CRC is one of the most common life-threatening malignancies in the world. It represents 8.5% of all cancer mortalities [18]. The initiation of CRC is a complex biological process that involves multiple genomic and epigenomic alterations. The escalating incidence and the poor unpleasant outcome of CRC has drawn scientific attention and efforts towards continuous trials aiming to discover the underlying pathological processes of CRC progression, in order to arrest these processes and thus CRC progression [19].
Ulcerative colitis (UC) which is known under the umbrella of inflammatory bowel disease (IBD), is a global disorder with a high incidence in developed countries (> 0.3% prevalence) and an accelerating incidence in developing countries, resulting in severe morbidity, substantial health care costs, and loss of productivity at work [20]. This chronic colonic inflammation, may precipitate colorectal cancer (CRC) as an expected and feared complication [8]. Analysis of CRC risk in patients with IBD showed a risk of 2% at 10 years after ulcerative colitis diagnosis, 8% at 20 years and 18% at 30 years after colitis onset [9].
Hence, the search for early, noninvasive convenient diagnostic biomarkers is required to be developed by scientists [21]. The diagnostic sensitivity of CEA is 50% with 80% specificity, thus, not considered as a reliable biomarker [22]. That’s why the eyes were directed towards non-coding RNAs as potential minimally invasive promising biomarkers and thus researches exploded in that rich field of genomics.
Herein, in our research we pursued two of non-coding RNAs namely the LncRNA H19 and the micro RNA-675 in 60 patients with CRC, 60 patients with UC and 30 totally free of diseases candidates as reference controls to assess the potential use of both H19 and miR-675 as biomarkers for early diagnosis of both UC and CRC.
In our study, demographic data which comprises age and gender showed a significant difference regarding age where CRC group showed a higher mean of age with statistical significance when compared to both control and UC groups who showed a lower mean in their ages, this comes in accordance with other studies [3, 23, 24] which stated that colorectal cancer is mainly a disease of the elderly [23]or above the age of 55[24, 10]. These results were previously challenged in an Egyptian study [25] that recognized a relatively high percentage of patients diagnosed with CRC at a younger age group (between 20–40 years).
Regarding UC it was recognized [26] that IBD have bimodal peak of incidence one at the 2nd decade which match our patients ages while another one after the 5th decade which is alleged to have a high mortality rate but does not match any of our patients’ data regarding age in the UC group. While it was mentioned in a study conducted in 2019 [8] that the peak age for UC is from 15–30 years another study [27] declared that the peak age of disease onset is between ages 30 years and 40 years, while onset after age 60 years tend to have milder disease compared with younger patients.
Gender did not vary significantly among groups although the male gender was slightly predominant in all groups of the study. Being more than half of patients or candidates in controls almost equal halves with slight increase in males while the increase in number of males was more recognized among CRC group followed by UC group which may be interpreted as previously stated in a previous study [28] that some social and cultural barriers such as embarrassment within females to undergo colonoscopy which delays screening and thus diagnosis.
microRNAs are responsible for negative modulating of up to 60% of protein-coding gene expression [29]. miRNA expression is related to the increase of tumor mass size, metastasis, increased malignancy of tumor cells. Several reports have demonstrated that miRNAs can be a potential biomarker for the diagnosis and prognosis of CRC [30]. The dysregulated non coding miRNA- 675 may be used as a potential biomarker for detecting carcinogenesis in multiple types of cancer. It promotes CRC cells proliferation through its negative regulation of tumor suppressor DMTF1 [29].
In our study, gene expression analysis of miR-675 revealed that it was under expressed in both groups. However, expression was higher in CRC compared to UC group with a significant difference. Downregulation of miR-675 may result from reduced conversion of H19 into pre-miR-675 and/or reduced conversion of pre-miR-675 into mature miR-675 [11] for which the underlying cause needs to be elucidated.
In contrary to our study, miR-675 was found to be upregulated in gastric cancer [31] in a study which revealed that the overexpression of H19 and miR-675 promoted cell proliferation and inhibited cell apoptosis.
lncRNA H19 showed an enhanced expression in both UC and CRC groups. Expression was higher in CRC group with a statistically significant difference.
Through accumulating researches and by analyzing the public database, lncRNA H19 was found as one of most overexpressed lncRNAs in primary tumors and metastatic tissues when compared with lncRNA H19 levels in the adjacent normal tissues in CRC [32], which align with our current study findings.
lncRNA H19 was found to be overexpressed in CRC tissues compared with adjacent normal tissues [33]. The expression level was correlated with tumor differentiation and advanced TNM stage of cancer [34]. In addition, another study [35] revealed that H19 expression in CRC in Egyptian patients reached a 1.38-fold increase compared to the controls with a statistical significant difference.
