LncRNA‐422 suppresses the proliferation and growth of colorectal cancer cells by targeting SFPQ

In this study, the biological function of lncRNA-422 was investigated in colorectal cancer, which suggested that down-regulated lncRNA-422 suppressed cell proliferation and inhibited tumor growth in cellular and xenograft models. Mechanistically, lncRNA-422 could interact directly with splicing factor proline and glutamine rich (SFPQ) to activate downstream pathways. SFPQ drives cancer progression through diverse roles in RNA transcriptional activity, mRNA processing, splicing regulation and innate immune response in hepatocellular carcinoma,1 breast cancer,2 ovarian cancer3 and colorectal cancer.4 Moreover, SFPQ promoted the proliferation and onset of colorectal cancer. LncRNA-422 was previously identified5 and selected for further investigation in this study (Figure 1A). We found that lncRNA-422 expression varied across multiple tissues but was enriched in colorectal tissues and cells (Figure 1B,C). LncRNA-422 was significantly downregulated in colorectal cancer tissues from The Cancer Genome Atlas (TCGA), GSE104836, quantitative reverse transcription PCR (qRT-PCR) and in-house RNA-Seq. Eighty-six per cent (45 of 52) of the colorectal cancer tissues demonstrated down-regulation of lncRNA-422 expression compared to that in the normal tissues (Figure 1D-H). Subsequent analysis showed that lncRNA-422 expressionwas significantly correlatedwith ages<60 years (Table S1). We evaluated the lncRNA-422 transcript in primates with extremely high homology with the chimp and gorilla genomes (Figure S1A). In addition, we found that the coding potential of lncRNA-422 for each transcript was negative, indicating that it is a noncoding RNA (Figure S1B-E). Furthermore, full-length lncRNA-422 (855 bp in total) was identified on RNA isolated from HCT116 cells by using rapid amplification of cDNA ends assays (Figure S1F-H). Taken together, the results support the lack of proteincoding activity for lncRNA-422. Afterwards, we examined the biological significance of lncRNA-422 down-regulation in HCT116 and SW620 cells

