor location, tumor size, presence of peritumoral inflammatory infiltrate, presence of desmoplastic reaction, degree of tumor differentiation, presence of venous/perineural/lymphatic invasion, and CRC staging by the tumor node metastasis (TNM) classification of 2010.³⁴

Table 1.

Clinicopathological data of patients with colorectal carcinoma.

Variables	n	%
Gender
Male	34	55.7
Female	27	44.3
Location
Right colon	14	22.9
Left colon	22	36.1
Rectum	25	41.0
Desmoplastic reaction
Absent	48	78.7
Present	13	21.3
Differentiation
Little	1	1.6
Moderately	52	85.3
Well differentiated	8	13.1
Inflammatory infiltration
Minimum	48	78.7
Moderate	13	21.3
Vascular infiltration
Absent	44	72.1
Present	17	21.9
Perineural infiltration
Absent	49	80.3
Present	12	19.7
Lymphatic infiltration
Absent	43	70.5
Present	18	29.5
MLH1 expression
Negative	1	1.6
Positive	60	98.4
MSH2 expression
Negative	1	1.6
Positive	60	98.4
MSH6 expression
Negative	2	3.3
Positive	59	96.7
PMS2 expression
Negative	1	1.6
Positive	60	98.4
Staging
I	13	21.3
II	21	34.4
III	19	31.5
IV	8	13.1
	Mean (SD)	Minimum–maximum
Age (years)	65.4 (10.7)	38.0–88.0
Size	5.2 (2.2)	1.0–12.0

CRC specimens with more than 95%, between 50% and 95%, or less than 50% glandular structures were considered well differentiated, moderately differentiated, or poorly differentiated, respectively. Only cases in which anatomopathological examination identified 12 or more lymph nodes without metastatic invasion were considered N0. Peritumoral lymphocyte infiltration was determined after qualitatively and quantitatively evaluating the lymphomononuclear leukocytes that were in close contact with neoplastic and/or adjacent cells, and this infiltration was classified as either minimum (less than 30 cells/field) or moderate (30–60 cells/field).

The desmoplastic stromal reaction was evaluated after quantitative and qualitative determination of fibroconjunctive tissue in contact with neoplastic cells, and it was classified as absent or present.

Quantitative reverse transcription polymerase chain reaction

Reverse transcription polymerase chain reaction (RT-PCR) was performed using CRC tissue fixed in paraffin to detect the presence or absence of the hMLH1, hMSH2, hMSH6, PMS2, TNF-α, IL-2, IL-6, and IL-10 genes.

To prepare 2.0 µg of cDNA, 0.5 µg of RNA was used per case studied. The primers were designed in exon-splicing regions and presented validity between 95% and 105%, ranging in size from 123 to 250 base pairs (Table 2).

Table 2.

Sequence and size of primers of MMR system genes, inflammatory marker genes, and the GAPDH gene used in samples of patients with colorectal carcinoma.

Primer	Sequence (5′ and 3′)	Base pairs
hMSH2	F-CCTTGTAAAACCTTCATTTGATCC R-ATCCAAACTGTGCACTGGAA	157
hMSH6	F-GAACATTCATCCGCGAGAAA R-TGAGGGCTCATCACAAACTG	250
hMLH1	F-CGGTTACTACCCAATGCCTC R-TTCTCGACTAACAGCATTTC	138
PMS2	F-ATCGGCGAAGGTTGGAACTC R-CGGATGCCTGCTGAAATG	156
TNF-α	F-AGAGGGAAGAGTTCCCCAGG R-CCTCAGCTTGAGGGTTTGCT	123
IL-2	F-CCCAAGAAGGCCACAGAACT R-TTGCTGATTAAGTCCCTGGGT	125
IL-6	F-CCAGAGCTGTGCAGATGAGT R-AGCTGCGCAGAATGAGATGA	174
IL-10	F-GCTGAGAACCAAGACCCAGA R-ATTCTTCACCTGCTCCACGG	141
GAPDH	F-GACCACAGTCCATGCCATGA R-CAGCTCAGGGATGACCTTGC	148

Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; CFX96 Touch™; Real-Time PCR; Detection System; BioRad, Hercules, CA, USA) was used to guarantee the quality of the genetic material. MMR gene expression levels were evaluated in relation to the relative expression and normalized by expression of the internal gene, GAPDH, to measure RNA quality and integrity. All cases in which the GAPDH gene presented negative RNA expression were excluded from this study.

