LARP6 expression is down-regulated in CRC, and lower expression associates with worse prognosis
To explore whether LARP6 engaged in the occurrence and development of CRC, we firstly analyzed LARP6 expression profile through GEPIA, and were surprised to discover that LARP6 expression was down-regulated in CRC (Figure. 1A). Further KEGG enrichment analysis revealed that some cancer-related cellular processes were significantly enriched in the lower LARP6 expression group, such as apoptosis, oxidative phosphorylation, fatty acid metabolism, and sphingolipid metabolism (Figure. 1B). These findings together indicated that LARP6 may functioned in CRC.
On the purpose of verifying our hypothesis, quantitative real-time PCR (qPCR) and western blotting (WB) were firstly performed. In agreement with above online analysis, our qPCR and WB results also suggested that LARP6 expression was lower in CRC tissues than in normal tissues (Figure. 1C, 1E). In addition, we noticed that, compared with non-metastatic tissues, LARP6 expression was significantly lower in metastatic CRC tissues (Figure. 1D). Next, immunohistochemistry (IHC) staining was performed on 204 pairs of human CRC and paracancer normal tissues, and down-regulated expression of LARP6 in CRC had been confirmed again (Figure. 1F, S1A). Further analysis showed that LARP6 expression was negatively correlated with T-stage, lymph node status, distant metastasis and clinical stage (Figure. 1G-J, Table. S1), but not associated with age, gender, tumor size, and tumor site (Table. S1). Besides that, online survival analysis indicated that CRC patients with lower LARP6 expression present a poorer overall and relapse-free survival (Figure. 1K-L). Taken together, these results suggest that LARP6 is lower expressed in CRC, and lower LARP6 expression is associated with worse prognosis.
Larp6 Inhibits Crc Cell Invasion And Metastasis In Vitro And In Vivo
To evaluate the effect of LARP6 on biological behavior of CRC cell, we firstly detected LARP6 expression level in 7 CRC cells (Figure. S1B, S1E). SW480 and DLD1, two lower LARP6 expression cells, were selected to construct stable LARP6 overexpressed cell lines (Figure. S1C, S1F), while SW620 and CACO2 were used to construct stable interference cell lines (Figure. S1D, S1F). CCK8 and clone formation assays showed that LARP6 had no significant effect on CRC cells proliferation in vitro (Figure. S1G-L). However, for transwell assay, LARP6 overexpression markedly weakened migration and invasion ability of SW480 and DLD1 cells (Figure. 2A, 2C), while knockdown exerted an enhanced effect (Figure. 2B, 2D).
Based on results in vitro, we constructed an orthotopic CRC mice model, to investigate whether LARP6 suppresses CRC metastasis in vivo likewise. On the 60th day after surgery, fluorescein substrate was injected into abdominal cavity of mice, and then liver metastasis of CRC was observed (Figure. 2E). Combined with results of intestinal and liver tissues separation and H&E staining assay (Figure. 2F, 2H-I), we exhibited a higher liver metastasis rate in LARP6 interference group than control group (Figure. 2G), indicating a stronger metastatic ability in LARP6 interference group.
By Binding To Znf267 Mrna, Larp6 Up-regulates Znf267 Expression
RBPs mediate post-transcriptional regulation of their target genes through various mechanisms. To further characterize the function of LARP6 as a RBP, SW620 cells were collected for high-throughput RNA immunoprecipitation sequencing (RIP-seq), and RNA potentially bound to LARP6 were identified (Supplementary material. 1). Interestingly, these genes were significantly enriched in some cancer-related cellular processes, such as: Phagosome, Cell adhesion molecules (CAMs), Adherens junction, Regulation of actin cytoskeleton, and etc (Supplementary material. 1), which further supports the association of LARP6 with CRC metastasis.
