Samples were collected from the Affiliated Hospital of Nantong University (Nantong, China) between February 2020 and June 2021, which were the remaining serum samples for routine tests. A total of 222 patients were recruited, including 171 patients clinically defined as CRC, 51 patients diagnosed as benign adenomas or adenomatous polyposis coli. CRC patients were diagnosed by histopathology, and tumors were staged and graded according to the 2017 AJCC/UICC TNM classification. A total of 138 control participants were recruited from a large crowd of individuals from the Health and Disease Management Center of the Affiliated Hospital of Nantong University. The demographic and clinical characteristics of researched patients and healthy folks are summarized in Table 1.This study was approved by the Human Research Ethical Committee of the Affiliated Hospital of Nantong University (Ethics Review Report No. 2018-L055), and the serum was stored at −80°C prior to further analysis.

Gene expression data (HTSeq-FPKM) with clinical information of CRC samples (COAD/READ) were collected from TCGA (https://cancergenome.nih.gov/). The R Project for Statistical Computing (https://www.R-project.org) was applied to generate the lncRNA expression matrix. The overall survival data from TCGA database were also downloaded and analyzed. The software of RNAhirbd and RNAhybrid database (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) were utilized for the exploration of downstream ceRNA network on the development and prognosis in CRC.

Serum RNA was extracted using Total RNA Fast Extraction Kit for Blood or Liquid Sample (BioTeke, Wuxi, China), and RNA in cells was extracted by TRIzol reagent. The nuclear and cytoplasmic fractions were isolated utilizing Ambion^® PARIS^™ Kit (Thermo Scientific, United States). All the extraction procedures were strictly under the manufacturer’s protocol. The concentration and integrity of RNA samples were assured by NanoDrop spectrophotometer (Thermo Fisher Scientific, United States). The OD260/280 ratio between 1.8 and 2.0 indicated good purity, and RNA samples were maintained at −80°C before use.

Extracted RNA was reversely transcribed into cDNA by the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States) following the manufacturer’s protocol. The 20-μl reaction mixture was centrifuged briefly, followed by incubation at 42°C for 60 min and subsequently at 70°C for 5 min.

The relative expression level of Linc01836 was determined by qRT-PCR, which was conducted using the LightCycler 480 (Roche, Switzerland). The sequences of primers are in Table 2, and the PCR procedure was set as follows: 95°C for 30 s, followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) was used as DNA-specific fluorescent dye, 18S rRNA was selected as a housekeeping gene, and the result for each sample was normalized to 18S rRNA expression. Relative expression was determined by the comparative Ct method (2^−ΔΔCt), and all experiments were carried out in three replications.

TAE solution and GelRed dye were used to prepare the 2% agarose gel. A mixture of 5 μl of PCR product and 1 μl of 6× loading buffer was loaded to the given position of solidified agarose gel, and electrophoresis was performed at a voltage of 120 V for 40 min. Images were obtained by a GelDoc XR Imaging System (Bio-rad, United States ).

The serum CEA, CA19-9, Cyfra21-1, and carbohydrate antigen 72-4 (CA72-4) concentrations in CRC patients and healthy controls were measured by Architect I4000 SR (Abbott, Chicago, IL, United States ).

All data obtained from three or more independent experiments were displayed as mean ± standard deviation (SD). The software of SPSS 20.0 (IBM SPSS Statistics, Chicago, IL, United States) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, United States) were used for statistical analysis. Student’s t-test was performed to do the comparison between two groups when the dataset obeyed Gaussian’s distribution; otherwise, Mann–Whitney U-test could be used. One-way ANOVA was applied for comparison between more groups. The diagnostic value of Linc01836 and other tumor markers for differentiating CRC patients from the healthy folks was evaluated by receiver operating characteristic (ROC) curves. The relationship between serum Linc01836 expression and clinicopathological characteristics of CRC cases was analyzed via chi-square test. Survival analysis using data from TCGA database was performed. Values of p < 0.05 were deemed as statistically significant.

CRC-related transcriptome sequencing data from TCGA database were downloaded and analyzed. A total of 599 lncRNAs were found to be differentially expressed between CRC tissues and adjacent normal tissues, including 472 upregulated and 127 downregulated. Linc01836 was highly expressed in CRC tissues (Figure 1A). Thereafter, we further analyzed the clinicopathological data and survival data of CRC patients from TCGA database, and found that upregulated Linc01836 was associated with short survival time and poor prognosis (Figure 3D). Analysis of qRT-PCR was primitively used to determine the serum expression level of Linc01836 in 20 CRC patients as well as in 20 healthy controls. The expression of Linc01836 was significantly upregulated in CRC (1.451 ± 0.628), compared with healthy samples (0.982 ± 0.341) (Figure 1B). The results indicated that circulating Linc01836 might increase in CRC patients.