It has been questioned in a previous study [36] that how lncRNA H19 and miR-675 were both up-regulated in many types of cancers and both were up-regulated by common triggers given that miR-675 is supposed to be processed at the expense of lncRNA H19. Such inquiry may support our results; since we found lncRNA H19 overexpressed while miR-675 under expressed indicating that lncRNA H19 was not consumed in expressing miR-675.
The role of lncRNA H19 in the pathogenesis of UC was mentioned in previous studies; which stated that the expression level of lncRNA H19 was significantly higher in UC tissues compared with paired normal tissues. It was found that it represses the tight junctions and adherent junction’s expression and results in the epithelial barrier dysfunction by acting as a primary miR-675 precursor, which after maturation inhibits TJ/AJ expression at the post-transcription level [37]. Another study [38] found that intestinal lncRNA H19 was dramatically upregulated in mice colitis models, as well as in inflamed colonic tissues from patients with IBD and that highly expressed lncRNA H19 repressed the function of mRNAs encoding TJ protein ZO-1 and AJ protein E-cadherin by releasing miR-675, leading to epithelial barrier damage.
Regarding laboratory studies in the CRC group, anemia was noted in CRC patients as evidenced by decreased mean hemoglobin concentration, with a marginal significant difference compared to control. This comes in accordance with another study [39]. There was also a significant difference in platelet count which was increased in CRC cases compared to control. In a study conducted in 2018 [40], platelet indices were investigated as diagnostic markers for CRC. Platelet count was found to be elevated compared to patients with colon adenoma. This comes in agreement with our results regarding platelets count compared between CRC patients and controls. No correlation was detected between the level of gene expression of lncRNA H19 and any of the measured laboratory parameters in our study.
Recto-sigmoid lesions represented the major site of lesions in our study. The highest expression of lncRNA H19 was found in caecum and ascending colonic lesions with significant difference compared to transverse and flexures & to recto-sigmoid lesions.
lncRNA H19 expression did not vary significantly with any of the CT findings in CRC, including mass lesion, wall thickening, regional lymph nodes involvement or liver metastasis. Also, no statistically significant difference was found between lncRNA H19 expression level and histopathological types or grades.
In a previous work [20], lncRNA H19 overexpression was directly proportionate to disease progression, pretreatment metastasis, poor differentiation level and advanced TNM stages.
No significant correlation was noted between miR-675 and lncRNA H19 expression levels. However, logistic regression analysis revealed that lncRNA H19 can be used in predicting CRC.
Laboratory investigations of UC patients with UC showed anemia, manifested by low hemoglobin and low hematocrit value, elevated CRP and ESR. Chronic diarrhea affects iron to be absorbed from intestine thus anemia occurs [41]. An elevated CRP or ESR is associated with increased risk of CRC in UC patients [42]. Serum albumin was relatively lower in UC and varied significantly compared to control. The main cause of hypoalbuminemia in UC patients is the malabsorption of food protein constituents [42].
Other laboratory parameters were not significant. No correlation was detected between the level of gene expression of lncRNA H19 and any of the measured laboratory parameters.
Severity of UC in our group of patients was categorized as: more than one third of cases, it was considered mild while the other two thirds were divided between moderate and severe, while only two patients were on remission based upon Mayo score. The score was classified into 4 different activities: Remission (0–2), Mild (3–5), Moderate (6–10) & Severe (> 10).
There was a significant increase in lncRNA H19 expression in the group with moderate & severe UC compared to cases with remission, and mild cases.
According to colonoscopy in our studied group, most of cases were either left sided colitis or pancolitis and a small percent was diagnosed with proctitis only.
lncRNA H19 showed the highest expression in left sided lesions, which was significant from pancolitis lesions (p = 0.05).
Spearman correlation indicated that there was no significant correlation between miR-675 and H19 expression levels in UC cases. By Logistic regression analysis, lncRNA H19 was examined for its potentiality to predict UC and proved potential as a diagnostic test.
ROC curve analysis showed that lncRNA H19 can be used in diagnosis of UC; with a sensitivity of 91.7%, specificity of 99.3% (AUC: 0.917, 95% CI: 0.852–0.959, P < 0.0001) at a cut off value = 2.659 with accuracy 95.5%.
It can also be used as diagnostic tests for CRC; a sensitivity of 90.0% and a specificity of 99.8% (AUC: 0.90, 95% CI:0.7919–0.9667, P < 0.0001) at a cut off value of 8.73 with 96.85% accuracy.