It is a common functional manner of RBPs to regulate mRNA ability [37–40]. Through literature research, we preliminarily screened several genes closely related to tumor metastasis in our RIP-seq list, and studied influence of LARP6 on these mRNA abundance. Among these six candidates, ZNF267 shared a most evident effect (Figure. 3A-D). Zinc finger protein 267 (ZNF267), belonging to Kruppel-like zinc finger family, can promote HCC cells proliferation and migration. Besides, it also has a effect on expression of MMP and ADAM protease family members [41–43]. More importantly, using TCGA dataset, GO analysis of ZNF267 in CRC also revealed its association with cell migration, adhesion, and etc (Supplementary material. 2).
In order to further characterize relationship between LARP6 and ZNF267, we firstly verified our RIP-seq result. RIP-qPCR and RNA-pull down assays collectively suggested that LARP6 could bind to ZNF267 mRNA (Figure. 3E-F, S2A). In addition, LARP6 over-expression increased, while LARP6 interference reduced ZNF267 protein level (Figure. 3G), which is compatible with mRNA result (Figure. 3A-D).
RBPs are important regulators of mRNA stability and translation. To clarify specific mechanism of above expression regulation, on the one hand, we treated cells with actinomycin D (ActD), and found that ZNF267 transcripts showed a better stability in LARP6 over-expressed cells, while opposite result was observed in cells with LARP6 knockdown (Figure. 3H-K). On the other hand, with sucrose gradient fractionation, ZNF267 mRNA distribution in polysomes (translationally active ribosome fractions) was examined. Specifically, we observed a more ZNF267 mRNA accumulation in polysomes in LARP6-overexpressed cells, while LARP6 knockdown increased an accumulation of ZNF267 mRNA in lower translational activity portion (Figure. 3L-O). In conclusion, these results suggest that LARP6 binds to ZNF267 mRNA and increases ZNF267 expression in a post-transcriptional manner.
Znf267 Bridges The Suppression Of Larp6 On Crc Metastasis
Although the correlation between LARP6 and ZNF267 had been well established in our research, it was obscure whether the latter involves in CRC metastasis. We then experimented functional assays, and found that ZNF267 over-expression inhibited CRC cells migration and invasion, while ZNF267 interference obtained opposite result (Figure. 4A-H, S2B). Based on above results, we believed that ZNF267 may play an important part in LARP6 inhibiting CRC invasion and metastasis. Matrigel transwell result confirmed our hypothesis, in which LARP6 knockdown promoted cell invasion, while it was reversed by ZNF267 reintroduction (Figure. 4I, S2C).
By Up-regulating Znf267 Expression, Larp6 Inhibits Sgms2-mediated Sphingomyelin Synthesis
In order to explore biological processes relevant to LARP6/ZNF267 axis inhibition on CRC metastasis, we firstly focused on our previous KEGG enrichment analysis (Figure. 1B). As results suggested, LARP6 is extremely possible to be involved in sphingolipid metabolism (Figure. 5A-B, S2D). Furthermore, ZNF267 is also been presented to associate with cellular metabolism, even lipid metabolism (Supplementary material. 2). Considering the characteristic of ZNF267 as a transcription factor, Cistrome DB (http://cistrome.org/db/#/) was used to mine downstream of ZNF267 (GSM2466511, CistromeDB: 77152). Among those candidate targets of ZNF267, SGMS2, a key enzymes in sphingomyelin synthesis, caught our attention. It transfers phosphocholine from phosphatidylcholine to ceramide to produce sphingomyelin, playing an important role in maintaining cell sphingolipid homeostasis, especially ceramide and sphingomyelin. More importantly, multiple data analysis consistently revealed a correlated expression between LARP6 and SGMS2 in CRC (Figure. 5C-D, S2E). As a matter of course, SGMS2 was chosen to be studied further.
Chromatin immunoprecipitation (ChIP) analysis was firstly performed, and confirmed combination between ZNF267 and SGMS2 promoter (Figure. S2F). Meanwhile, we found ZNF267 has a negative effect on SGMS2 expression, both mRNA and protein (Figure. 5E-G, S2G-H). What interests us is that, LARP6 also inhibited SGMS2 expression, but this suppression was dependent on ZNF267 (Figure. 5H-M).