Given that there was no unified internal reference for serum lncRNA detection in CRC, 18S rRNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two most commonly used reference genes were taken into consideration. We detected their expression in 12 mixed sera, and finally, 18S rRNA was chosen for the reference gene in later research, which showed lower Ct value and acceptable repeatability (Supplementary Table S1). To verify whether the self-established qRT-PCR method for the detection of Linc01836 was available for clinical laboratory analysis, methodology evaluation, including specificity, linearity, intra- and interassay imprecision and stability, was performed. Specific single peak was exhibited in the qRT-PCR melting curves (Figure 2A). A single band also appeared in the agarose gel electrophoresis of the PCR products (Figure 2B). Sequence alignment confirmed that the amplification product was exactly Linc01836 (Figure 2C). Serial dilutions (1:1, 1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000) of cDNA from serum sample with high Linc01836 expression were used to assess the linearity. The regression equation of 18S rRNA was y = −3.832x + 7.486, and the R² of the standard curve was 0.9970. Meanwhile, the regression equation of Linc01836 was y = −2.338x + 22.6, and the R² of the standard curve was 0.9532. When expurgating the data from the maximum dilution factor (1:100,000), the R² of the Linc01836 standard curve could be raised up to 0.9810, which meant that the qRT-PCR method we established exhibited good linearity and could be applicable for serum Linc01836 detection at a very low concentration (Figure 2D). For the evaluation of imprecision, intra-assay imprecision was evaluated by 12 aliquots of mixed serum within one experimental process, and the inter-assay imprecision was performed on 12 separate days using the same samples. Related data are listed in Table 3, suggesting satisfactory precision for clinical detection. Next, we investigated the stability of serum Linc01836 by both the room temperature incubation test and the freeze–thaw experiment. In the room temperature incubation test, serum aliquots were maintained for 0, 6, 12, 18, and 24 h at room temperature. In the freeze–thaw experiment, serum aliquots were frozen and thawed 0, 1, 3, 5, and 10 times. No significant changes were found for the expressions of Linc01836 and 18S rRNA (Figures 2E, F). Taken together, all these results indicated that the performance of the self-established qRT-PCR method for Linc01836 detection in serum was satisfactory and suitable for further research.

Serum Linc01836 expression levels in 137 CRC patients, 51 patients with benign adenomas or adenomatous polyposis coli, and 138 healthy donors were detected by the abovementioned qRT-PCR, and normalized to the reference gene 18S rRNA. Since serum Linc01836 expressions did not fit the normal distribution, data were shown in the format of median and 95% confidence limit (CI). It was found that serum Linc01836 expression in CRC patients, benign patients, and healthy donors were 2.208 (1.765, 2.567), 1.104 (0.714, 1.320), and 0.990 (0.901.1.110), respectively. As illustrated in Figure 3A, serum Linc01836 expression in CRC patients was significantly higher than that in the patients with benign colorectal diseases (p < 0.0001) and healthy controls (p < 0.0001), but there was no significant difference between benign and healthy groups (p = 0.4934). These results suggested that serum Linc01836 could be conducive in the auxiliary diagnosis of CRC.

According to previous studies, some circulating noncoding RNAs with upregulated expressions in tumor patients could return to a favorable level, some even showing no significance with expressions in healthy people. To verify whether detection of serum Linc01836 expression was helpful for tumor dynamic monitoring, a comparison of Linc01836 expressions between 137 preoperational sera and 82 postoperational sera was carried out (Figure 3B). Results manifested that the level of serum Linc01836 decreased significantly after surgical resection (p = 0.0001). Moreover, we analyzed circulating Linc01836 expressions from 48 pairs of CRC patients before and after surgery. As illustrated in Figure 3C, results from pre- and postoperation groups also confirmed the same conclusion that serum Linc01836 levels declined after surgery (p = 0.0082). Moreover, to better understand the relationship between overall survival rate and Linc01836 expression in CRC patients, we downloaded the survival data from TCGA database and classified CRC patients into high-expression group and low-expression group by the median of Linc01836 expression in tissues. The survival curve showed that the high-expression group possessed lower overall survival rate (Figure 3D). To sum up, serum Linc01836 could be served as a new tumor marker for early diagnosis of CRC and might be used for cancer surveillance.