Total RNA was extracted from tumor tissue samples, and RT-PCR was performed on cDNA for the hMLH1, hMSH2, hMSH6, and PMS2 genes.

Immunohistochemical staining

Paraffin blocks were sectioned (4 µm thick) and stained using the hematoxylin and eosin (HE) method. Five slides used in the IHC assays (four slides for expression of MMR gene proteins and one slide for control) were prepared from each block with the representative sample of the tumor fixed in paraffin, and an additional five CRC tissue samples were used for RT-PCR.

For the IHC study, anti-MSH2 clone H300 (code sc-22771, lot #I1704), anti-MLH1 clone C20 (code sc-582, lot #E2511), anti-MSH6 clone E8 (code GTBP (H141), lot #G0604), and anti-PMS2 clone C20 (code sc-618, lot #F2712) monoclonal primary antibodies were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 1:100 dilution.

The expression of the proteins encoded by the studied genes was evaluated according to the method proposed by de Jong et al.,³⁵ in which the results of the IHC assays are classified exclusively as positive (presence of protein labeling) or negative (absence of protein labeling) in the cell nucleus. Thus, the result was considered positive when there was unambiguous deposition of the chromogen product in the nuclei of CRC cells, whereas the result was considered negative when unlabeled CRC cell nuclei were present.

A CRC tissue slide staining positive for the studied antibody was used as the positive control, and a similar slide without the primary antibody of the IHC reaction was used as the negative control.

Statistical analysis

Chi-square and Student’s t-tests were used to analyze the association of clinical and anatomopathological variables with the mRNA and protein expression of the DNA repair genes hMLH1, hMSH2, hMSH6, and PMS2 as well as mRNA expression of the inflammatory markers IL-2, IL -6, IL-10, and TNF-α. The Kruskal–Wallis test was applied for the analysis of the association of factors with TNM staging. Stata 11.0 software (Stata Corporation LLC, College Station, TX, USA) was used for statistical analysis.

Results

Pathological CRC findings

A total of 61 patients were enrolled in this study. Of these, CRC was found in the colon in 36 patients (59.0%) and in the rectum in 25 patients (41.0%). CRC localization in the right neck (up to the left flexure) was observed in 14 cases (22.9%) and in the left neck (after the left flexure) in 22 cases (36.1%) (Table 1). Of the 61 patients evaluated, 34 (55.7%) had stage I/II by the TNM classification of CRC. Moderate peritumoral inflammatory infiltration was observed in 16 patients (21.9%). In 18 patients (24.7%), desmoplastic reaction was present in the tumor, and vascular infiltration was present in 13 patients (21.3%); 12 patients (19.7%) had perineural infiltration, and 18 patients (29.5%) showed lymphatic infiltration. CRC was characterized as moderately differentiated in 52 patients (85.3%) (Table 1).

Immunohistochemical staining of DNA repair system (MMR) proteins

For the tumor tissue samples evaluated, the following results were obtained: four (6.6%) presented absence of expression for at least one of the proteins of the MMR system; three (4.9%) showed negative expression of a protein (MSH6, MLH1, or PMS2); and one (1.7%) patient showed negative expression for two proteins (MSH2 and MSH6).

Expression of the hMLH1, hMSH2, hMSH6, PMS2, TNF-α, IL-2, IL-6, and IL-10 genes in CRC and nontumor tissues

RT-PCR analysis of the MMR system genes indicated that hMLH1 and PMS2 gene expression was observed in all cases. The hMSH2 gene was not expressed in 10 (16.4%) cases, and there was no expression of the hMSH6 gene in 2 (3.3%) patients (Table 2). The TNF-α gene was expressed in 12 (19.7%) cases, and IL-2 was expressed in 18 (29.5%) cases. Expression of IL-6 was not identified in any of the patients, and IL-10 was expressed in 5 (8.2%) cases (Table 2).

