The long noncoding RNA LINC00341 suppresses colorectal carcinoma by preventing cell migration and apoptosis

Long noncoding RNAs (lncRNAs) are ubiquitous transcripts that play key roles in regulating gene expression at the levels of transcription, RNA processing, and translation. Aberrant expression and mutations of lncRNAs represent a driving force behind oncogenesis and development of tumours. However, most of the lncRNAs are still being undiscovered, and conclusive experimental evidence for their functional relevance continues to be lacking for most malignancies. We have found that lncRNA long intergenic non–protein‐coding RNA 341 (LINC00341) is aberrantly downregulated by microarray‐based screenings on nonmetastatic and metastatic colorectal carcinoma (CRC) specimens; LINC00341 is a novel long intergenic non–protein‐coding RNA with unknown functions. LINC00341 overexpression restricts tumour growth and promotes its apoptosis. Instead, LINC00341 silencing accelerates CRC cell proliferation and migration. RNA‐pulldown assay identifies LINC00341 physically binds to HMGB2 and stabilizes the localization of HMGB2 in the cytoplasm. Notably, LINC00341 knockdown leads to the shift of HMGB2 into nuclear, in which it triggers epithelial to mesenchymal transition (EMT) programming. Moreover, LINC00341 can also promote apoptosis. Significance of the study LncRNAs are ubiquitous transcripts that play key roles in regulating gene expression at the levels of transcription, RNA processing, and translation. Aberrant expression and mutations of lncRNAs represent a driving force behind oncogenesis and development of tumours. However, the function of lncRNA still needs further exploration. Our study has revealed a new noncoding RNA‐mediated regulatory network that highly likely protects colorectal carcinoma by preventing migration and apoptosis. The results will help further explore the molecular details about the progression of colorectal carcinoma and stimulate efforts to develop effective therapies.

Long noncoding RNAs (lncRNAs) are a group of non-proteincoding transcripts that are longer than 200 nucleotides. [5][6][7] Despite of fast turnover rates and low copy numbers, lncRNAs have been widely accepted as truly functional biomolecules. 8 Recent studies have shown that long-chain noncoding RNA (lncRNAs) plays a major role in human cancer. 9 Their discovery and research have changed our understanding of cancer cell biology to a certain extent. At present, some lncRNAs are thought to activate tumour suppressor genes or oncogenes in common cancers and participate in the occurrence and development of cancers, 10 and their molecular mechanisms are also elucidated. Nevertheless, there are still many unknown or controversial functional mechanisms of lncRNAs at the forefront of cancer research. 11 It is imperative to further study its identification, function, and mechanism, which may be brilliant in the basic and clinical research on cancer. LINC00341 is a novel long intergenic non-protein-coding RNA with unknown functions. In this report, we demonstrate that lncRNA long intergenic non-proteincoding RNA 341 (LINC00341) is aberrantly downregulated in CRC.
Low expression of LINC00341 promoted patient's poor survival, as well as cancer metastasis. Epithelial to mesenchymal transition (EMT) is widely regarded as a key step for CRC to acquire an invasive or metastatic phenotype. It has been reported that TGFβ triggers EMT process by dampening E-cadherin and upregulating vimentin in multiple CRC cell lines. 12 Recent studies have also shown that lncRNAs activated by TGFβ is upregulated in serum of CRC patients, and EMT is induced by reducing the expression of epithelial markers E-cadherin, ZO-1, and increasing the expression of interstitial markers ZEB1 and N-cadherin (n-cad). 13,14 In our study, we concluded that LINC00341 can bind to HMGB2 to inhibit tumour migration. In this report, we also demonstrate that partially restoring  Figure 1A). Next, total RNAs were isolated from the colorectal tissues, and RNA levels were determined by qRT-PCR. We found that the levels of lncRNA LINC00341 in the colorectal tissues were much lower in tumour colorectal tissues ( Figure 1B). To validate this result, we also performed quantitative real-time PCR.
Total RNAs were isolated from the adjacent colorectal tissues and F I G U R E 1 Differential expression of LINC00341 in colorectal cancer. A, Heat map and hierarchical clustering (log2 fold change and q value <0.05) are presented to show the variation in lncRNAs between paired colorectal cancer and adjacent nontumor colorectal tissues. Red indicates high expression, and green indicates low relative expression. B, LINC00341 expression in normal samples and CRC samples was measured by qRT-PCR. C, LINC00341 expression in 36 paired with colorectal carcinoma tissues was determined using real-time PCR assays, compared with the adjacent tissues. LINC00341 expression level was normalized to GAPDH. Results are shown as means ± SD by two-tailed Student's t test. *P<0.01. **P<0.001. ***P<0.0001 colorectal tissues. We can also find that LINC00341 is downregulated ( Figure 1C).

| Decreased LINC00341 expression correlated with tumour progression and poor prognosis of CRC patients
Based on the decreased LINC00341 expression in colorectal cancer, we next evaluate the relationship between LINC00341 and CRC progression, and we analysed the correlation between high LINC00341 expression and clinicopathological features of CRC; the data are summarized in (Table 1). No significant association was found between LINC00341 expression and age (P = .534), gender (P = .362), and location (P = .364). However, LINC00341 expression significantly correlated with TNM stage (P = .012), differentiation (P = .021), AJCC Stage I/II (P = .041), AJCC Stage III/IV (P = .009), and lymph node metastasis (P = .018). Next, we evaluated the prognostic effect of LINC00341 on overall survival by comparing the overall survival of CRC patients with high or low LINC00341 levels. A total of 34 paired cases of CRC patients were divided into two groups: a high LINC00341 expression group (above the median LINC00341 expression, n = 17) and a low LINC00341 expression group (below the median LINC00341 expression, n = 17). Among the participants, patients with high LINC00341 expression were associated with a significantly higher survival rate than those with a low expression, according to Kaplan-Meier curve assessment (P = .016, log-rank test; Figure 2B). The above findings suggested that a decreased LINC00341 expression was significantly correlated with progression, metastasis, and poor outcome in CRC patients.

