A micropeptide JunBP regulated by TGF-β promotes hepatocellular carcinoma metastasis

LINC02551 is upregulated by TGF-β stimulation

This study was initiated in an attempt to verify lincRNAs which were regulated by TGF-β. Thus, we applied RNA-sequencing (RNA-seq) in HLF cells stimulated with TGF-β (5 ng/ml for 12 h) (Fig. 1A and Supplementary Table S1). Five lincRNAs significantly upregulated upon TGF-β were verify through qRT-PCR in HLF and Hep3B cell lines. LINC02551 appeared to be the top hit due to its concurrent regulation by time-dependent TGF-β stimulation in the two cell lines (Fig. 1B and Supplementary Fig. S1C). And it was also upregulated upon TGF-β stimulation in a dose-dependent manner. (Supplementary Fig. S1D). Compared with LINC02551, the other four lincRNAs did not have accordant responses upon TGF-β stimulation in the two cell lines (Supplementary Fig. S1A, B). To explore how LINC02551 was upregulated by TGF-β, we used different inhibitors in TGF-β signaling pathway to stimulate HLF and Hep3B and found that its upregulation upon TGF-β stimulation was totally abrogated by TGFBR1 inhibitor LY364947 but not other inhibitors (Fig. 1C and Supplementary Fig. S1E). To further confirm the role of SMAD proteins, we tested the effects of ectopic expression of SMAD2/3/4 alone or together and found that SMAD3 overexpression significantly increased the level of LINC02551 upon TGF-β stimulation and the level was maximal when SMAD2/3/4 were all overexpressed (Fig. 1D and Supplementary Fig. S1F). Furthermore, LINC02551 expression was upregulated upon TGF-β stimulation in SMAD3-overexpressed Hep3B (Fig. 1E), while the upregulation was interrupted in SMAD3-knockdown LM3 (Supplementary Fig. S1G).

Fig. 1: LINC02551 is upregulated upon TGF-β stimulation.figure 1

A Volcano plot of the analysis of the RNA-seq of HLF treated without and with TGF-β. Red plots mean the upregulated genes; green plot means the downregulated genes. B qRT-PCR analysis of LINC02551 expression treated with TGF-β for in the HLF cell line. C qRT-PCR analysis of LINC02551 levels in HLF cells treated with TGF-β together with different inhibitors in TGF-β signaling. D qRT-PCR analysis of LINC02551 expression level in HLF cells transfected with SMAD2/3/4 alone or together. E qRT-PCR results of LINC02551 levels in Hep3B-SMAD3 stable cells treated with TGF-β. F Luciferase activities of LINC02551 promoter in HLF cells treated with TGF-β. G The activity of truncated mutants of LINC02551 promoter in HLF cells. H The activity of SBE mutants of LINC02551 promoter in HLF cells. I Anti-SMAD3 chromatin immunoprecipitation assays followed by quantitative real-time PCR and reverse transcription PCR analyses was conducted in HLF cells. Anti-H3 is used as a positive control antibody and PRL30 is used as a positive control gene. (mean ± SD, *P < 0.05; **P < 0.01, ***P < 0.001 and ns, P > 0.05).

SMAD3 is reported to be a canonical transcriptional factor together with SMAD2 and SMAD4. To explore whether the regulation was occurred in the transcriptional level, we used the reporter assay and found that LINC02551 is transcriptionally upregulated upon TGF-β stimulation (Fig. 1F and Supplementary Fig. S1H). According to the JASPAR analysis (http://jaspar.genereg.net/), LINC02551 promoter contained 4 putative SMAD3/4-binding elements (SBE) on the minus or plus strand at positions -936, -902, -879 and -639, respectively. After constructing different point and truncated mutants, the reporter assay indicated that SBE-3 and SBE-4 might be responsible for the upregulation of LINC02551 induced by TGF-β stimulation (Fig. 1G, H). Chromatin immunoprecipitation (ChIP) followed by qRT-PCR analyses showed that SMAD3 bound to SBE-3 and SBE-4 regions (Fig. 1I).