Significant association was observed between PMS2 expression and vascular infiltration (p = 0.012) and lymphatic infiltration (p = 0.029). No other significant association was found between the clinicopathological data and expression of the MMR system genes (hMLH1, hMSH2, hMSH6, and PMS2) or the expression levels of the inflammatory marker genes (TNF-α, IL-2, IL-6, and IL-10) (Tables 3 and 4).

Table 3.

Association between clinicopathological data with hMLH1 and PMS2 gene expression in patients with colorectal carcinoma.

Variables	Desmoplastic reaction		p-value*	Vascular invasion		p-value*	Neural invasion		p-value*	Lymphatic invasion		p-value*	Peritumoral inflammatory infiltration		p-value*
	Absent	Present		Absent	Present		Absent	Present		Absent	Present		Absent	Present
	Mean (95% CI)			Mean (95% CI)			Mean (95% CI)			Mean (95% CI)			Mean (95% CI)
hMSH1 expression	1.4 (1.5, 1.5)	1.5 (1.5, 1.6)	0.469	1.5 (1.4, 1.5)	1.5 (1.3, 1.6)	0.484	1.5 (1.5, 1.5)	1.4 (1.4, 1.5)	0.098	1.6 (1.4, 1,6)	1.5 (1.4, 1.6)	0.280	1.4 (1.4, 1.5)	1.5 (1.4, 1.6)	0.911
PMS2 expression	1.5 (1.4, 1.6)	1.4 (1.4, 1.5)	0.523	1.5 (1.5, 1.6)	1.7 (1.6, 1.7)	0.012**	1.5 (1.5, 1.6)	1.5 (1.5, 1.6)	0.114	1.5 (1.4, 1.5)	1.6 (1.6, 1.6)	0.029**	1.5 (1.4, 1.5)	1.5 (1.3, 1.6)	0.250

Student’s t-test, 95% confidence interval.

Significant.

Table 4.

Association between clinicopathological data of expression of the hMSH2 and hMSH6 genes and the expression of IL-2, IL-10, and TNF-α inflammatory genes in patients with colorectal carcinoma.

Variables	IL-2		p-value*	IL-10		p-value*	TNF-α		p-value*	hMSH2		P-value*	hMSH6		p-value*
	Present	Absent		Present	Absent		Present	Absent		Present	Absent		Present	Absent
	n (%)			n (%)			n (%)			n (%)			n (%)
Desmoplastic reaction
Absent	32 (74.4)	16 (88.9)	0.208	44 (78.6)	4 (80.0)	0.940	10 (83.3)	38 (77.5)	0.661	40 (80.0)	7 (70.0)	0.483	46 (78.0)	2 (100)	0.454
Present	11 (25.6)	2 (11.1)		12 (21.4)	1 (20.0)		2 (16.7)	11 (22.5)		10 (20.0)	3 (30.0)		13 (22.0)	0 (0.0)	0.454
Vascular invasion
Absent	34 (79.1)	44 (72.1)	0.062	40 (71.4)	4 (80.0)	0.682	8 (66.7)	36 (73.5)	0.638	34 (68.0)	9 (90.0)	0.159	43 (72.9)	1 (50.0)	0.478
Present	9 (20.9)	8 (44.4)		16 (28.6)	1 (20.0)		4 (33.3)	13 (26.5)		16 (32.0)	1 (10.0)		16 (27.1)	1 (50.0)	0.478
Perineural invasion
Absent	37 (86.0)	12 (66.7)	0.082	44 (78.6)	5 (100)	0.248	9 (75.0)	40 (81.6)	0.604	38 (76.0)	10 (100)	0.083	48 (81.4)	1 (50.0)	0.273
Present	6 (14.0)	6 (33.3)		12 (21.4)	0 (0.0)		3 (25.0)	9 (18.4)		12 (24.0)	0 (0.0)		11 (18.6)	1 (50.0)	0.273
Lymphatic invasion
Absent	33 (76.7)	10 (55.6)	0.098	39 (69.6)	4 (80.0)	0.627	8 (66.7)	35 (71.4)	0.746	33 (66.0)	9 (90.0)	0.131	42 (71.2)	1 (50.00	0.518
Present	10 (23.3)	8 (44.4)		17 (30.4)	1 (20.0)		4 (33.3)	14 (28.6)		17 (34.0)	1 (10.0)		17 (28.8)	1 (50.0)	0.518
Inflammatory infiltration
Minimum	32 (74.4)	16 (88.9)	0.208	44 (78.6)	4 (80.0)	0.940	10 (83.3)	38 (77.5)	0.661	42 (84.0)	6 (60.0)	0.083	46 (78.0)	2 (100)	0.454
Moderate	11 (25.6)	2 (11.1)		12 (21.4)	1 (20.0)		2 (16.7)	11 (22.5)		8 (16.0)	4 (40.0)		13 (22.0)	0 (0.0)	0.454