| Downregulated and upregulated LINC00341 in CRC cells in vitro
The expression of LINC00341 was analysed in five CRC cell lines and a normal human intestinal mucosal cell line NCM460. As shown in Figure 2A, lower expression of LINC00341 was found in all CRC cell lines, 15 Figure 2C). On the contrary, SW620 cells were transfected with siRNA for LINC00341 to knock down LINC00341 expression. Among the three siRNA, we select the most efficient one for subsequent experiments ( Figure 2D).

| LINC00341 inhibits aggressive phenotypes and reverses EMT process of CRC cells in vitro
To  Figure 3E). Conversely, SW620 cells were transfected with siRNA for LINC00341 to knock down LINC00341 expression significantly promoted the cell proliferation of the SW620 ( Figure 3F). As showed above, LINC00341 strikingly reduced the potential of cell migration, invasion, and motility, respectively.

| LINC00341 promotes cell apoptosis process of CRC cells in vitro
We found that the levels of lncRNA LINC00341 in the CRC tissue were much lower than those in the normal tissues ( Figure 1B). This  HMGB2 is a key EMT-associated transcription factors. To seek proteins that are interacted with LINC00341, RNA-pulldown assay was conducted in SW620 cells. RNA-associated proteins were identified by SDS-PAGE and subjected to mass spectrometry. Among all proteins identified by mass spectrometry, HMGB2 was successfully validated by Western blot with RNA-pulldown analysis. After pulldown operation with the sense sequence of LINC00341, significant enrichment of HMGB2 was detected ( Figure 5A). But no enrichment was observed with the antisense LINC00341 as blotted by HMGB2 antibody. We further use the HMGB2 antibody as a bait to carry out RNA immunoprecipitation (RIP) with cell extracts from the SW620 tumour cell lines. We observed that the LINC00341 enrichment, with GAPDH mRNA, remains unchanged, using the HMGB2 antibody vs a nonspecific antibody (IgG control) ( Figure 5B). HMGB2 is a nuclear protein that binds to DNA and plays a role in chromatin remodelling. LINC00341 overexpression significantly decreased its level in the nucleus and increased the level of HMGB2 in the cytoplasm. In contrast, LINC00341 silencing led to the enrichment of HMGB2 in the nuclear ( Figure 5C,D). To further prove the interaction of LINC00341 with HMGB2. We overexpressed HMGB2 at the cellular level, and we found HMGB2 can upregulate mRNA expression E-cadherin and fibronectin, however, the mRNA expression of ZO-1 was decreased.
Meanwhile, we further overexpress LINC00341. As we expected, the increase E-cadherin and fibronectin range of HMGB2 is weakened due to the overexpression of LINC00341. Under previous results, LINC00341 reversed the decrease of ZO-1 mRNA level caused by overexpression of HMGB2 ( Figure 5E). The same results were verified on Western blot ( Figure 5F). So we came to this conclusion.

| DISCUSSION
LncRNA imbalance has been found in many human cancers, including CRC, which is the third most common cancer in the world with poor In recent years, many studies have shown that lncRNAs were associated with the occurrence and development of various tumours.
Among them, lncRNAs have been increasingly reported being involved in colorectal cancer. 25 However, the expression and biological function of lncRNA LINC00341 in colorectal cancer is not illuminated. In our study, we first found that the LINC00341 level in colorectal cancer was significantly lower than that in normal adjacent tissues. Overexpression of LINC00341 can suppress the proliferation and invasion/migration and promote apoptosis in CRC cells. In summary, LINC00341 is an effective tumour suppressor, which is related to a better prognosis of CRC patients. By stabilizing the cytoplasmic localization of HMGB2, it can play an antimetastasis role. Furthermore, we found that LINC00341 can promote cell apoptosis, but the molecular biological mechanism of LINC00341 has not been well studied, and the underlying molecular mechanism needs further study.

| RNA-pulldown analysis and RIP assay
The biotinylated LINC00341, antisense LINC00341, or mutated LINC00341 was mixed with proteins obtained from cancer cells for overnight at 4 C. The complex of biotinylated lncRNA and proteins was purified using streptavidin-agarose for 4 hours at 4 C. The proteins are then eluted from the RNA-protein complex and detected by Western blotting analysis. RIP assay was performed using RIP RNAbinding protein immunoprecipitation kit (Merck Millipore) and following the manufacturer's protocol.

| Statistical analysis
Data were analysed using GraphPad Prism 5. Differences between two comparisons were evaluated using Student's t test. Analysis of variance method was performed to analyse the differences in multiple comparisons. Log-rank analysis was applied in survival comparisons.
Statistical significance was established at P < .05.

This work was supported by The Foundation of Tianjin Union Medical
Center (2016rmnk002).

CONFLICT OF INTEREST
The authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT
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