These data suggested that SMAD3 might promote the transcription of LINC02551 upon TGF-β stimulation.

LINC02551 encodes a micropeptide, which is naturally, endogenously produced

To figure out the subcellular localization of LINC02551, we used nuclear/cytoplasm fractionation and confocal microscopy analysis of fluorescent in situ hybridization and found that more than half of LINC02551 was located in the cytoplasm (Fig. 2A, B), suggesting that it might be translated into protein. The rapid-amplification of cDNA ends (RACE) assay confirmed that LINC02551, located on human chromosome 11, was composed of 1455 nucleotides and consisted of six exons (Supplementary Fig. S2A). To determine whether the in-frame ATG initiation codon of LINC02551 was functional, we constructed plasmids by fusing the wild-type (WT) ORFs of LINC02551 and Flag, as well as the mutant versions that ATG initiation codons were mutated to ATT (Fig. 2C). The expression of Flag fusion protein was observed in the 293T cells transfected with ORF-Flag and 5’UTR-ORF-Flag plasmids but not the cells transfected with 5’UTR-ORF-mut plasmid, suggesting that LINC02551 ATG codon was functional (Fig. 2D, E and Supplementary Fig. S2C). Then we got the detailed amino acids sequence of JunBP and after blasting the sequence in NCBI, we found that it was not conservative and only expressed in human and pan paniscus (Supplementary Fig. S2B). To determine whether JunBP was endogenously expressed, we generated a rabbit polyclonal antibody against JunBP (Anti-JunBP). LINC02551, LINC02551-Flag, and ORF-Flag were transfected into 97H cells and all detected by Anti-JunBP (Fig. 2F–H and Supplementary Fig. S2D). When LINC02551 was knocked down by Smart Silencer (RiboBio company), JunBP detected by Anti-JunBP was much lower (Fig. 2H and Supplementary Fig. S2E). These data confirmed the specificity of Anti-JunBP. To further confirm that JunBP was translated from LINC02551 naturally, LY364947 and LINC02551 Smart Silencer were used and we found that the upregulation of JunBP upon TGF-β stimulation was blocked with the inhibition of either TGFBR1 or LINC02551 (Fig. 2J, K and Supplementary Fig. S2G).

Fig. 2: A micropeptide JunBP is naturally and endogenously translated by LINC02551.figure 2

A LINC02551-FISH (fluorescence in situ hybridization) with indicated probes (Cy3 is added to LINC02551) in HCC cells. B qRT-PCR analysis of LINC02551 subcellular localization followed by subcellular fractionation (representative from n = 3 biological replicates). GAPDH and MALAT1 were used as controls for the cytoplasm and nucleus, respectively. C The diagram of the Flag fusion constructs used for transfection. The initiation codon ATG in LINC02551 was mutated to ATT (ORFM). D WB results of the three plasmids expression in 97H and Hep3B cells. E IF (immunofluorescence) assays in 97H cells. F 97H cells were transfected with ORF-Flag in concentration gradients. G 97H cells were transfected with LINC02551 in concentration gradients. H 97H cells were transfected with Smart Silencer. I 97H cells were transfected with LINC02551 or ORFM. J The expression of JunBP in 97H cells treated with LY364947 and TGF-β for indicated times. K The expression of JunBP in HCC cells transfected with Smart Silencer and stimulated by TGF-β.

Altogether, our results showed that JunBP was naturally and endogenously produced by LICN02551 in HCC cells and was upregulated upon TGF-β stimulation.