Chi-square test.

The hMLH1 and PMS2 repair genes were expressed in all cases. In contrast, no DNA repair gene expression was identified in 11 patients (18%); 10 patients (16.4%) showed no hMSH2 expression, and 2 patients (3.3%) showed no hMSH6 expression. Only one case (1.6%) lacked expression of both hMSH2 and hMSH6.

Significant associations (p = 0.026) were found between hMSH6 expression and IL-2 expression (Table 5) and between TNF-α expression and hMLH1 and hMSH2 protein expression (p = 0.042) (Table 6).

Table 5.

Association between the expression of inflammatory marker genes—mRNA—(TNF-α, IL-2, and IL-10) with the expression of MMR genes—mRNA—(hMLH1, hMSH2, hMSH6, and PMS2) in patients with colorectal carcinoma.

Variables	IL-2		p-value*	IL-10		p-value*	TNF-α		p-value*
	Negativeexpression	Positiveexpression		Negativeexpression	Positiveexpression		Negativeexpression	Positiveexpression
	n (%)			n (%)			n (%)
hMSH2
Negative expression	7 (16.7)	3 (16.7)	1.00	8 (14.5)	2 (40.0)	0.144	9 (18.7)	1 (8.3)	0.386
Positive expression	35 (83.3)	15 (83.3)		47 (85.5)	3 (60.0)		39 (81.3)	11 (91.7)	0.386
hMSH6
Negative expression	0 (0.0)	2 (11.1)	0.026	2 (3.6)	0 (0.0)	0.667	2 (4.1)	0 (0.0)	0.477
Positive expression	43 (100)	16 (88.9)		54 (96.4)	5 (100)		47 (95.9)	12 (100)	0.477
	Mean (95% CI)		p-value**	Mean (95% CI)		p-value**	Mean (95% CI)		p-value**
hMSH1 expression	1.4 (1.4, 1.5)	1.6 (1.5, 1.6)	0.541	1.5 (1.4, 1.6)	1.4 (1.2, 1.7)	0.210	1.4 (1.4, 1.5)	1.6 (1.5, 1.6)	0.618
PMS2 expression	1.6 (1.5, 1.6)	1.6 (1.5, 1.6)	0.727	1.5 (1.4, 1.6)	1.4 (1.3, 1.6)	0.129	1.5 (1.5, 1.5)	1.5 (1.5, 1.6)	0.416

Chi-square test.

Student’s t-test, 95% confidence interval.

Table 6.

Association between MMR (MLH1, MSH2, MSH6, and PMS2) protein expression levels and the level of mRNA expression of inflammatory markers (tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), interleukin-6 (IL-6), and interleukin-10 (IL-10)) and mRNA expression of MMR (hMLH1, hMSH2, hMSH6, and PMS2) genes in patients with colorectal carcinoma.

