LncRNA不能编码蛋白多肽？您又OUT了！！！

本文原文是今年11月发表在J Hepatol 杂志上的一篇基础研究论著，该杂志最新影响因子为 20.582，第一作者是第二军医大学长海医院Yanan Pang和Zhiyong Liu，通讯作者是第二军医大学长海医院Shanrong Liu 教授和俄克拉何马大学斯蒂芬森生命科学研究中心、浙江大学材料科学与工程学院 Chuanbin Mao教授。

本期文献解读作者为介入小崔哥。

SMIM30肽通过诱导SRC/YES1膜锚定

和MAPK通路激活促进肝癌的发生发展

人们对肽如何激活在癌症等疾病中起关键作用的信号通路知之甚少。本研究报道了LncRNA LINC00998编码的一种小的保守内源性多肽（SMIM30），而不是LINC00998本身，通过调节细胞增殖和迁移来促进肝细胞癌的发生。值得注意的是，它结合非受体酪氨酸激酶SRC/YES1，驱动其膜锚定和磷酸化，激活下游MAPK信号通路。本研究不仅阐明了肽促进HCC肿瘤发生的新机制，也展示了肽如何激活MAPK信号通路。

背景与目的：越来越多的证据表明，一些非编码RNA(NcRNAs)含有可翻译成短肽的小开放阅读框(SmORFs)。在这里，我们的目标是确定这些短肽在哪里以及如何促进肝细胞癌(HCC)的发展。

方法：用抗核糖体蛋白S6(RPS6)抗体对4种癌细胞进行RNA免疫沉淀和高通量测序(RIP-SEQ)。以长非编码RNA(LncRNA)LINC00998为研究对象，利用qPCR和公共数据库对其在肝癌患者中的表达水平进行了研究。构建了特殊载体以确定其编码潜力。我们还探讨了LINC00998编码的多肽在肿瘤生长和转移中的作用和机制。

结果：我们发现在癌细胞中有许多lncRNA与RPS6结合。其中一个lncRNA，LINC00998，编码一种小的内源性多肽，称为SMIM30。SMIM30，而不是RNA本身，通过调节细胞增殖和迁移促进了肝癌的发生，其水平与肝癌患者的低存活率相关。此外，SMIM30被c-Myc转录，然后驱动非受体酪氨酸激酶SRC/YES1的膜锚定。此外，SRC/YES1激活了下游的MAPK信号通路。

结论：我们的研究结果不仅揭示了ncRNA编码的多肽促进肝癌发生的新机制，而且提示这些多肽可以作为肝癌治疗的新靶点和肝癌诊断和预后的新生物标志物。

主要研究内容：

1.SMIM30肽在HCC组织中高表达。

2.SMIM30肽在体内外均能促进HCC细胞的增殖和迁移。

3.SMIM30是SRC/YES1膜锚定和激活状态的重要适配器。

4.MAPK信号通路被SRC/YES1-SMIM30复合物激活。

主要研究结果：

Fig. 1

Frequent upregulation of LINC00998 in HCC tissues and its peptide coding potential.

(A). Fold enrichment of U1 snRNA in SNRNP70 pull-downs (left panel) and ACTB in RPS6 pull-downs (right panel). (B). Hierarchical clustering analysis of significantly enriched 105 lncRNAs co-existed in Huh7, DU145, HCT116 and SW1990 cell lines (left panel) and the top 10 enriched lncRNAs were shown as enlarged heat-map (right upper panel). Furthermore, graphic (right lower panel) showed the smORFs of the top 10 enriched lncRNAs. (C) Boxplots show that the expression of LINC00998 was significantly increased in patients with HCC (T = 369, N = 160) and CHOL (T = 36, N = 9) from GEPIA (T tumor; N normal; ∗p <0.05). (D) High expression of LINC00998 was significantly correlated with the shorter OS and DFS of HCC in GEPIA cohort. Red line indicates high expression and blue line indicates low expression. (E). Relative expression levels of LINC00998 in HCC tissues (n = 24) compared with corresponding NT tissues (p = 0.018). (F). LINC00998 expression levels were compared between primary HCC tissues and corresponding adjacent tissues (NT) in the indicated cases by gel electrophoresis. (G). Fold enrichment of LINC00998n in RPS6 pull-downs. ∗∗p <0.01, ∗p <0.05. Statistical analysis was determined by Student's t test. CHOL, cholangiocarcinoma; DFS, disease-free survival; GEPIA, Gene Expression Profiling Interactive Analysis; HCC, hepatocellular carcinoma; HR, hazard ratio; lncRNA, long non-coding RNA; smORFs, small open reading frames; snRNA, small nuclear RNA; NT, non-tumor; OS, overall survival. (This figure appears in color on the web.)