JunBP promoted HCC metastasis in vitro and in vivo

To determine the function of JunBP in HCC progression, we checked its expression in different HCC cell lines (Supplementary Fig. S2F). In Hep3B and 97H cells, we overexpressed JunBP stably (Fig. 3A), which led to increased cell migration and invasion and similar effects were observed in wound healing assay (Fig. 3B–D). And also, western blot analysis indicated upregulation of epithelial-mesenchymal transition (EMT) markers in JunBP overexpression cells (Supplementary Fig. S3A). To evaluate the tumor promotive role of JunBP in HCC in vivo, we applied orthotopic xenograft models to examine intrahepatic metastasis of 97H cells after 5 weeks post inoculation (Fig. 3E). Injection of JunBP-overexpressed cells substantially increased the capacity of HCC cells to form secondary lesions in the liver and mice in overexpressed group went so far as to develop lung metastasis in 5 weeks (Fig. 3F, G). Before these mice were executed, their livers were separated and the IHC staining showed strengthened expression of epithelial-mesenchymal transition (EMT) markers in the JunBP-overexpressed group (Fig. 3H and Supplementary Fig. S3B).

Fig. 3: The effect of JunBP overexpression on HCC metastasis.figure 3

A The identification of JunBP-Flag expression in Hep3B and 97H cells. B The migration and invasion results of JunBP overexpression in Hep3B cells (M: Migration; I: Invasion). C The migration and invasion results of JunBP overexpression in 97H cells. D The wound healing assay of JunBP overexpression in Hep3B and 97H cells. E Before executed, the mice were injected with luciferase substrate. And the luciferase activities were analyzed in the right graph. F The representative images of the mouse liver. And the in situ metastasis nodules were counted. G The lungs were separated and perfused by luciferase substrate. The H&E staining of the lung were presented. H EMT markers IHC staining in the mouse livers.

In 97H and HLF cells, we knocked down JunBP. And to evaluate the role of JunBP upon TGF-β stimulation, we treated JunBP knockdown cells with TGF--β stimulation. The results showed that the knockdown of JunBP could partially rescue the role of TGF-β (Supplementary Fig. S4A). To further investigate the rescue effects in vivo, we firstly inoculated 97H vector cells or 97H shJunBP cells into the left lobe of livers of nude mice or injected the above two kinds of cells through tail vein in the nude mice. After inoculation, we administered TGF-β (4 μg/kg) once a week for four times. In the fifth week, we separated the mice livers in the orthotopic xenograft groups and examined the fluorescence intensity of the lungs in the tail vein injection groups. H&E staining analyses of the livers and the lungs showed that TGF-β treatment significantly increased the intrahepatic metastasis and the lung metastasis of HCC and knockdown of JunBP could partially rescue the effects of TGF-β (Supplementary Fig. S4B, C).

JunBP interacted with c-Jun

We next explored the underlying mechanism of JunBP-induced promotion of HCC metastasis. Immunoprecipitation (IP) followed by mass spectrometry (MS) assay was used to identify potential JunBP interaction partners (Fig. 4A and Supplementary Table S2). c-Jun was verified to interact with JunBP but did not lead to the change of c-Jun RNA level (Fig. 4B, C). Immunostaining showed that JunBP associated with c-Jun in the nucleus (Fig. 4D). It is reported that c-Jun is composed up of transactivation domain (TAD) and basic-leucine zipper domain (bZIP) [31]. We then constructed two truncated mutants of c-Jun and further analysis showed that JunBP interacted with the bZIP domain of c-Jun (Fig. 4E).

Fig. 4: JunBP interacts with c-Jun.figure 4

A CoIP with anti-FLAG, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and silver staining. B CoIP analysis between Flag-c-Jun and HA-JunBP in HEK293 cells. C qRT-PCR analysis of c-Jun mRNA in Hep3B and 97H cells with JunBP overexpressed. D IF results in 293T and HCC cells transfected with Flag-JunBP and HA-c-Jun (the upper panel). The fluorescence intensities in the nucleus were quantified (the lower panel). E Upper: the diagram of truncated mutants of c-Jun. Lower: the IP results of JunBP and truncated mutants of c-Jun.

