LINC 01436 is overexpressed in colorectal cancer and promotes cancer cell proliferation by suppressing tumor-suppressive miR-466 maturation

LINC 01436 (lncRNA) promotes lung and gastric cancers. However, it is unclear whether it participates in colorectal cancer (CRC) progression. Therefore, the study was carried out to analyze the role of LINC 01436 in CRC. LINC 01436 expressions in CRC tissues were analyzed by RT-qPCR, and its prognostic value was investigated in a follow-up study. Correlation between LINC 01436 and mature miR-466 or miR-466 precursor was analyzed by linear regression. Mature miR-466 and miR-466 precursor levels in CRC cells with LINC 01436 overexpression were studied using RT-qPCR. CRC cell proliferation was evaluated using CCK-8 assay. LINC 01436 was upregulated in CRC and predicted poor survival. LINC 01436 was inversely correlated with mature miR-466, but not miR-466 precursor. LINC 01436 was predicted to bind with miR-466 precursor. Their interaction was further verified by dual-luciferase activity assay. In CRC cells, LINC 01436 overexpression downregulated mature miR-466 but not miR-466 precursor. Cell proliferation analysis showed that LINC 01436 overexpression rescued cell proliferation reduced by miR-466. LINC 01436 is overexpressed in CRC and promotes cancer cell proliferation by suppressing miR-466 maturation.


Introduction
Colorectal cancer (CRC), also known as colon cancer, bowel cancer, or rectal cancer, is a type of malignancy originating from the rectum or colon (Siegel et al. 2017a, b;Bray et al. 2018). With people's lifespan increasing and lifestyle changing, the burden of CRC is expected to increase by 60% by 2030 worldwide (Arnold et al. 2017). It is estimated that more than 90% of patients with localized CRC can survive 5 yr after initial diagnosis (Favoriti et al. 2016). CRC is rarely diagnosed at early stages, and the 5-yr survival rate of metastatic CRC is below 15% (Mahasneh et al. 2017;Siegel et al. 2017a, b). Therefore, novel therapeutic approaches are needed.
It has been well established that physical inactivity, obesity, smoking, and alcohol abuse are the major risk factors for CRC (Vargas and Thompson 2012;Johnson et al. 2013). Besides that, molecular factors also contribute to CRC development and progression (Pritchard and Grady 2011). Understanding the function of these factors provides novel insights into the development of targeted anti-CRC approaches (Heinemann et al. 2013;Mitchell 2013). However, the development of targeted therapy is limited by lacking promising targets. Although lncRNAs are non-coding RNAs, they regulate cancers by affecting gene expression (Prensner and Chinnaiyan 2011;Xie et al 2021). LINC 01436 promotes lung and gastric cancers (Yuan et al. 2019;Zhang et al. 2020). It is hardly known whether this lncRNA also participates in CRC. We have previously performed a preliminary deep sequencing analysis and observed altered LINC 01436 expression and its inverse correlation with mature miR-466, a cancer-related miRNA (Colden et al. 2017). Therefore, in this study, we further explored the potential interaction between LINC 01436 and miR-466 in CRC.