Fig. 2

Membrane peptide SMIM30 is endogenously produced and highly expressed in HCC tissues.

(A). A smORF is contained within the exon 3 of an annotated lncRNA – LINC00998 in human genomes. The position of the smORF is indicated in red. (B) Cellular localization analysis: RNA-FISH assays of LINC00998 in Huh7 cells. Scale bars, 20 μm. (C) Diagram of the constructs used for in vitro translation of the SMIM30 peptide. The full-length of smORF sequence with Flag tag was cloned into the pSPT19 vector containing the SP6 phage RNA polymerase promoter (SP6-SMIM30). Coupled in vitro transcription and translation reaction of the SP6-SMIM30 vector produced a ~7.3 kDa peptide (SMIM30-Flag) visualized by SDS-PAGE. (D) Diagram of the constructs used for in vivo translation of the SMIM30 peptide. The full-length smORF sequence with Flag tag was cloned into the pcDNA3.1 vector containing the CMV phage RNA polymerase promoter (CMV-SMIM30). Coupled in vivo transcription and translation of the CMV-SMIM30 vector produced a ~7.3 kDa peptide (SMIM30-Flag) also visualized by SDS-PAGE. (E) The unique SMIM30 peptide was identified using mass spectrometry. (F) The SMIM30 peptide in the indicated liver cancer cells (Hep3B, Huh7 and SMMC7721) was directly detected with the anti-SMIM30 antibody. Moreover, the expression of the SMIM30 peptide can be abolished by blocking translation with cycloheximide at the indicated time (upper panel). Otherwise, SMIM30 peptide levels were detected in liver cancer tissues (T) and their corresponding adjacent non-tumor (NT) tissues. SMIM30 peptide in Huh7 cells was a positive control (lower panel). (G) Huh7 cells were transfected with the SMIM30-GFP fusion constructs, SMIM30-GFP fusion constructs with ΔATG mutants and pEGFP-N1 control plasmids. Transfected cells were examined by western blot (upper panel) and fluorescence microscopy (lower panel). (H) Representative immunofluorescence images of SMIM30 peptide in Huh7 cells detected by imaging flow cytometry analysis (left panel) and immunostained using the SMIM30 antibody in different protein extractions (membrane and cytoplasm) by western blot analysis (right panel). Scale bars, 20 μm. (I) SMIM30 is conserved in vertebrates. ClustalW2 multiple protein sequence alignment of SMIM30 peptide sequences from 5 vertebrates. Darker shading indicates higher percentage identity of the amino acid. The signal peptide cleavage sites predicted by Signal IP-4.1 were also indicated (left panel). SMIM30 signal sequence drives membrane anchoring. Transfection of SMIM30-GFP construct revealed that the wild-type SMIM30 signal sequence drove membrane anchoring (membrane localization of SMIM30-GFP fusion protein), whereas mutation of V→W, G→W in the signal peptide cleavage sites caused SMIM30-GFP to diffuse intracellularly. Scale bars, 50 μm. CMV, cytomegalovirus; FISH, fluorescence in situ hybridization HCC, hepatocellular carcinoma; lncRNA, long non-coding RNA; smORFs, small open reading frames. (This figure appears in color on the web.)

Fig. 3

The SMIM30 peptide, not LINC00998, promotes HCC cell proliferation, migration and invasion in vitro and in vivo.

