CCDC144NL-AS1/Hsa-miR-143-3p/HMGA2 Interaction: In-silico and Clinically Implicated in CRC Progression, Correlated to Tumor Stage and Size in Case-controlled Study; Step Toward ncRNA Precision

1. Introduction 1.1. Background Colorectal cancer (CRC) is one of the malicious malignancies worldwide, accounting for nearly 8 % of all annual deaths [1]. It is considered Egypt's 7th most prevalent cancer, representing about 3.47 % of male tumors and 3 % of female tumors, respectively [2]. By 2030, there will be an estimated 60 % increase in incidence and mortality for CRC globally [3]. Early-stage CRC is usually asymptomatic, but when symptoms do manifest, timely detection is essential because any delay in the diagnosis may increase mortality rates [4].

1.2. Problem Surgical resection could cure 90 % of CRCs in the early stages. Nevertheless, the majority of patients frequently have poor prognosis since they are detected at an advanced stage [5]. Although colonoscopy tissue biopsy is commonly used for CRC diagnosis, yet, it is an invasive high-risk test, not convenient to be implemented in routine medical examination for longitudinal monitoring or prognosis and is considered partially representative of inter-metastatic or tumoral genetic heterogeneity [6]. The classical circulating tumor biomarkers (TMs) as carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) [7] are used as follow-up or prognosis markers, despite of their limited sensitivity and specificity [8]. Therefore, there is an urgent need for more efficient prognostic molecular biomarkers which could be "epi/genetic molecular marker(s)" that have 2 benefits, first, harbor potential therapeutic target(s) and second, augment classical circulating TMs, in order to improve CRC precision. Liquid biopsy has emerged as a minimally invasive diagnostic tool to analyze tumoral genetic and epigenetic molecular markers released into the circulation. Liquid biopsy captures, better, tumor genetic heterogeneity, reflecting the dynamic picture of tumor molecular landscape with lower processing time and lower cost than tissue biopsy [9]. Long non-coding RNAs (lncRNAs) are RNA molecules with >200 nucleotides length, that regulate gene expression at the transcriptional, post-transcriptional, and translational levels, but cannot code for proteins synthesis [10-12]. Multiple cancer types exhibit deregulations of lncRNAs, which are involved in all cancer hallmarks, including cancer genesis, progression, and metastasis [13,14]. Emerging studies revealed several lncRNAs are implicated in CRC tumorigenesis, metastasis, and progression [15,16]. Beyond their potential for diagnosis, lncRNAs would also serve as possible therapeutic targets [17]. Coiled-lncRNA Coil Domain Containing 144 N-Terminal-Like antisense 1 (CCDC144NL-AS1) located on the 17p11.2 human chromosome, is a novel oncogenic lncRNA recently reported to be involved in carcinogenesis [18]. LncRNA CCDC144NL-AS1 was found to be upregulated in various cancers including gastric cancer (GC) [18], hepatocellular carcinoma (HCC) [19], non-small cell lung cancer (NSCLC) [20], ovarian cancer (OC) [21], osteosarcoma [22], and CRC [23]. However, its clinical role as biomarker for CRC needs to be elucidated. MicroRNAs (miRNAs or miRs) are single-stranded, small RNA molecules that are 18-25 nucleotides length [24]. They have a regulatory function in a variety of physiological processes, involving cell differentiation, growth, apoptosis, immunological response, hematopoiesis, and proliferation [25]. Several studies have shown miRs have crucial role in both initiation and progression of CRC, besides their potential as molecular biomarkers and possible therapeutic hits [26-28]. Hsa-miR-143 located on the human chromosome 5q32 [29] is a tumor-suppressor miR reported to be down-regulated via miR-mediated post-transcriptional gene silencing in several human cancers including prostate cancer [30], cervical cancer [31], OC [32], and B-cell lymphoma [33]. The 3' arm of the miR precursor product, hsa-miR-143-3p is down regulated in CRC and if it would contribute to CRC initiation [34] will be studied currently clinically. LncRNA-miR interaction plays an essential role during various cancer development [35,36]. CCDC144NL-AS1 was reported to act as competing endogenous RNA (ceRNA) for hsa-miR-143-3p during GC progression, via competing with the common binding regions of miRs, in order to sequester them and therefore, alter the expression of miRs downstream target genes or proteins [18]. Being approved experimentally by Fan et al. [37] in GC lncRNA CCDC144NL-AS1 upregulation in GC tissues and sponging hsa-miR-143-3p followed by upregulated expression of its direct endogenous target protein. Similarly, the clinical correlation between lncRNA CCDC144NL-AS1 and hsa-miR-143-3p in CRC patients' peripheral blood samples, that haven't been estimated previously, could further clarify our understanding of CRC molecular pathogenesis and proof the cancer findings documented in silico. High Mobility Group AT-hook 2 (HMGA2) gene encoded by 5 exons and an open reading frame of 330 base pair, is found at human chromosome band 12q13-15 [38]. The adult normal cell's HMGA2 protein concentration is minimal or absent under normal physiological conditions, but it is highly expressed during embryogenesis [38] and carcinogenesis [39,40]. Patients with CRC who have HMGA2 overexpression are experiencing worse prognosis [41]. HMGA2 takes part in almost every stage of biological activity of CRC, including cell division, proliferation, apoptosis, senescence, tumor invasion, epithelial-tomesenchymal transition (EMT), DNA repairing mechanism, and stem cell ability of self-renewal [42]. HMGA2 in osteosarcoma, was reported to be positively modulated by CCDC144NL-AS1 [22].