JunBP promoted the JNK-dependent activation of c-Jun

c-Jun is a vital mediate molecule of mitogen-activated protein kinases (MAPK) signaling pathway [32] and TGF-β could act as one of the extracellular ligands that activate this pathway [33]. When Ser63 and Ser73 of c-Jun are phosphorylated by its canonical upstream kinase JNK upon TGF-β stimulation, it is activated and functions as a transcriptional factor for its many downstream target genes [34]. We next explored whether JunBP has effect on the biological function of c-Jun. JunBP was overexpressed in a dose-dependent manner in HCC cells upon TGF-β stimulation and the results showed that JunBP promoted the activation of c-Jun but not its total protein level (Fig. 5A and Supplementary Fig. S5A). And in JunBP stably overexpressed cell lines, the activation of c-Jun was in line with the TGF-β treatment in a time-dependent manner (Fig. 5B and Supplementary Fig. S5B). When JNK was knocked down (Supplementary Fig. S5C) or its activation was blocked via SP600125 treatment, the upregulation of phosphorylated c-Jun caused by JunBP upon TGF-β stimulation was attenuated (Fig. 5C, D and Supplementary Fig. S5D, E). To investigate whether JNK also interacted with JunBP, endogenous CoIP analyses were conducted in 97H cells and the results indicated that JunBP interacted with c-Jun and JNK in 97H cells (Supplementary Fig. S5F). Further CoIP result showed that JNK had higher binding affinity to c-Jun in the presence of JunBP (Fig. 5E).

Fig. 5: JunBP promotes the JNK-dependent activation of c-Jun upon TGF-β stimulation.figure 5

A The upregulation of phosphorylated c-Jun (p-c-Jun) when transfected with JunBP upon TGF-β stimulation. B The upregulation of p-c-Jun in JunBP stably overexpressed Hep3B stimulated with TGF-β in a time-dependent manner. C siRNA-mediated knockdown of JNK leads to no change of the expression of p-c-Jun. D SP600125 treatment diminished the upregulation of p-c-Jun caused by JunBP. E IP analysis between JNK and c-Jun when JunBP was overexpressed.

Together with these observations, JunBP acts as a bridge for JNK/c-Jun interaction and leads to the more activation of c-Jun upon TGF-β stimulation.

JunBP promoted the transcription of LINC02551 in a SMAD3-dependent manner

It is reported that SMAD3 can physically interact with AP-1 family members (JunB,c-Jun, and JunD), but the specific modification sites in c-Jun that affect its interaction with SMAD3 remain unstudied [35]. Immunostaining showed that JunBP promoted the interaction between SMAD3 and c-Jun in the nucleus upon TGF-β stimulation (Fig. 6A and Supplementary Fig. S6A). Further IP analysis indicated that the higher binding affinity between SMAD3 and c-Jun caused by JunBP overexpression or TGF-β stimulation was attenuated when the cells used for IP analysis were stimulated by LY364947 (TGFBR1 inhibitor) or SP600125 (JNK inhibitor) (Fig. 6B). To further verify the modification sites in c-Jun that affect its binding affinity with SMAD3, we generated three c-Jun point mutants (S63A, S73A, and S63/73A). The results showed that S63/73A mutant nearly did not interact with SMAD3 upon TGF-β stimulation (Fig. 6C) and additional JunBP expression could not raise the binding affinity between c-Jun and SMAD3 (Supplementary Fig. S6B), which suggested that the activation of c-Jun upon TGF-β stimulation was important for its interaction with SMAD3 and the promotive effect of JunBP was partly dependent on the activation status of c-Jun. AP-1 complex is also reported to act as transcription cofactor for SMAD3 [35]. Based on our previous finding that SMAD3 interacted with the promoter region of LINC02551 to increase its transcription, we did re-ChIP assay using anti-c-Jun antibody and anti-SMAD3 antibody and found that SBE-4 but not SBE-3 was the right binding sequence with c-Jun and SMAD3 in LINC02551 promoter region (Supplementary Fig. S6C).

