Clinical Significance and Function of MALAT1 Gene Expression and the rs619586 Polymorphism in Colorectal Cancer

Colorectal cancer (CRC) is a malignant tumor of the digestive system that poses a serious threat to human health. In 2018, around 1.8 million people were newly diagnosed with CRC, and 881,000 people died from the disease^[1]. The identification of CRC-related genes and genetic polymorphisms will aid in the prevention and treatment of this disease.

Metastasis is associated with the lung adenocarcinoma transcript 1 (MALAT1) gene located on human chromosome 11q13.1, which encodes a lncRNA that participates in the malignant progression of multiple cancers, including CRC^[2-4]. Several studies have found that the rs619586 A>G polymorphism in the MALAT1 gene was associated with the risk of multiple cancers, suggesting that the polymorphism might serve as a potential indicator for cancer risk^[5]. The current study aims to investigate the relationship of MALAT1 expression with survival prognosis and immune infiltrates in CRC patients and drug sensitivity, as well as the role of the rs619586 polymorphism in CRC risk, which will help search for new CRC biomarkers.

TIMER (cistrome.shinyapps.io/timer) was utilized to explore the relationship of MALAT1 expression with survival prognosis and immune infiltrates in CRC patients. GSCA (http://bioinfo.life.hust.edu.cn/GSCA/#/) was used to analyze the correlation between MALAT1 expression and drug IC50 by Pearson correlation analysis. A false discovery rate (FDR) < 0.05 and |r| > 0.1 were considered significant. LinkedOmics (http://www.linked omics.org/) was utilized to obtain the genes coexpressed with MALAT1 in CRC. The co-expression conditions were as follows: |r| > 0.3 and FDR < 0.05. Functional enrichment analysis and protein–protein association networks for coexpressed genes were conducted by using the DAVID tool and STRING database. Protein–protein association networks were further analyzed by using Cytoscape software.

We collected peripheral blood samples from 300 CRC patients, 300 healthy individuals, and 27 pairs of CRC and normal paracancerous tissues (Supplementary Table S1, available in www.besjournal.com). The study protocol (No. 047-001) was approved by the Ethics Committee at Shanghai’s Xuhui District Central Hospital. The TIANamp genomic DNA Kit was used to extract DNA from peripheral blood and tissue samples. The polymerase chain reaction (PCR) method was used to amplify the sequence containing the rs619586 polymorphism. The PCR reaction conditions and primer sequences were as follows: 95 ℃ 5 min; 35 cycles of 94 ℃ 30 sec, 57 ℃ 30 s, 72 ℃ 30 s; 72 ℃ 10 min;

F: 5′-GGGAGAAAGTCCGCCATTTTGCCAC-3′;

R: 5′-ACGGGTCATCAAACACCC-3′.

Genotyping was performed by Sanger sequencing.

PubMed, Embase, and the China National Knowledge Infrastructure databases were used to search for case-control studies on the association of the MALAT1 rs619586 polymorphism with CRC risk in the Chinese population. The last search was conducted on February 10, 2022. Two researchers independently collected information from the included studies. Any differences were resolved through discussion.

TRIzol reagent was used to extract total RNA. A reverse transcription kit was used to convert mRNA into cDNA. The cDNA was then amplified using the Applied Biosystems 7500 Real-Time PCR System. Syber green was utilized to detect fluorescence signals. The sequences of the primers were as follows: MALAT1 forward: 5′-TGACGGAGGTTGAGATGAAGCT-3′ and reverse: 5′-TAATTCGGGG CTCTGTAGTCCT-3′; GAPDH forward: 5′-GTCTCCTCTGACTTCAACA-3′ and reverse: 5′-TGAGGGTCTCTCT CTTCCT-3′. Relative expression of the MALAT1 gene was calculated using the 2^−∆∆Ct method. All the experiments were repeated in triplicate.

The miRNASNP-v3 database was utilized to investigate whether the rs619586 polymorphism affected the binding of miRNA to MALAT1. The psiCHECK2 vector was used to create recombinant dual-luciferase reporters. A 200-bp sequence containing the rs619586 A or G allele was synthesized and inserted into the psiCHECK2 vector to generate the wild-type vector (psiCHECK2-WT) containing the A allele and the mutant vector (psiCHECK2-MT) containing the G allele. The 293 T cell line was grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37 °C. In the logarithmic growth phase, 293 T cells were seeded into 24-well plates at a density of 10⁵ cells/well. According to the protocol, 16 hours after plating, the recombinant dual-luciferase vectors were cotransfected with miR-214-3p mimics or miRNA-NC into 293 T cells using Lipofectamine 2000. The transfected cells were collected 48 hours after transfection, and their luciferase activity was evaluated using a dual-luciferase assay system. All experiments were performed independently in triplicate.

