All analysed samples (Table I) were obtained from consecutive patients who underwent a laparoscopic low anterior resection with total mesorectal excision for rectal adenocarcinoma. The anastomosis was constructed by the double stapling method (27). Preoperative staging was performed by either computed tomography or magnetic resonance imaging of the abdomen and the pelvis. Patients with locally advanced tumours, T3 tumours and T4 tumours with or without involvement of the lymph nodes received preoperative full course chemoradiotherapy (CHRT) to downstage the tumour.

If patients had undergone CHRT, restaging was performed within 6 weeks of CHRT completion and surgery was performed 10 weeks after CHRT completion. The standard CHRT regimen involved irradiation with 45 Gy in 25 fractions to the pelvic area, the rectum and the pararectal, presacral and internal iliac lymph nodes. A supplementary boost dose of radiation was applied to the tumour itself (5.4 Gy in 3 fractions) for a total dose of 50.4 Gy. Radiotherapy was potentiated by chemotherapy with continuous 5-fluorouracil infusion at a dose of 200 mg/m²/24 h for the entire duration of radiotherapy.

Surgical procedures took place in the between March 2019 and December 2020 at two medical institutions: The University Hospital Martin and Faculty Hospital with an outpatient clinic in Zilina. After terminating the resection phase of the surgical procedure, two small samples of the intestinal wall (normal adjacent tissue) were cut from the specimen by an operating surgeon. Adjacent rectal tissues (ARTs) were obtained 10.0-15.00 cm away from the primary tumour and immediately frozen and stored at -80˚C for quantitative polymerase chain reaction (qPCR) and estimation of hydroxyproline. The total number recruited for the present study (adjacent rectal tissues of patients with rectal adenocarcinoma) was 29, of which 12 were without therapy and 17 with neo-adjuvant therapy. The present study was performed according to the Guidelines of the World Medical Association Declaration of Helsinki. The Ethics committee of Jessenius Faculty of Medicine CU in Martin with certification code no. IRB00005636 approved the study and written informed consent was given by all patients.

Hydroxyproline, a major component of collagen, was quantified as a measure of the collagen content using the Total collagen Colorimetric Assay kit (BioVision, Inc.). The assay is based on the acid hydrolysis of samples to form hydroxyproline. Adjacent rectal samples were homogenized in dH₂O, using 100 µl H₂O for every 10 mg of tissue (a of total 80 mg of tissue was used).

To a 100 µl sample of homogenate, 100 µl of concentrated HCl (~12 M) was added in a pressure-tight, Teflon-capped vial and hydrolysed at 120˚C for 3 h. A total of 10 µl of each hydrolysed sample was transferred to a 96-well plate and evaporated to dryness under a vacuum. A total of 100 µl of the chloramine T reagent was added to each sample and standard and incubated at room temperature for 5 min. Then, 100 µl of the p-dimethylaminobenzaldehyde (DMAB) reagent was added to each well and incubated for 90 min at 60˚C. Finally, absorbance at 560 nm was measured by Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Inc.). The collagen concentration was expressed in total amount of collagen µg to mg dry tissue.

Colorectal tissue samples stored frozen at -80˚C in stabilizing reagent (RNAlater; Qiagen, Inc.) were used for total RNA isolation. The samples ~25 mg were disrupted in QIAZOL Lysis reagent (Qiagen, Inc.) by Polytron PT 1600 E homogeniser (Kinematica AG). The RNA was isolated by using RNeasy Plus Universal kit (Qiagen, Inc.) and measured by spectrometry using a Implen P300 NanoPhotometer (Implen GmbH). A total of 2 µg of purified cellular RNA was converted to single stranded cDNA using RT² First Strand kit (Qiagen, Inc.) according to protocol supplied by the manufacturer. The newly synthesized and diluted cDNA was used for studying multigene expression MMPs, tissue inhibitors of MMPs and collagens using the RT² Profiler PCR Array (Qiagen, Inc.). Three endogenous control genes; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB) and β2-microglobulin (B2M) present on the PCR array were used for normalization. The set of primers for all human genes were acquired from RT² qPCR Primer Assays database (https://geneglobe.qiagen.com/sk/product-groups/rt2-qpcr-primer-assays; Table II).