Considering the importance of SGMS2 in sphingomyelin synthesis, we next examined several sphingolipid metabolism indexes, mainly ceramide and sphingomyelin. As is shown in our research, LARP6 over-expression increased ceramide accumulation (Figure. 6A) but decreased sphingomyelin content in CRC cells (Figure. 6B), and LARP6 interference is opposite (Figure. 6C-D). Moreover, further studies indicated that these effects of LARP6 on sphingolipid metabolism is relied on its expression regulation on ZNF267 and SGMS2 (Figure. 6E-I, S2I-N).
As study reported, ceramide and sphingomyelin, as bioactive lipids, have more or less effects on cell survival, apoptosis, autophagy, migration and other processes [44–49]. Therefore, there was a good reason to believe that, in LARP6 suppression on CRC metastasis, ZNF267/SGMS2 axis-mediated sphingomyelin metabolism may play a important role. As expected, matrigel transwell assays confirmed our thought (Figure. 6J-M, S3A).
Larp6 Upregulates Autophagy Activity Of Crc Cells
Ceramide, center of sphingolipid metabolism, is a valid inducer of apoptosis and autophagy [50–53]. With the affection of LARP6 on ceramide and sphingomyelin content in CRC cells, we tentatively investigated whether LARP6 influences cell autophagy activity. As results showed, LARP6 over-expression increased abundance of microtubule-associated protein 1 light chain 3 (LC3B)-II but decreased P62 protein level, and with the presence of BafA1, an inhibitor of autophagosome and lysosome fusion, LARP6 over-expressed cells presented a more higher LC3B-II accumulation (Figure. S3B). Correspondingly, LARP6 knockdown resulted in a contrary trend (Figure. 7A). To verify above findings, transmission electron microscopy (TEM) indicated that LARP6 increased the number of autophagic vesicles in CRC cells (Figure. 7B-C, S3D-E). In addition, we transfected a RFP-GFP-LC3 reporter to measure autophagy flux. Abundant red dots (autophagolysosomes) and occasional yellow dots (autophagosomes) were more observed in LARP6 over-expressed cells (Figure. S3F). However, the number of red dots and yellow dots found in LARP6 knockdown cells were less than in control cells (Figure. 7E-F), indicating a decrease in autophagy flux. Together, these data all agree that LARP6 upregulates CRC cells autophagy activity.
To illustrate the relevance between LARP6-mediated sphingolipid imbalance and autophagy, we used a selective sphingomyelin synthase 2 inhibitor, Ly93 [54–55], and found that inhibitory effect of LARP6 interference on autophagy activity could be eliminated by this inhibitor (Figure. 7D and 7G, S3C and S3G), indicating that SGMS2-mediated sphingomyelin synthesis is crucial for LARP6 to regulate autophagy activity of CRC cells.
Related to cell survival, death, migration, metastasis and other numerous cell processes, autophagy is thought to be engaged in cancer development. In our research, we also found enhanced autophagy activity is crucial to LARP6 suppression on CRC metastasis (Figure. 7H).
Larp6 Inhibits Crc Metastasis Through Znf267/sgms2 Axis
Combined with above results, we believe that ZNF267/SGMS2 axis is vital for LARP6 to inhibit CRC metastasis. In vitro rescue experiment firstly proved our conjecture (Figure. 6J-M, S3A). We then detected expression of these three genes in primary tumor tissues of orthotopic CRC mice model (Figure. 2E-I), and found that compared with control mice, representing a weaker metastatic ability, LARP6 knockdown group shared a lower LARP6 and ZNF267 expression, but a higher SGMS2 expression (Figure. 8A). In addition, IHC staining analysis of 30 clinical CRC samples indicated that, in those samples with a higher LARP6 expression, ZNF267 staining was stronger, but it was opposite for SGMS2 (Figure. 8B). Statistical analysis further verified that, LARP6 expression was positively correlated with ZNF267, while negative with SGMS2 (Figure. 8C).