Fig. 6: JunBP promotes the activation of SMAD3 and c-Jun upon TGF-β stimulation.figure 6

A IF analysis of the colocalization of SMAD3 and c-Jun when JunBP was overexpressed upon TGF-β stimulation. B IP analysis of the combination between SMAD3 and c-Jun when added with LY364947/ SP600125/ TGF-β alone or together. C IP analysis of the combination of SMAD3 and different point mutants of c-Jun upon TGF-β stimulation. D The luciferase activity of LINC02551 promoter region treated with LY364947 or SP600125 upon TGF-β stimulation. E ChIP assays in SBE-4 followed by rt-PCR in 97H cells added with LY364947 or SP600125 upon TGF-β stimulation.

Above all, we hypothesized that there is a positive feedback among SMAD3, JunBP, and c-Jun upon TGF-β stimulation. We then did reporter assay and found JunBP x’enhanced the luciferase activity of LINC02551; on the contrary, LY364947 or SP600125 treatment led to decreased activity despite the presence of TGF-β stimulation (Fig. 6D). This result was confirmed in the further ChIP assay (Fig. 6E).

These data suggest that JunBP-induced activation of c-Jun in turn leads to the higher binding affinity of SMAD3 to the promoter region of LINC02551 and there is a positive feedback among them.

JunBP is positively correlated with c-Jun activation in HCC patients

Immunohistochemistry (IHC) analysis combined with in situ hybridization (ISH) analysis of HCC tissues from Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China) indicated that the expression of JunBP in HCC samples was positively correlated with the expression of the activation status of c-Jun and the expression of LINC02551 (Fig. 7A–C and Supplementary Fig. S7A). After further correlation analyses, the results showed that LINC02551 was positively correlated with SMAD3 and c-Jun (Supplementary Fig. S7A); JunBP was also positively correlated with SMAD3 and c-Jun (Supplementary Fig. S7B, C). Patients with high expression of JunBP had worse overall survival (OS) and disease-free survival (DFS) (Supplementary Fig. S7B). All 126 patients were then divided into different groups based on p-c-Jun (Ser63) and JunBP expression or p-c-Jun (Ser73) and JunBP expression: double-high p-c-Jun (Ser63)/JunBP expression (n = 54); double-low p-c-Jun (Ser63)/JunBP expression (n = 40); double-high p-c-Jun (Ser73)/JunBP expression (n = 50); double-low p-c-Jun (Ser73)/JunBP expression (n = 38). After analyzing the prognosis of these patients, we found that HCC patients with high JunBP /high p-c-Jun (Ser63) expression and high JunBP/high p-c-Jun (Ser73) expression had worse DFS and OS (Fig. 7D, E). Combined with the specific information of HCC patients, univariate regression analysis was conducted on the clinicopathological features containing JunBP expression in the cohort of HCC patients from Tongji Hospital, and the results showed BCLC stage, tumor cirrhosis, ALT level, tumor size, vascular invasion, portal vein tumor thrombus (PVTT) and JunBP expression were all correlated with the OS. After multivariate regression analysis of the above factors, we found that JunBP expression was an independent factor associated with OS (Fig. 7F).

Fig. 7: The clinical correlation between JunBP and activated c-Jun.figure 7

A IHC staining of JunBP and activated c-Jun on paraffin-embedded hepatocellular carcinoma tissue from Tongji Hospital. B The expression relevance between JunBP and activated c-Jun. C The expression relevance of JunBP/p-c-Jun (Ser63) and JunBP/p-c-Jun (Ser73). D OS and DFS of patients with high JunBP expression and high p-c-Jun (Ser73) expression. E OS and DFS of patients with high JunBP expression and high p-c-Jun (Ser63) expression. F Multivariate regression analysis with clinicopathological features significantly associated with prognosis in univariate regression analysis in HCC samples from Tongji hospital. G Schematic illustration showing the roles of JunBP in HCC cells.

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