(A). Relative expression of LINC00998 in Huh7 and SMMC7721 cells after transfected with LINC00998 shRNAs compared with controls. Thus, LINC00998/SMIM30-silenced stable cell lines were established. Data are mean ± SD (n = 3) (lower panel). Meanwhile, the expression level of SMIM30 peptide was also detected by western blot after LINC00998/SMIM30 silencing (upper panel). (B). CCK8 assays were performed to measure cell proliferation ability of Huh7 (upper panel) and SMMC7721 (lower panel) after LINC00998/SMIM30 silencing at the indicated time points. (C). Transwell assays were performed to evaluate cell migration ability following LINC00998/SMIM30 knockdown in Huh7 (left panel) and SMMC7721 (right panel) cells. Scale bars, 100 μm. (D). Representative images of the wound-healing migration assays in LINC00998/SMIM30-silenced Huh7 and SMMC7721 cells compared with negative controls. Scale bars, 200 μm. (E). The statistical graphs represented the percentage of LINC00998/SMIM30-silenced Huh7 and SMMC7721 cells in G0/G1, S or G2/M phase, as indicated. (F). CCK8 assays were performed to measure cell proliferation ability of SMIM30-silenced Huh7 cells after transfected with indicated constructs. (G). The statistical graphs indicate the cell numbers of Transwell assays performed in SMIM30-silenced Huh7 cells after transfected with indicated constructs. Data are mean ± s.d. (n = 3). (H). Representative images of the wound-healing migration assays in SMIM30-silenced Huh7 cells after transfected with indicated constructs, compared with shRNA1. Scale bars, 200 μm. (I). The statistical graphs represented the percentage of SMIM30-silenced Huh7 cells after transfected with indicated constructs in G0/G1, S or G2/M phase, as indicated. (J). Tumors formed from the Huh7 cells with stable SMIM30 knockdown and the control cells in nude mice are shown. (K). Representative H&E staining images of metastatic nodules in the lungs of nude mice (SMIM30 stably knockdown Huh7 cells and control group). The metastatic nodules are indicated by black arrows (left panel). Images of nude mice lungs (middle panel). The numbers of metastatic tumor in nude mice lungs were calculated and compared between the 2 groups (right panel). Scale bars, 200 μm and 50 μm.

∗∗p <0.01, ∗p <0.05. Statistical analysis was determined by Student's t test. HCC, hepatocellular carcinoma; shRNA, short hairpin RNA. (This figure appears in color on the web.)

 

Fig. 4

SMIM30 peptide drives the membrane anchoring of SRC/YES1.

(A) Proteins interacted with the SMIM30 peptide were identified by combining co-immunoprecipitation and mass spectrometry. (B) Protein-protein interaction assay indicated that proteins interacted with SMIM30 peptide participated in the metabolic pathways, focal adhesion, endocytosis, cell tight junction, protein binding and ATP binding etc. (C) Details of some potential proteins interacted with SMIM30 peptide. (D) SMIM30 peptide knocking down led to the reduced phosphorylation level of P-416 (SRC/YES1) and increased P-527 phosphorylation level (SRC/YES1) in Huh7 cells and SMMC7721 cells. But this effect could be reversed by transfection with SMIM30-Flag (OF) plasmid. (E) Immunoprecipitation after SMIM30-Flag (OF) plasmid was transfected into Huh7 cells and cellular lysates were treated with 30 μg/ml RNase A for 30 min or not. First, SMIM30-Flag complexes were co-immunoprecipitated by anti-Flag antibody, then SRC and YES1 were detected respectively; SRC or YES1 complexes were also co-immunoprecipitated by anti-SRC or anti-YES1 antibodies directly, then SMIM30 peptide was detected. (F) Immunofluorescence assay of double-labeled SRC/YES1 and SMIM30 was performed. SMIM30-Flag plasmid was transfected into Huh7 cells with SRC-HA plasmid and YES1-HA plasmid separately, then SMIM30 (Flag)-SRC/YES1 (HA) complexes were immunostained with anti-Flag and anti-HA antibodies. (G) Diagram of SRC/YES1 wild-type and mutations constructs with the different domains. (H) Co-immunoprecipitation of SRC/YES1(NH2) part and SMIM30 peptide. (I) Domains of SRC/YES1 interacted with the SMIM30 peptide were identified. The indicated SRC/YES1-HA plasmids together with SMIM30-Flag plasmid were co-transfected into Huh7 cells, then SRC/YES1-HA complexes and SMIM30-Flag complexes were co-immunoprecipitated by anti-HA and anti-Flag antibodies, respectively. SMIM30-Flag and SRC/YES1-HA were then detected using anti-Flag and anti-HA antibodies, respectively. (This figure appears in color on the web.)

