Prognostic Lnc-S100B-2 Affects Cell Apoptosis and Microenvironment of Colorectal Cancer through MLLT10 Signaling

Long noncoding RNA (LncRNA) is closely associated with the development of colorectal cancer (CRC). The chip data and clinical information of GSE104364 and GSE151021 were downloaded by GEOquery. Limma and Kaplan–Meier analysis were performed. Lnc-S100B-2 was obtained, and high expression of Lnc-S100B-2 was predicted to be associated with a lower survival rate. Online software was adopted to predict downstream regulatory genes, and miR-331-3p and Mixed Lineage Leukemia Translocated to 10 (MLLT10) were screened and verified. After silencing Lnc-S100B-2 and MLLT10, the proliferative activity of CRC cells decreased, and the apoptosis rate increased. At the gene and protein levels, the expressions of PCNA, Ki67, and Bcl-2 were decreased in the sh-Lnc-S100B-2 group, sh-MLLT10 group, and sh-Lnc-S100B-2 + sh-MLLT10 group, while the expressions of cleaved caspase 3, caspase 9, and Bax were increased. In vivo, the volume and mass of the tumor decreased in the sh-Lnc-S100B-2 + sh-MLLT10 group. Proliferation and apoptosis-related index (PCNA, Ki67, cleaved caspase 3, caspase 9, Bax, and Bcl-2) expression level was also altered. Meanwhile, the infiltration of immune cells (CD3 (-), CD16 (+), and CD11b (+) cells) decreased. The expressions of epithelial-mesenchymal transformation (EMT) related indicators (E-cadherin, N-cadherin, Vimentin, β-catenin, Snail, and Slug) were changed. E-cadherin and β-catenin were increased in the sh-Lnc-S100B-2 + sh-MLLT10 group, while N-cadherin, vimentin, snail, and slug were decreased. In conclusion, our study found that the expression of Lnc-S100B-2 was dysregulated in CRC. Lnc-S100B-2 could affect cell apoptosis and the microenvironment of CRC through regulating MLLT10.


Introduction
Colorectal cancer (CRC) is one of the most common malignant tumors in humans and the fourth deadliest cancer in the world, with nearly 900,000 deaths every year [1]. CRC has become a major global public health problem [2]. As previously described, CRC might develop in patients with distinct intestinal diseases such as inflammatory bowel diseases, microscopic colitis, and irritable bowel syndrome [3]. It might bring some difficulties to the diagnosis of CRC. Studies have shown that some progress has been made in diagnosing, treating, and preventing CRC. For example, colonoscopy's targeted screening and surveillance policy will curb the rising incidence of CRC [4]. Allium constituents are shown to modify the risk of colon cancer and reduce the mortality rates associated with this malignancy [5]. e poor prognosis of CRC patients remains a major problem [6]. CRC patients are usually diagnosed as advanced, with a poor prognosis and a low 5-year survival rate [7]. Previous studies have shown that the poor prognosis of CRC is related to molecular and gene changes [8]. Differential genes and molecules have essential research value in CRC [9,10].
Long noncoding RNAs (LncRNAs) are more than 200 nucleotides in length without protein-coding potential. LncRNAs are involved in regulating biological processes such as cell proliferation, differentiation, migration, and invasion [11][12][13] via mediating interactions between DNA and proteins, adsorbing microRNAs, and binding to proteins as decoys [14,15]. In recent years, studies on LncRNAs have attracted widespread attention. LncRNA interacts with cell metabolism (glucose metabolism, mitochondrial function, and oxidative stress) to affect cancer development [16]. In breast cancer and bladder cancer studies, LncRNAs can be used as prognostic markers for patients [17,18]. Similarly, in studies on CRC, prognostic LncRNAs have been found to promote or inhibit the growth, metastasis, invasion, and affect the microenvironment of CRC [9,19,20]. However, the role of Lnc-S100B-2 played in CRC cells, and the CRC microenvironment has never been reported previously.
Mixed Lineage Leukemia Translocated to 10 (MLLT10) is a transcriptional activator of gene expression. MLLT10 rearrangement is closely related to the development of leukemia. MLLT10 is one of the most common fusion partners of mixed-lineage leukemia (MLL, also known as KMT2A) in acute leukemia [21]. MLLT10 and IL3 are involved in gene rearrangement in patients with early T-cell precursor acute lymphoblastic leukemia [22]. Meanwhile, MLLT10 might be involved in the metastasis of non-small cell lung cancer [23]. e expression of MLLT10 is different in CRC [24]. However, the regulatory pathway of MLLT10 in CRC remains unclear.
In the study, we aimed to obtain prognostic LncRNA and their downstream regulatory genes through database screening and bioinformatics prediction. e expression of genes and their interrelationships were verified by experiments. Its functions were verified by in vitro and in vivo experiments. e study was expected to provide a biomarker and a promising therapeutic target for the treatment of CRC.
Limma was used to analyze LncRNA differentially expressed on chip data [25], selection criteria for | logFC | > 1 and P < 0.05. e R-package pheatmap was used to cluster the expression patterns of differentially expressed LncRNAs in the two groups, and a heatmap was drawn for visualization.