In this study, the biological function of lncRNA-422 was investigated in colorectal cancer, which suggested that down-regulated lncRNA-422 suppressed cell proliferation and inhibited tumor growth in cellular and xenograft models. Mechanistically, lncRNA-422 could interact directly with splicing factor proline and glutamine rich (SFPQ) to activate downstream pathways. SFPQ drives cancer progression through diverse roles in RNA transcriptional activity, mRNA processing, splicing regulation and innate immune response in hepatocellular carcinoma, 1 breast cancer, 2 ovarian cancer 3 and colorectal cancer. 4 Moreover, SFPQ promoted the proliferation and onset of colorectal cancer.
LncRNA-422 was previously identified 5 and selected for further investigation in this study ( Figure 1A). We found that lncRNA-422 expression varied across multiple tissues but was enriched in colorectal tissues and cells ( Figure 1B,C). LncRNA-422 was significantly downregulated in colorectal cancer tissues from The Cancer Genome Atlas (TCGA), GSE104836, quantitative reverse transcription PCR (qRT-PCR) and in-house RNA-Seq. Eighty-six per cent (45 of 52) of the colorectal cancer tissues demonstrated down-regulation of lncRNA-422 expression compared to that in the normal tissues . Subsequent analysis showed that lncRNA-422 expression was significantly correlated with ages <60 years (Table S1).
We evaluated the lncRNA-422 transcript in primates with extremely high homology with the chimp and gorilla genomes ( Figure S1A). In addition, we found that the coding potential of lncRNA-422 for each transcript was negative, indicating that it is a noncoding RNA (Figure S1B-E). Furthermore, full-length lncRNA-422 (855 bp in total) was identified on RNA isolated from HCT116 cells by using rapid amplification of cDNA ends assays ( Figure S1F-H). Taken together, the results support the lack of proteincoding activity for lncRNA-422.
Afterwards, we examined the biological significance of lncRNA-422 down-regulation in HCT116 and SW620 cells We first identified that lncRNA-422 is predominantly localized in the cell nucleus 7 ( Figure 3A,B). We then used unbiased approaches to identify intracellular lncRNA-422-interacting proteins, 8 including RNA pull-down, silver staining and mass spectrometry analysis 9 ( Figure 3C). Three potential interacting proteins were identified based on peptide number >2 and sequence coverage number >10 in the sense group and were absent in the antisense group (Table S2). Furthermore, independent western blotting confirmed that sense but not antisense lncRNA-422 was significantly bound to SFPQ ( Figure 3D). 10 Importantly, RIP-qPCR indicated that immunoprecipitation with an anti-SFPQ antibody specifically retrieved lncRNA-422 ( Figure 3E). We confirmed that lncRNA-422 can directly affect the protein level of SFPQ ( Figure 3F). Collectively, these results suggest that lncRNA-422 physically interacts with SFPQ in colorectal cancer cells.
Specific small interfering RNA (siRNA) for SFPQ remarkably invoked proliferation defects, inhibited colony formation and induced apoptosis abilities, while SFPQ overexpression significantly increased the cellular functions in two colorectal cancer cells ( Figure 3G SFPQ knockdown mimicked the influence of lncRNA-422, whereas SFPQ overexpression prevented lncRNA-422induced proliferation and colony formation, and SFPQ knockdown restored the cell apoptosis effects reduced by lentivirus-based knockdown of lncRNA-422 ( Figure 4A-D, Figure S5). Together, these results suggest that lncRNA-422 exerts tumor-suppressing effects at least partially by binding to and inhibiting SFPQ activity in colorectal cancer.
We assessed the clinical significance of SFPQ in TCGA, GEO, in-house RNA-Seq and combined datasets ( Figure  S6A-G). Remarkably, we first assessed the protein level of SFPQ in 30 paired in-house microarrays using IHC and found that more than half of colorectal cancer tis-sues showed increased SFPQ expression compared with that in paired normal tissues ( Figure S6H). Strong correlations were also identified in colorectal cancer tissues (r = -0.16, P = 3.00 × 10 -4 ; Figure S6I). Intriguingly, the age at colorectal cancer diagnosis was decreased even further in patients with high SFPQ and low lncRNA-422 expression ( Figure S6J,K). Together, these integrated analyses indicate that the effects of SFPQ and lncRNA-422 lead to colorectal cancer at a younger age.
We then tested the co-expression of SFPQ and lncRNA-422 in the TCGA cohort and in-house RNA-Seq on colorectal cancer tissues (P < 0.05, Figure 4E,F, Table S3). Further Kyoto Encyclopedia of Genes and Genomes analysis found that a total of 116 genes were enriched in RNA degradation and other metabolic pathways ( Figure 4G, Figure S7). We validated the co-expression of the candidate genes in the Genotype-Tissue Expression (GTEx) databases (r > 0.20,  Figure 4H) and targeted three genes, MEX3A, SLC41A1 and UBAP2L, which was confirmed with qRT-PCR ( Figure 4I).
In conclusion, this study revealed a novel lncRNA, lncRNA-422, that regulates tumor cell proliferation and growth by targeting SFPQ. We focused on exploring the histological, molecular and cellular functions of lncRNA-422 and discovered that lncRNA-422 accelerates proliferation and tumor growth in cells and nude mouse models. RNA pull-down, mass spectrometry and RIP assay demonstrated that lncRNA-422 directly binds to SFPQ, leading to the change of malignancy. The lack of our mutation data and cells limits further validation, while we consider SFPQ mutants to affect interaction. LncRNA-422 and SFPQ knockdown shared highly similar patterns in terms of cell proliferation and apoptosis, which supports the combined evaluation of lncRNA-422 and SFPQ may be an efficient indicator for colorectal cancer prognosis. These results implicate lncRNA-422/SFPQ as a novel target in colorectal cancer development and provide a basic understanding of lncRNA-targeted molecular therapy.

A C K N O W L E D G E M E N T S
This study was partly supported by the National Natural Science Foundation of China (81773516 and 82073631) and the Jiangsu Province Graduate Student Scientific Research Innovation Plan Project ( KYCX20_1410).

C O N F L I C T O F I N T E R E S T
The authors declare that they have no conflict of interest.