A reaction mixture prepared according to manufacturer's protocol containing cDNA template (800 ng) and particular volume (1,240 µl) of SYBR Green ROX qPCR Mastermix (Qiagen, Inc.) was immediately loaded into each well of 96-well plate of RT² Profiler PCR Array. Each plate was sealed and centrifuged for 1 min at 1,000 x g at room temperature and loaded into the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Amplification of cDNA was initiated by denaturation at 95˚C for 10 min followed by 40 PCR stage (95˚C, 15 min; 60˚C, 1 min) and melting curve stage (95˚C, 15 min; 60˚C, 1 min; 95˚C, 15 min). The relative changes in gene expression were analysed by using the 2^-ΔΔCq method (28), where ΔΔCt = (Ct_GOI-Ct_HKG)_{TESTING GROUP} - (Ct_GOI-Ct_HKG)_{CONTROL GROUP}. Commercially available total RNAs from normal rectum tissue were used as a control group for quantitative PCR (Ambsbio; R1234206-50_Human Adult Normal Rectum Tissue and HR-312_Normal Human Rectum Tissue).

The relative mRNA levels of ART (both with/without pre-therapy) were compared with normal rectal mRNA by the Welch's ANOVA and Tukey's HSD post hoc test. Relations between gene expression and clinicobiochemical parameters were modelled by multivariate linear regression with all continuous predictors, except for age, and were log-transformed to bring the distributions closer to a Gaussian one. The Akaike Information Criterion was used to reduce the full regression model. The correlation between the log-transformed gene expressions was assessed by the Pearson correlation coefficient. The data were analysed in R (R Development Core Team, R: A Language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria, URL http://www.R-project.org, 2006). Data are represented as means ± standard error of mean. All statistical comparisons were performed two-sided in the sense of an exploratory data analysis using ^*P<0.05, ^**P<0.01 and ^***P<0.001 as levels of significance.

Gene expression and fold-change calculations were performed using RT2 Profiler PCR Arrays & Assays Data Analysis Webportal (https://geneglobe.qiagen.com/us/analyze, Qiagen, Inc.). The genes with a significant difference in expression were those with an average fold-change of ≤-2.0 or ≥2.0. P<0.05 was considered to indicate a statistically significant difference.

The absolute pattern of relevant MMPs, TIMPs and COLs was analysed through mRNA expression (Fig. 1). When comparing the absolute levels of gene expression, we decided to represent them in log(10) of 2^-ΔΔCq values. It should be added that the average Ct of the control genes (ACTB, GAPDH and B2M) was subtracted from the expression value of the determined gene for each sample separately. Fig. 1 shows the transcriptomic levels of the genes of interest in all sample types [control mRNA from normal human rectum (Ctrl); adjacent rectal tissue of patients with pre-therapy (ART+) and adjacent rectal tissue of patients without pre-therapy (ART-)]. It can be clearly stated that the highest amplification was recorded for COL3A1, TIMP2 and COL1A1. Among MMPs, the highest levels in MMP2 were detected in both the control and patient samples. An absolute lack of amplification signal was accompanied by control of the human rectum for the MMP8 and MMP13 genes. In patient samples, amplification of MMP8 was detected in 65.5% of cases and MMP13 in 89% of cases. No statistical differences were observed when comparing gene levels between both patient groups. On the contrary, significant differences in the TIMP1, TIMP2, TIMP4 and COL3A1 genes were demonstrated when comparing patient samples with the control group (Fig. 1). Significantly increased MMP2 levels were also detected in patients without pre-therapy compared with controls (-3.07 vs. -2.48, P=0.025).