1.3. Aim The oncogenic lncRNA CCDC144NL-AS1 if being involved in CRC pathogenesis, by acting as a ceRNA/sponging the tumor suppressor hsamiR-143-3p, and further, upregulating the expression of its endogenous target HMGA2 as an interaction arm will be highlighted in the current study.

1.4. Objectives Elucidate the role of lncRNA CCDC144NL-AS1, hsa-miR-143-3p, and HMGA2 protein as non-invasive epigenetic molecular biomarkers in liquid biopsy of CRC Egyptian patients, individually or as an interaction arm and in comparison, to the conventional protein TMs. In addition, we investigated the potential role of lncRNA CCDC144NL-AS1 as a mediator for development and/or progression of the cancer phenotype as well as CRC metastasis and its relation to both hsa-miR-143-3p and HMGA2, clinically and in silico. 2. Subjects 2.1. Sample size and power of the study Based on the previous study by Zhang et al. in 2019 lncRNA CCDC144NL-AS1 was normally distributed with a standard deviation (3.2) and large effect size (0.85) [43]. If the true differences between the CRC group and the control group means are 1 and 3.3, respectively, the study group sizes are 34 patients and 34 control subjects. Total sample size was 68 cases, which was increased by 25 % for expected losses, and total sample was finally 90 subjects, 60 CRC subjects and 30 control (2:1). This is to be able to reject the null hypothesis, that the population means of the experimental groups are equal with probability (power) of 0.8. The Type I error probability associated with this test of this null hypothesis is (0.05). Sample size estimation was performed by G power* sample size online calculator http://www.gpower.hhu.de/en.html, depending on two-sided confidence level 95 %.

2.2. Study design Case-controlled retrospective mono-center study. 2.3. Institutional Review Board (IRB) statement The Research Ethics Committee of the Faculty of Pharmacy, Ain Shams University, granted ethical permission to conduct the study, 2022. All participants (controls or patients) were fully cognizant of the purpose of the study and signed a written, ethically-approved, informed consent (I⋅C) form. This study was conducted in accordance to the Declaration of Helsinki Guidelines approved in 2013 [44]. 2.4. Study participants 2.4.1. Patients group A total of 60 primary CRC treatment-naïve Egyptian patients admitted to the Dar Al Shefa Hospital, Cairo, Egypt, were enrolled in the study.