To further understand the clinical application of serum Linc01836 detection, the clinicopathological features of the 137 CRC patients are summarized in Table 4. CRC patients were divided into two groups according to the median (2.028) of serum Linc01836 expression: relative high group (expression >2.028, n = 68) and relative low group (expression ≤2.028, n = 69). Chi-square test showed that high serum Linc01836 expression levels were significantly associated with histological stage (p = 0.002) and lymph node metastasis (p = 0.006), but had no statistical significance with other parameters, such as gender (p = 0.349), age (p = 0.932), tumor size (p = 0.797), tumor location (p = 0.671), nerve/vascular invasion (p = 0.917), and morphological characteristics (p = 0.808). It was worthy to mention that the T stage represented the depth of tumor invasion; even though no statistical significance was found in the T stage and TNM stage in general, it displayed obvious correlation between increased serum Linc01836 expression and lymph node metastasis. We then compared the serum Linc01836 expression in 137 CRC patients at different stages of lymph node metastasis, and the results manifested a significant difference between N0 and N2 stages (p = 0.0144). Although the difference between N0 and N1 stages (p = 0.621) and the difference between N1 and N2 stages (p = 0.113) did not show any significance, we still could intuitively see that serum Linc01836 levels increased gradually with the escalating degrees of lymph node metastasis (Figure 3E).

Although the sensitivity and specificity of commonly used serum tumor biomarkers were undesirable, they still were most widely used in clinics for the early screening of malignancies. Therefore, we also examined serum CEA, CA19-9, Cyfra21-1, and CA72-4 levels in CRC patients and healthy donors. Results are shown in Supplementary Table S2, exhibiting higher concentrations in the CRC group (Figures 4A–D). ROC curves were generated to assess the potential of Linc01836 along with these serum tumor biomarkers as diagnostic biomarkers for CRC, which demonstrated that serum Linc01836 could effectively differentiate CRC patients from the healthy folks, with the favorable area under the curve (AUC) of 0.809 (95% CI: 0.757–0.861, p < 0.001, Figure 4E). CEA, as the most commonly used tumor biomarker in the clinic, exhibited similar diagnostic value, with an AUC of 0.804 (95% CI: 0.754–0.855, p < 0.001). Cyfra21-1 was usually used for the diagnosis of lung cancer, non-small cell lung cancer (NSCLC), in particular. Surprisingly, it also possessed acceptable diagnostic value, with an AUC of 0.702 (95% CI: 0.639–0.764, p < 0.001) in our research. However, CA19-9 and CA72-4, as commonly accepted gastrointestinal tumor markers, did not perform well with AUCs of 0.569 (95% CI: 0.500–0.637, p = 0.049) and 0.424 (95% CI: 0.356–0.492, p = 0.030), respectively (Figure 4F). Cutoff value was often set by the expression level at the maximum value of the Youden index. As for the relative Linc01836 expression in serum samples, the cutoff value was defined as 1.63, offering sensitivity of 65.0% and specificity of 87.0%, and the accuracy of differentiating CRC from a healthy population could reach 76.0%, which was superior to CEA (73.8%), Cyfra21-1 (68.7%), CA19-9 (59.3%), and CA72-4 (53.1%). Subsequently, we drew the ROC curves for the joint diagnosis and evaluated their efficacy concomitantly. As illustrated in Figure 4G and Table 5, combined detection of Linc01836, CEA, and Cyfra21-1 displayed the best diagnostic efficacy, harboring an AUC of 0.916 (95% CI: 0.881–0.950, p < 0.001), higher than that of a combination of Linc01836 and CEA [AUC = 0.905 (95% CI: 0.871–0.939, p < 0.001)] and a combination of Linc01836 and Cyfra21-1 [AUC = 0.864 (95% CI: 0.820–0.909, p < 0.001)]. Our results confirmed the diagnostic utility of serum Linc01836, CEA, and Cyfra21-1 and their combination in differentiating CRC patients from healthy population.

To investigate the biological function and underlying mechanisms, we extracted RNA from NCM460 and SW480 cells by nucleoplasm separation, and qRT-PCR results manifested that Linc01836 was averagely located in the nucleus and cytoplasm, suggesting that it might participate in CRC progression through both transcriptional and posttranscriptional regulation (Figure 5A). Crosstalk among lncRNAs, miRNAs, and mRNAs plays a pivotal role in the initiation and progression of CRC (Wang et al., 2019; Xu et al., 2019). Subsequently, the potential lincRNA–miRNA–mRNA regulatory axis in CRC was predicted by bioinformatics analysis. As shown in Figures 5B, C, eight miRNAs (hsa-miR-663b, hsa-miR-503-5p, hsa-miR-1231, hsa-miR-4665-5p, hsa-miR-1299, hsa-miR-133a-5p, hsa-miR-488-5p, and hsa-miR-1275) and their corresponding target mRNAs associated with prognosis were depicted, which might offer a new guidance in exploring the regulatory network of Linc01836 in CRC in the future.