Zheng et al. found that the expression level of MALAT1 was higher in CRC tissues than in noncancerous tissues^[6]. A higher expression of MALAT1 might act as a negative prognostic marker in patients with stage II/III CRC. Yang et al. revealed that MALAT1 overexpression in primary CRC could increase cell proliferation, invasion, and migration via PRKA kinase anchor protein 9^[7]. In addition, our research found that high MALAT1 expression was associated with poor survival of patients with colon cancer (Supplementary Figure S1, available in www.besjournal.com). These findings indicated that the MALAT1 gene is capable of acting as an oncogene in CRC. The presence of tumor-infiltrating B cells is associated with poor outcomes in several cancers^[8]. Our research showed that MALAT1 expression was positively correlated with B-cell infiltration in colon cancer (Supplementary Figure S2, available in www.besjournal.com). Furthermore, MALAT1 expression was also correlated with the sensitivity of multiple anti-CRC drugs, such as camptothecin and cetuximab (Supplementary Table S2, available in www.besjournal.com).

Figure S1.  The association of MALAT1 gene expression with the survival of CRC patients. CRC: colorectal cancer.

Figure S2.  The correlation of MALAT1 gene expression with immune infiltration in CRC. CRC: colorectal cancer.

There were 1,270 genes coexpressed with MALAT1, including 106 negatively related genes and 1164 positively related genes. These coexpressed genes were significantly enriched in multiple biological processes, molecular functions, and cellular components, such as GO:0003676~nucleic acid binding, GO:0005634~nucleus, and GO:0005622~intracellular (Supplementary Table S3, available in www.besjournal.com). Protein–protein association networks of the coexpressed genes showed that node CDC42, with the most edges, was the hub gene (Supplementary Figure S3, available in www.besjournal.com). CDC42 was a small GTPase of the Rho subfamily, which regulated signaling pathways that controlled diverse cellular functions, including cell morphology, migration, endocytosis, and cell cycle progression. CDC42 gene expression dysregulation involved several pathogenic processes of CRC^[9]. Therefore, MALAT1 may be involved in the progression of CRC by regulating the expression of the CDC42 gene.

Figure S3.  Protein-protein association networks of the genes co-expressed with MALAT1.

The current case-control study showed that the MALAT1 rs619586 polymorphism was significantly associated with CRC risk [AG vs. AA: OR = 0.64, 95% CI = 0.43–0.96, P = 0.03; (AG + GG) vs. AA: OR = 0.62, 95% CI = 0.42–0.91, P = 0.02; G vs. A: OR = 0.62, 95% CI = 0.44–0.89, P = 0.01] (Table 1). A similar result was also observed in the pooled analysis of 1266 CRC cases and 1288 healthy controls [GG vs. AA: OR = 0.46, 95% CI = 0.25–0.84, P = 0.01; AG vs. AA: OR = 0.73, 95% CI = 0.60–0.89, P = 0.002; (AG + GG) vs. AA: OR = 0.71, 95% CI = 0.58–0.85, P = 0.0003; GG) vs. (AG + AA): OR = 0.49, 95% CI = 0.27–0.89, P = 0.02; G vs. A: OR = 0.71, 95% CI = 0.60–0.84, P < 0.0001] (Supplementary Table S4 and Supplementary Figure S4, available in www.besjournal.com). Further genotype-tissue expression analysis showed that the expression level of MALAT1 was significantly lower in the AG + GG genotype than in the AA genotype in CRC and normal paracancerous tissues (Figure 1). Bioinformatics analysis showed that the rs619586 G allele contributed to the binding of several miRNAs, such as miR-214-3p, miR-3619-5p, and miR-761, to MALAT1 (Supplementary Table S5 available in www.besjournal.com). Among these miRNAs, miR-214-3p could inhibit tumor proliferation and metastasis in CRC by targeting the PLAGL2-MYH9 axis. The dual-luciferase assay showed that the rs619586 G allele facilitated the binding of miR-214-3p to MALAT1 (Figure 2), which was consistent with previous research findings^[10]. Thus, the rs619586 G allele might reduce CRC risk by facilitating the binding of miR-214-3p to MALAT1 and thus reducing the expression of the cancer-promoting molecule MALAT1. Additionally, the rs619586 polymorphism might also affect the survival prognosis and anti-cancer drug sensitivity of CRC patients; however, this hypothesis awaits confirmation by future studies.

Figure S4.  Meta-analysis of the association of MALAT1 rs619586 polymorphism with CRC risk in the Chinese population.

Figure 1.  Association of the rs619586 polymorphism with MALAT1 expression (Error bars indicate Standard Deviation). ^***P < 0.001.

Figure 2.  Influence of the rs619586 polymorphism on the binding of miR-214-3p to MALAT1 (A: Bioinformatics analysis; B: Dual-luciferase assay; Error bars indicate Standard Deviation). ^***P < 0.001.

Although the current study has yielded some interesting findings, there remain some shortcomings. For example, the specific molecular mechanisms by which MALAT1 expression is correlated with B-cell infiltration and anti-CRC drug sensitivity were not revealed. The risk analysis did not correct for several confounding factors, including smoking, alcohol consumption, red meat intake, etc.

In conclusion, our study suggests that MALAT1 expression is associated with survival prognosis and B-cell infiltration in patients with colon cancer and anti-CRC drug sensitivity, and the rs619586 polymorphism is associated with CRC risk. Thus, MALAT1 expression and the rs619586 polymorphism may act as biomarkers for assessing CRC risk, prognosis, and anti-CRC drug sensitivity.