Variables	IL-2		p-value*	IL-10		p-value*	TNF-α		p-value*	hMSH2		p-value*	hMSH6		p-value*
	Did notexpress	Expressed		Did notexpress	Expressed		Did notexpress	Expressed		Did notexpress	Expressed		Did notexpress	Expressed
	n (%)			n (%)			n (%)			n (%)			n (%)
MLH1 expression
Absent	1 (2.3)	0 (0.0)	0.514	1 (1.8)	0 (0.0)	0.763	0 (0.0)	1 (8.3)	0.042	0 (0.0)	1 (2.0)	0.652	0 (0.0)	1 (1.7)	0.853
Present	42 (97.7)	18 (100)		55 (98.2)	5 (100)		49 (100)	11 (91.7)		10 (100)	49 (98.0)		2 (100)	58 (98.3)	0.853
MSH2 expression
Absent	1 (2.3)	0 (0.0)	0.514	1 (1.8)	0 (0.0)	0.763	0 (0.0)	1 (8.3)	0.042	0 (0.0)	1 (2.0)	0.652	0 (0.0)	1 (1.7)	0.853
Present	42 (97.7)	18 (100)		55 (98.2)	5 (100)		49 (100)	11 (91.7)		10 (100)	49 (98.0)		2 (100)	58 (98.3)	0.853
MSH6 expression
Absent	2 (4.6)	0 (0.0)	0.352	2 (3.6)	0 (0.0)	0.667	1 (2.0)	1 (8.3)	0.273	0 (0.0)	2 (4.0)	0.520	0 (0.0)	2 (3.4)	0.791
Present	41 (95.3)	18 (100)		54 (96.4)	5 (100)		48 (98.0)	11 (91.7)		10 (100)	48 (96.0)		2 (100)	57 (96.6)	0.791
PMS2 expression
Absent	1 (2.3)	0 (0.0)	0.514	1 (1.8)	0 (0.0)	0.763	1 (2.0)	0 (0.0)	0.618	0 (0.0)	1 (2.0)	0.652	0 (0.0)	1 (1.7)	0.853
Present	42 (97.7)	18 (100)		55 (98.2)	5 (100)		48 (98.0)	12 (100)		10 (100)	49 (98.0)		2 (100)	58 (98.3)	0.853

Chi-square, 95% confidence interval.

Discussion

Significant association was observed between the expression of the PMS2 gene with the presence of vascular and lymphatic invasion of CRC. These findings were consistent with a study by Lynch et al.,³⁶ who associated more advanced stages in CRCs with mutations in DNA repair genes compared with other sporadic CRCs, suggesting a relationship among the DNA repair system, local inflammatory response, and tumor staging.³⁷

Low serum levels of IL-2 are related to the poor prognosis of cancer patients because IL-2 contributes to the maintenance of antitumor cells.^38,39 Rosenberg⁴⁰ showed that IL-2 leads to durable and even curative regressions in patients with melanoma and renal tumors. This study showed an association between IL-2 expression and hMSH6 expression, which may suggest the influence of chronic inflammation on DNA repair system genes in CRC patients. The expression of IL-2 promotes the control of chronic inflammation and the mechanisms of cellular and mutagenic aggression, which favors the preservation of DNA repair through the expression of DNA repair genes.^24,41

In a qualitative study on TNF-α expression in 119 CRC tissue samples, Stanilov et al.⁴² observed a twofold increase in TNF-α serum gene expression in CRC patients compared with control cases (individuals without a diagnosis of cancer or inflammatory bowel disease). These authors concluded that higher levels of TNF-α may be related to more advanced staging and to poor prognosis of the neoplasia.^42,43 In the present series, TNF-α was identified in 19.7% of the cases, and no relationship between gene expression level and the clinicopathological aspects or CRC staging was observed.