Clinical
Specimens. CRC samples (N � 5) and matched adjacent tissues (N � 5) were randomly collected from Zhuzhou Central Hospital. Before participation, we obtained the informed consent of the study subjects.

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR).
e Trizol method was used to isolate the total RNA from tissues and HCT116 cells. Briefly, 0.02 g tissues or 5 × 10 6 cells were lysed with 1 mL Trizol. Isopropyl alcohol and ethanol were successively added for extraction and separation. 30 μL sterile enzyme-free water was used to dissolve RNA precipitates. After detecting the RNA concentration, HiFiScript cDNA Synthesis Kit (CW2569 M, CWBIO, China) and miRNA cDNA Synthesis Kit (CW2141S, CWBIO, China) were used reverse transcription with a 20 μL reverse transcription reaction system. SYBR-Green PCR Master Mix (CW2601S, CWBIO, China) was used for PCR amplification using the 30 μL amplification system. 40 cycles were amplified. 2 −ΔΔCt was applied to calculate RNA expression levels. e sequences of primers used in the study were listed at Table 1. e expression of U6 and β-actin was applied as control.
2.5. Plate Clone Formation Assay. As previously described, the plate clone formation assay was adopted to detect cell proliferation [26]. Briefly, cells were digested with 0.25% trypsin (C0201, Beyotime) and cultured for 14 days. e cells were fixed with 4% paraformaldehyde ((N1012, NCM Biotech) for 15 min and stained with crystal violet (G1062, Solarbio) for 30 min. A microplate reader (MB-530, HEALES) was adopted to measure the cell colony number, and pictures were taken.

Western Blot.
e RIPA buffer (P0013 B, Beyotime) was used to extract proteins by lysing cells and tissues. e SDS-PAGE gel was used to separate the proteins. e proteins were transferred to the nitrocellulose membrane. 5% skimmed milk was used to block the membrane at 4°C overnight. e membranes were incubated with primary antibodies or secondary antibodies at room temperature (RT) for 90 min. e antibodies used were as follows: anti 1000, #9585, CST), HRP goat anti-mouse IgG (1 : 5000, SA00001-1, proteintech), and HRP goat anti-rabbit IgG (1 : 6000, SA00001-2, proteintech). Proteins were detected by Western Bright ECL kit (K-12045-D50, advansta). e expression of β-actin was applied as control.

Flow
Cytometry. Apoptosis analysis was as follows. e cells were digested by trypsin without EDTA. Cells were washed twice by PBS and centrifuged at 2000 rpm for 5 min. 500 μL binding buffer was added to resuspend cells. After being mixed with 5 μL Annexin V-FITC, 5 μL propidium iodide (PI) was added to the cells and mixed and incubated for 10 min in the dark at RT. Flow cytometry (A00-1-1102, Beckman Coulter, USA) was used for observation and analysis.
Cell-cycle analysis was as follows. e cells were digested by trypsin and centrifuged at 800 rpm for 5 min. After being resuspended with 400 μL PBS, 1.2 mL of 100% precooled ethanol was added, and the cells were placed at 4°C overnight. Cells were washed twice with 1 mL precooled PBS. en, cells were fixed with 150 μL PI staining solution and incubated for 30 min in the dark at 4°C. Flow cytometry (A00-1-1102, Beckman Coulter, USA) was applied to analyze the cell cycle.

Animal Experiments.
Male BALB/c nude mice (N � 24) were purchased from Human SJA Laboratory Animal Co., Ltd. As previously mentioned [27], animal models were constructed. Briefly, mice were fed adaptively for a week with normal food, water, and light. Stable HCT116 cells were cultured after NC, sh-MLLT10, and sh-Lnc-S100B-2 transfection. After the cells had grown to about 80% fusion, they were digested with trypsin and counted. 200 µL PBS containing 2 × 10 6 HCT116 cells was injected into the right lower flank of 6-8 weeks old mice. ey were randomly divided into four groups: the NC group, the sh-MLLT10 group, the sh-Lnc-S100B-2 group, and the sh-Lnc-S100B-2 + sh-MLLT10 group, with 6 rats in each group. After 35 days of normal feeding, the mice were euthanized humanely. e tumor body was taken, and the tumor volume was measured (volume � (widths × width × length)/2).