CRC patients and follow-up
The study was approved by the Ethics Committee of People's Hospital of Baoan District. A total of 62 CRC patients (36 males and 26 females) with ages from 46 to 68 yr (57.1±5.6 yr) were enrolled from June 2013 to May 2015. CRC patients were diagnosed by histopathological examinations. Patients with initiated therapy and recurrent CRC were excluded. The 62 enrolled patients were grouped into American Joint Committee on Cancer (AJCC) stage I or II (n=26) and III or IV (n=36). Based on the AJCC stages, they were treated with different therapies. A follow-up (5 yr) was performed monthly to record their survival. Informed consent was signed by all patients.
CRC tissues and cells Prior to treatments, CRC and paired non-tumor tissues were obtained from all patients. Following confirmation by histopathological examinations, the tissue samples were stored in liquid nitrogen before use.
Human CRC cell lines WiDr (ATCC ® CCL-218™) and HT-29 (ATCC ® HTB-38™) purchased in 2020 from ATCC ( Manassas, VA) were used for all cell experiments and cultured at 37°C in McCoy's 5a Medium Modified (Catalog No. 30-2007) with 10% FBS following the instructions from ATCC. Cells were authenticated by STR analysis in 2021 and mycoplasma-free. RNA isolations Total RNAs were isolated using Ribozol reagent (Invitrogen) and treated with DNase I (Invitrogen) for 80 min at 37°C. RNA integrity was determined on 5% urea-PAGE gels (5%). RNA purity was determined by calculating OD 260/280 ratios.
Dual-luciferase reporter assay LINC 01436 luciferase vector was constructed by inserting the binding site of miR-466 on LINC 01436 into the region between SV40 promoter and Luc (+) of pGL3 luciferase reporter vector (Promega Corporation, Madison,WI). Cells were transfected with LINC 01436 luciferase vector combined with NC miRNA (NC group) or miR-466 precursor (miR-466 group), and luciferase activity was determined at 48 h post-transfection. After transcription, a fusion RNA composed of RUNX1-IT1 + Luc (+) was produced. The binding of miR-466 to the fusion RNA reduced the translation of Luc (+), resulting in decreased luciferase signal.
RNA pull-down assay Flag assaysion RNA redu RNA pull-down assay was conducted to determine the interaction between LINC 01436 and miR-466. Specifically, pcDNA3-FlagMS2 bp and pcDNA3-LINC 01436-MS2bs were co-transfected to cells. Two days after transfection, cells were collected. About 1 × 10 7 cells from each sample were dissolved in the soft lysis buffer plus 80 U/mL RNasin (Promega) and with 50 μL ANTI-FLAG M-280 Magnetic Beads (Invitrogen) for 4 h. After washing the beads with lysis buffer six times, RNAs were retrieved and subjected to qRT-PCR.
RT-qPCRs RNA samples with an OD 260/280 ratio close to 2.0 were used to synthesize cDNA samples and subjected to qPCRs with 18S rRNA as the internal control to study LINC 01436 expression. To determine miR-466 precursor expression, a sequence-specific reverse primer was used to perform reverse transcriptions (RTs), and sequence-specific forward and reverse primers were used to perform qPCRs. To determine mature miR-466 expression, miRNAs were subjected to RTs using poly (T) as the reverse primer after poly (A) addition. After that, qPCRs were performed using All-in-One™ miRNA qRT-PCR reagent kit (GeneCopoeia, Rockville, MD) with poly (T) and the sequence-specific forward primer. The Ct values were analyzed using the 2-ΔΔCt method. Primers are listed in supplemental Table 1.
Statistical analysis Gene expression levels in paired tissues were expressed as the average of three technical replicates and subjected to paired t test. Other data were expressed as mean ± standard deviation (SD) of three biological replicates and subjected to ANOVA Tukey's HSD test. Correlation analysis was performed using linear regression. Patients were classified into high and low LINC 01436 level groups with the median LINC 01436 level in CRC tissues as the cutoff value (n=31) and compared using log-rank test. p<0.05 was deemed statistically significant.

Results
LINC 01436 was highly expressed in CRC tissues and predicted poor survival LINC 01436 expressions in CRC and paired non-tumor tissues were determined by RT-qPCR. LINC 01436 was significantly upregulated in CRC tissues (Fig. 1A,p<0.001). Survival analysis illustrated that high LINC 01436 expression was closely correlated with poor survival (Fig. 1B). In addition, LINC 01436 levels were not correlated with cancer stages (Supplementary Fig. 1). These data suggest that LINC 01436 is an independent prognostic factor for CRC.
LINC 01436 was only correlated with mature miR-466 Mature miR-466 and miR-466 precursor in CRC tissues were detected by RT-qPCR. The expression of mature miR-466 and miR-466 precursor was determined by RT-qPCR. Mature miR-466 was significantly decreased in CRC tissues (Supplementary Fig. 2A, p<0.05). MiR-466 precursor level was not significantly different between CRC tissues and non-tumor tissues ( Supplementary Fig. 2B). Correlation analysis illustrated that LINC 01436 level was inversely correlated with mature miR-466 ( Fig. 2A) but not with miR-466 precursor (Fig. 2B). Therefore, LINC 01436 may suppress miR-466 maturation in CRC.

LINC 01436 directly interacted with miR-466
Nuclear and cytoplasm samples of WiDr cells were prepared and subjected to RNA isolations and RT-PCR to determine the subcellular location of LINC01436, mature miR-466, and miR-466 precursor in WiDr cells. LINC01436, mature miR-466, and miR-466 precursor were all detected in both nuclear and cytoplasm fractions (Fig. 3-A-C). The interaction between LINC 01436 and miR-466 precursor was predicted by IntaRNA 2.0 (http:// rna. infor matik. uni-freib urg. de/ IntaR NA/ Input. jsp). It showed that LINC 01436 and miR-466 precursor might form strong base pairing (Fig. 3D). Dual-luciferase reporter assay showed that miR-466 precursor expression significantly decreased luciferase activity compared to the NC group, suggesting a direct interaction between LINC Figure 1. LINC 01436 was overexpressed in CRC and correlated with patients' survival. Paired CRC and non-tumor tissues from 62 CRC patients were collected, and LINC 01436 expression levels in these tissue samples were determined by RT-qPCR. QPCRs were performed in three technical replicates, and the average values were presented. ***p<0.001 (A). Survival analysis was performed following the methods described in the "Methods" section using data of the 5-year follow-up study. The comparison of survival curves was performed using the log-rank test (B). 01436 and miR-466 precursor (Fig. 3E, p<0.05). In addition, we performed RNA to pull down using a biotinylated miR-466 probe. The data indicated that endogenous LINC 01436 was enriched specifically in miR-466 pull-down complexes compared with the control, suggesting that miR-466 is a direct inhibitory target of LINC 01436 (Fig. 3F, p<0.05).
In addition, miR-466 overexpression decreased and miR-466 inhibition increased WiDr and HT-29 cell proliferation. Moreover, LINC 01436 overexpression reduced the inhibitory effects of miR-466 overexpression on cell proliferation (Fig. 5, p<0.05). To detect the impact of LINC 01436 knockdown on mature miR-466 expression, LINC 01436 was inhibited in WiDr and HT-29 cells, and the mature miR-466 level was measured. The results showed that LINC 01436 knockdown increased mature miR-466 level and decreased WiDr and HT-29 cell proliferation (Supplementary Fig. 2A-D, p<0.05). In addition, we examined the changes in cell cycle and apoptosis. WiDr and HT-29 cells were stained with PI for FACS analysis. The results indicated that LINC01436 overexpression decreased the percentage of cells in G0/G1 phase and increased the percentage of cells in S phase ( Supplementary Fig. 3A-B, p<0.05). LINC01436 overexpression decreased the percentages of apoptotic WiDr and HT-29 cells ( Supplementary Fig. 3E-F, p<0.05). MiR-466 overexpression significantly increased the percentage of cells in the G0/G1 phase, decreased cells in the S phase ( Supplementary Fig. 3C-D, p<0.05), and promoted the percentages of apoptotic WiDr and HT-29 cells ( Supplementary Fig. 3G-H, p<0.05).