The comparison between gene levels and clinicobiochemical parameters of all patients is summarised in Table III, where only significant differences are shown.

It should be noted at the outset that none of the TIMP genes showed a relationship with the clinical parameters. MMP1, MMP8 and MMP10 gene expression levels in all samples of patients were negatively related to albumin concentration. In contrast, a positive significant association of albumin vs. COL3A1 mRNA level was detected. Moreover, MMP1 and MMP10 were negative trends to C-reactive protein values (Table III). The expression of MMP2 and COL3A1 correlated with increased age, while high levels of MMP8 were typical for younger patients. In addition, it was demonstrated that the level of MMP8 increased with body mass index (BMI) value and decreased with the collagen concentration in the examined biopsies. The high expression of the COL1A1 gene was significantly related to collagen concentration and was in overweight individuals (Table III). The weak effect of neoadjuvant therapy in association with MMP1, MMP2 and MMP10 genes was observed.

The present study investigated the correlations between individual levels of mRNA obtained from patient samples. The power of correlations (positive or negative) were categorised as low, medium and high according to their correlation coefficient and P-values (Fig. 2). The highest positive association of the MMP8 gene was significantly related to the TIMP1 gene (0.78; P=0.0001). The same strong effect was observed between TIMP1 and TIMP2 (0.7; P=0.00001). Among the significant positive correlations, the relationship between the two genes for collagen was considered. The remaining positive relationships are indicated by blue spots. A negative relationship (red spots) was detected between MMP1 and TIMP2 (-0.38; P=0.043), between MMP8 and both collagen genes (COL1A1: -0.52; P=0.020; COL3A1: -0.57, P=0.01) and between MMP9 and MMP13 (-0.42; P=0.032).

In this section, the present study focused on the regulatory changes of genes measured in the adjacent rectum tissue from carcinoma patients compared with control biopsies (RNAs from Human Adult Normal Rectum Tissue; Amsbio), where cancer status was not present. In addition, the patient samples were divided into two groups, with or without applied therapy. Table IV represents changes in fold regulation of individual genes. The data themselves were calculated as the difference of log₁₀ 2^-ΔCq between the groups of samples that are illustrated in Fig. 1. It was found that the present of therapy did not serve a significant role in the regulation of genes (Table IV). This was reflected in a parallel comparison of patient samples vs. the control samples. MMP2 (2.0 fold; P=0.025), TIMP1 (4.2; P=0.0022) and TIMP2 (3.9; P=0.0012) were significantly increased in patients without therapy (ART-), compared with the control. Similarly, the mRNA of COL1A1 (2.2; P=0.22) and COL3A1 (3.1; P=0.023) increased in the ART-group. TIMP1 showed the highest fold difference, while in the MMP1 gene higher downregulation was detected, but without statistical significance. The present study did not find fold-change differences in MMP regulation between patient samples with therapy compared with the control. In contrast, fold regulation for TIMPs and COLs had a similar trend as in patients without therapy.

The present study confirmed the similar profile of fold regulation when comparing all patient samples versus the control (ART/Ctrl; Table IV). As mentioned earlier, the therapy did not affect the change in gene regulation; although, the mRNA of MMP1 showed a non-significant upregulation in patients after therapy compared with patients without therapy (ART+/ART-).

The biopsies for collagen determination after storage at -80˚C weighed 75±5.2 mg wet tissue. The relationships between the collagen concentrations of each patient and the clinicobiochemical parameters were then examined. The collagen amounts in the biopsies were unrelated to gender, BMI, proteins in plasma, leukocytes and plasma iron. In 24 biopsy specimens of rectal tissue, a decrease in collagen concentration with the patient's age was observed. Combined with the third parameter (neoadjuvant therapy), it was found that 13 patients who underwent preoperative therapy almost doubled their collagen content in rectal tissue (Fig. 3).