Immunohistochemistry (IHC).
Briefly, after 12 hours of baking at 60°C, the paraffin slices were dewaxed. After heating for antigenic repair, 1% periodic acid was used to inactivate endogenous enzyme activity. After incubation with anti-caspase 3 (1 : 200, 19677-1-AP, proteintech) at 4°C overnight, 100 µL anti-rabbit IgG was inoculated at 37°C for 30 min. After DAB color development, the hematoxylin was counterstained for 10 min. en, the sections were sealed with the neutral resin and observed with a light microscope.

Statistics
Analysis. Data were analyzed using the GraphPad Prism 8.0.1 and presented as the mean ± SD. Kaplan-Meier analysis and log-rank test were adopted to analyze the survival time of patients. Correlation between the expression of miR-331-3p and Lnc-S100B-2 was analyzed by Pearson's correlation analysis. Paired t-test, oneway ANOVA or two-way ANOVA with Tukey's multiple comparisons test were performed to evaluate the statistical significance. P < 0.05 was considered to indicate a statistically significant difference.

Lnc-S100B-2 Is Highly Expressed with a Poor Prognosis in CRC.
To obtain differential LncRNAs in CRC, we analyzed the expression profiles of LncRNAs in the GSE104364 and GSE151021 datasets. We found a series of differentially expressed LncRNAs in CRC (Figure 1(a). Kaplan-Meier analysis showed that the survival curve of Lnc-CA14-1, Lnc-FABP2-4, Lnc-MYH11-1, and Lnc-S100B-2 was P < 0.05 (Figure 1(b)). Higher Lnc-S100B-2 level was associated with poorer survival. ese results suggested that Lnc-S100B-2 might be involved in the prognosis of CRC.

3.2.
Lnc-S100B-2 Affects the Proliferation and Apoptosis of HCT116 Cells. We randomly collected 5 pairs of tumor and matched adjacent tissues. Clinical samples were used to verify the level of Lnc-S100B-2.
e paired t-test (Figure 2(a)) were consistent with those of predicting results (Figure 1(a)). e expression of Lnc-S100B-2 was significantly upregulated in tumor tissues.
e results of plate clone formation assay showed that the activity of HCT116 cells was decreased when Lnc-S100B-2 was inhibited ( Figure 2(b)). Apoptosis results proved that Lnc-S100B-2 was positively correlated with CRC cell activity (Figure 2(c)). Knockdown of Lnc-S100B-2 resulted in cell stagnation in the G2 phase (Figure 2(d)). Expression of proliferation (PCNA and Ki67) and apoptosis-related indicators (cleaved caspase 3, Bax, and Bcl-2) at the gene and protein levels was identified (Figure 2(e) and 2(f )). Expressions of PCNA, Ki67, and Bcl-2 decreased in the sh-Lnc-S100B-2 group compared to the control group, while cleaved caspase 3 and Bax were the opposite. Combined with the above results, the expression of Lnc-S100B-2 in CRC might affect cell proliferation and apoptosis.
ese results suggested that Lnc-S100B-2 might regulate the expression of MLLT10 to affect cell apoptosis. At the gene and protein levels, the levels of proliferation (PCNA and Ki67) and apoptosis-related indexes (cleaved caspase 3, Bax, and Bcl-2) further suggested that Lnc-S100B-2 could affect the development of CRC by regulating the expression of MLLT10 (Figures 5(f )-5(k)).
ese results indicated that Lnc-S100B-2 might affect the proliferation and apoptosis of CRC cells by regulating MLLT10.

Effects of Lnc-S100B-2 and MLLT10 on Immune Cell
Invasion and EMT in CRC. Immune cell invasion and EMT are two essential components of the tumor microenvironment. To further explore the role of Lnc-S100B-2 and MLLT10 in CRC, we investigated the immune cell invasion and the degree of EMT in the tumor. CD3 expression was significantly decreased after sh-Lnc-S100B-2 and sh-MLLT10 treatment (Figure 6(a)). It suggested that the infiltration degree of lymphocytes in the tumor tissue was reduced. e number of CD3 (-) CD16 (+) cells and CD11b (+) cells were also significantly decreased with the silencing of Lnc-S100B-2 and MLLT10 (Figures 6(b) and 6(c)). ese results suggested that regulation of Lnc-S100B-2 and MLLT10 might affect the abundance of immune cells in tumor tissues. In addition, the expression of E-cadherin was significantly increased in the sh-MLLT10 group compared with the other three groups. Vimentin is the opposite (Figure 6(e)). We examined the expression levels of EMTrelated indicators (E-cadherin, N-cadherin, vvimentin, β-catenin, snail, and slug) at the gene and protein levels. e results showed (Figures 6(f )-6(l)) that E-cadherin and β-catenin were significantly increased in the sh-Lnc-S100B-2+sh-MLLT10 group, compared with the sh-Lnc-S100B-2 group and the sh-MLLT10 group, while N-cadherin, vimentin, snail, and slug were decreased considerably. It is suggested that Lnc-S100B-2 might affect the EMT of tumor cells through MLLT10, at least partially. Combined with the above experimental results, we found that the regulation of Lnc-S100B-2 and MLLT10 could affect the immune cell invasion and EMT in the tumor.