An association between TNF-α gene expression in the CRC samples and the absence of MLH1 and MSH2 proteins was verified in this study. A previous study⁴⁴ showed that TNF-α was increased in patients with chronic inflammatory processes and that this elevation in patients with CRC may be due to the presence of inflammation that may accompany carcinogenesis and the development of CRC. These findings suggest that chronic inflammation decreases DNA repair protein synthesis.

Patients with competent DNA repair genes may be more resistant to oncogenic factors, such as inflammatory processes of environmental origin that occur due to smoking, alcoholism, and obesity. Therefore, these patients would be less susceptible to developing neoplasias, such as CRC, than would individuals deficient of these genes.

In cases of hereditary nonpolyposis colorectal cancer (HNPCC) with autosomal dominant mutation, individuals inherit one mutated allele and one normal allele, requiring only a mutational event to inactivate the DNA repair gene. In these individuals, this mechanism may allow neoplasia to develop at earlier ages than those in the general population, thus provoking greater susceptibility to proinflammatory stimuli by increasing TNF-α expression.⁴⁵

In this study, a significant association was observed between TNF-α expression and hMSH2 protein level. This finding was corroborated by the results of Brentnall et al.,⁴⁵ who observed an association between the hMSH2 mutation in patients with chronic ulcerative colitis and the development of CRC. This finding may be related to the presence of chronic inflammation that is associated with decreased cell growth control capacity, which leads to genomic destabilization by the production of reactive oxygen species (hydrogen peroxide (H₂O₂), singlet dioxygen (1O₂), and hydroxyl radical (HO)) and nitrogen compounds (molecules derived from nitric oxide (NO) and superoxide (O₂−)) due to the action of phagocytic cells.^6,34,46

The significant association between gene expression and DNA repair proteins and expression of inflammatory markers (IL-2 and TNF-α genes) reinforced the idea that chronic inflammation plays a decisive role in the development of hereditary CRC,⁴⁷ and measures that reduce inflammatory stimuli may consequently play preventive roles in neoplastic development.

Analysis of the expression of inflammatory markers in patients with sporadic or hereditary CRC may identify subgroups of patients at high risk for the development of CRC recurrence, resulting in more efficient population screening.⁴⁸

This study sought to observe the relationship between inflammatory marker gene expression and DNA repair gene and protein expression. The lack of protein expression observed in the inflammatory markers in the CRC tissues was not characterized because the turnover of the studied mRNAs was variable and short-lived due to the regulatory mechanisms.^49,50

A better understanding of the influence of chronic inflammation on the onset and development of CRC and its influence on the DNA repair system may warrant the identification, treatment, and prevention of intestinal inflammation to reduce the development of neoplasms of the large intestine, where inflammation contributes to its genesis.

Conclusion

In this study, IL-2 and TNF-α gene expression in tissues is associated with hMSH6, hMLH1, and hMSH2 gene expression, suggesting that the presence of chronic inflammation, as represented by the IL-2 and TNF-α gene expression, could influence the DNA repair process in CRC.

Footnotes

Acknowledgements

The authors thank Vinicius Alcantara for his help and effort in the statistical study of this manuscript.

Author contributions

Demétrius Eduardo Germini contributed to protocol and data analysis, design of the study, literature review, and manuscript preparation; Fernando Luiz Affonso Fonseca contributed to technical analysis and data analysis; Beatriz da Costa Aguiar Alves Reis contributed to sample collection, data analysis, and medical records analysis; Leonardo Cardili contributed to technical analysis and medical records analysis; Thérèse Rachell Theodoro contributed to technical analysis and literature review; Maria Isete Fares Franco contributed to interpretation of analysis results; Celina Tizuko Fujiyama Oshima contributed to technical analysis, interpretation of analysis results, and reviewed the manuscript; and Jaques Waisberg contributed to final data analysis and final manuscript revision.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This study was approved by the Research Ethics Committee of State Civil Servant Hospital. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research Ethics Committee of State Civil Servant Hospital and with the 1964 Helsinki Declaration. This article does not contain any studies with animals performed by any authors.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Demétrius Eduardo Germini

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