Discussion
We studied the crosstalk between LINC 01436 and miR-466 in CRC. The results showed that LINC 01436 was overexpressed in CRC and LINC 01436 expression levels predicted the survival of CRC patients. In addition, LINC 01436 may suppress miR-466 maturation to promote CRC cell proliferation.
Previous studies have reported that LINC 01436 plays an oncogenic role in both lung cancer and gastric cancer (Yuan et al. 2019;Zhang et al. 2020). In lung cancer, LINC 01436 is a hypoxia-sensitive lncRNA regulated by E2F6. In addition, LINC 01436 overexpression increases cancer cell proliferation, invasion, and migration by targeting tumor-suppressive miR-30a-3p (Yuan et al. 2019). LINC 01436 is also overexpressed in gastric cancer. It sponges miR-585 and suppresses FBXO11 translation by binding to its promoter region to promote tumor growth and metastasis (Zhang et al. 2020). In this study, we first reported LINC 01436 upregulation in CRC. Moreover, LINC 01436 overexpression increases CRC cell proliferation. Therefore, LINC 01436 plays an oncogenic role in CRC by increasing cancer cell proliferation. CRC treatment is limited by the low early diagnostic rate, which is unlikely to be significantly improved in the near future, mainly due to the lack of sensitive biomarkers (Mahasneh et al. 2017). We found that LINC 01436 predicts CRC patients' poor survival. Therefore, LINC 01436 may serve as a biomarker to guide the determination of therapies to improve the survival of CRC patients. However, clinical trials are needed to test our hypothesis.
MiR-466 is a tumor suppressor in several cancers (Colden et al. 2017;Tong et al. 2018). It is downregulated in CRC, and its overexpression suppresses cancer proliferation. This study confirmed the inhibitory effects of miR-466 on CRC cell proliferation. Previous studies mainly investigated the interactions between LINC 01436 and miRNAs (Yuan et al. 2019;Zhang et al. 2020). In this study, we showed that LINC 01436 could downregulate mature miR-466, but not miR-466 precursor. Therefore, LINC 01436 may suppress miR-466 maturation to participate in cancer biology. A recent study showed that lncRNA CCAT2 could suppress miR-145 maturation by suppressing the transportation of miR-145 precursor (Yu et al. 2017). LINC 01436 may also suppress miR-466 transportation from the nucleus to the cytoplasm, where mature miR-466 is produced from its precursor. However, some limitations should be noted. The study did not explore the target genes of miR-466. More studies are needed to validate the target genes of miR-466 and explore other possible mechanisms.

Conclusion
LINC 01436 is overexpressed in CRC and predicts poor survival of CRC patients. In addition, LINC 01436 may suppress miR-466 maturation to promote CRC cell proliferation.
Author contribution HL, DH: study concepts, literature research, clinical studies, data analysis, experimental studies, manuscript writing, and review; HL, WD: study design, literature research, experimental studies, and manuscript editing; JH: data acquisition, manuscript preparation, and data analysis.
All authors have read and approved the submission of the manuscript.

Availability of supporting data
The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants, but are available on reasonable request from the corresponding author. Figure 5. LINC 01436 overexpression increased WiDr and HT-29 cell proliferation through miR-466. The roles of LINC 01436 and miR-466 in regulating WiDr and HT-29 cell proliferation were analyzed by CCK-8 assay. Cell proliferation was reflected by OD values at 450 nm, which were measured every 24h until 96h. The mean±SD values of three biological replicates were presented. *p<0.05.

Declarations
Ethics approval and consent to participate Informed consent was obtained from all individual participants included in the study. All procedures were approved by the Ethics Committee of People's Hospital of Baoan District and operated in keeping with the standards set out in the Announcement of Helsinki and laboratory guidelines of research in China.

Consent for publication Not applicable
Competing interests The authors declare no competing interests.