Discussion
In our study, Lnc-S100B-2 has obtained through Limma and Kaplan-Meier analysis in the CRC datasets (GSE104364 and GSE151021). At the cellular and animal levels, the effects of Lnc-S100B-2 and its downstream MLLT10 signaling on CRC have been identified.
Lnc-S100B-2 is a long noncoding RNA. Our study found that Lnc-S100B-2 was overexpressed in CRC. e expression of Lnc-S100B-2 could affect the proliferation, apoptosis, and EMT of CRC cells. e prognosis of CRC is closely related to EMT. Kaplan-Meier analysis showed that the overexpression of Lnc-S100B-2 predicted a poor prognosis in CRC. EMT is closely associated with poor prognosis of cancer patients, including gastric cancer [28], glioma [29], and bile duct cancer [30]. In bladder cancer, Cao R. et al. found that EMT, as a negative independent prognostic factor, had a tumor-promoting effect due to its related genetic characteristics [31]. ese findings suggest that EMT in CRC may affect patient prognosis. At the same time, this verified our results from the side that Lnc-S100B-2 affected the prognosis of CRC through EMT of CRC cells.
Our study found that Lnc-S100B-2 might regulate the expression of MLLT10 through miR-331-3p. miRNA is also involved in CRC development and prognosis [32]. Lin et al. showed that miR-195-5p/NOTCH2 signaling could affect the polarization of M2-like tumor-associated macrophages by mediating tumor cell EMT [33]. Zhang Y et al. found that miR-17-5P could activate cancer-associated fibroblasts by regulating RUNX3/MYC/TGF-β1 signaling, influencing tumor microenvironment and promoting CRC development [34]. ese results suggest that miRNA might influence the tumor microenvironment and CRC development by regulating the expression of downstream target genes.
Studies have shown that MLLT10 is often observed in acute myeloid and lymphoid leukemia, affecting its treatment and prognosis [35,36]. Previous studies have shown that inhibition of MLLT10 expression can affect the proliferation, migration, and invasion of non-small cell lung cancer cells [23]. It is similar to our findings. MLLT10 could affect the apoptosis level of CRC cells. e expression of apoptosis-related indicators (cleaved caspase 3, caspase 9, Bax, and Bcl-2) was altered with the silence of MLLT10. MLLT10 also has a particular regulatory effect on cell EMT and immune cell infiltration. After inhibiting the expression of MLLT10, the expression levels of EMT-related indicators (E-cadherin, N-cadherin, vimentin, β-catenin, snail, and Slug) changed. EMT is involved in the migration, invasion, and metastasis of cancer cells [37]. EMT is closely related to cell apoptosis. A negative correlation between apoptosis and EMT has been reported in ovarian cancer [38]. Vimentin can affect the apoptosis of SMMC-7721 cells in liver cancer studies [39]. Regulation of Snail1 expression can restore EMT and prevent ethanol-induced apoptosis of neural crest cells [40]. All these proved from the side that MLLT10 affects CRC cell apoptosis and EMT, with sure accuracy. Jing et al.'s study further proved our results, knockdown of MLLT10 could also inhibit EMT and affect the development of colorectal cancer [24].
In our study, MLLT10 expression could affect the degree of infiltration of immune cells. After regulating the expression of MLLT10, the proportion of CD16 and CD11b positive cells decreased. e abundance of tumor-infiltrating immune cells is highly correlated with the progression of 12 Journal of Oncology CRC [41]. Our study found that the proportion of CD3 positive cells (T cells) decreased after MLLT10 silencing. It is suggested that MLLT10 could affect the infiltration degree of T cells in CRC. Studies have shown that the proportions of T cells, NK cells, and macrophages in CRC are higher than those in normal tissues [41]. CD3 (-) CD16 (+) are cytotoxic natural killer cells (NK) that can directly kill tumor cells [42].
In the peripheral blood of CRC patients, it was identified that CRC patients with high CD16 (+) NKT-like cells had shorter disease-free survival [43]. at is, relative CD16 (+) NKTlike cells are reduced in patients with high survival. ese findings are similar to ours. Low levels of MLLT10 have a low degree of immune cell infiltration.

Conclusion
Lnc-S100B-2 was screened out in this study, which is closely associated with a poor prognosis of CRC. Regulation of Lnc-S100B-2 and its downstream MLLT10 can affect CRC cell apoptosis. Lnc-S100B-2 and MLLT10 are associated with EMT and immune cell infiltration in CRC cells. It might provide a potential biomarker for CRC prognosis.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.