Fig. 5

Activation of the MAPK signaling pathway by c-Myc transcribed SMIM30 peptide.

(A). Hierarchical clustering analysis of differentially expressed genes between SMIM30-silenced (LINC00998 shRNA1) and control Huh7 cells. (B). MA-plot of differentially expressed genes in SMIM30-silenced Huh7 cells compared with control cells. log2 fold >1, p <0.05. (C). Most significantly enriched GO terms of differentially expressed genes in SMIM30-silenced Huh7 cells. (D). GSEA results indicated that SMIM30 peptide expression is positively correlated with protein-targeting to membrane and anchoring component of membrane. (E). Top 20 enrichment KEGG pathways of the differentially expressed genes in SMIM30-silenced Huh7 cells. (F) Protein-protein interaction network of differentially expressed genes in SMIM30-silenced Huh7 cells compared with control cells. Genes in MAPK pathway were highlighted in red. (G). Relative expression of RPS6KA1, FOS, ATF2, TGFB1, HSPA1B, RRAS genes in SMIM30-silenced (Huh7 and SMMC7721) cells compared with control cells. (H). Western blot analysis of indicated proteins in SMIM30-silenced (Huh7 and SMMC7721) cells, control cells and both transfected with (smORF+Flag) OF plasmid. (I). c-Myc biding site prediction in the LINC00998 promoter region using JASPAR (upper panel) and chromatin immunoprecipitation assays with Huh7 and SMMC7721 cells (lower panel). B site: potential biding site of c-Myc; N site: nonbinding site of c-Myc. (J). Luciferase reporter assays: The LINC00998 promoter region (−1507 to −210 nt relative to TSS) of deletion variants with (MUT) or without (WT) mutation in the c-Myc binding site was cloned upstream of the firefly luciferase coding region. Their luciferase activities were tested in both Huh7 and SMMC7721 cells co-transfected with c-Myc expression vector (c-Myc) or empty vector (Vector). Data were normalized to renilla luciferase. ∗∗p <0.01, ∗p <0.05. Statistical analysis was determined by Student's t test. GO, gene ontology; GSEA, gene set enrichment analysis; NES, normalized enrichment score; shRNA, short hairpin RNA. (This figure appears in color on the web.)

原文链接：https://sci-hub.se/10.1016/j.jhep.2020.05.028

补充材料链接：https:// doi.org/10.1016/j.jhep.2020.05.028.

小崔哥有话说：为什么这是一篇20+的文章？

加分项：肿瘤非编码RNA研究是热点；LncRNA编码保守多肽SMIM30促进肿瘤的作用报道很少，创新性强；MAPK通路作为SRC/YES1下游是新发现；有整体动物实验结果和临床数据分析

减分项：LncRNA有些过气了，上游机制和功能的连接稍薄弱。

结论：热点领域+新方向新发现+新机制+临床生信分析 = 20+分SCI论文

预计工作量：3-5年

技术难度：常规实验操作（功能实验、RIP、高通量测序、mRNA、蛋白表达检测、FISHI、Co-IP、质谱等）+  在体功能实验（裸鼠肿瘤模型）+蛋白表达组分析（芯片公司）+临床数据、生信分析

难点：选题思路设计，选定下游的SRC/YES1分子和MAPK通路

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