LINC00493-encoded microprotein SMIM26 exerts anti-metastatic activity in renal cell carcinoma.

Human microproteins encoded by long non-coding RNAs (lncRNA) have been increasingly discovered, however, complete functional characterization of these emerging proteins is scattered. Here, we show that LINC00493-encoded SMIM26, an understudied microprotein localized in mitochondria, is tendentiously downregulated in clear cell renal cell carcinoma (ccRCC) and correlated with poor overall survival. LINC00493 is recognized by RNA-binding protein PABPC4 and transferred to ribosomes for translation of a 95-amino-acid protein SMIM26. SMIM26, but not LINC00493, suppresses ccRCC growth and metastatic lung colonization by interacting with acylglycerol kinase (AGK) and glutathione transport regulator SLC25A11 via its N-terminus. This interaction increases the mitochondrial localization of AGK and subsequently inhibits AGK-mediated AKT phosphorylation. Moreover, the formation of the SMIM26-AGK-SCL25A11 complex maintains mitochondrial glutathione import and respiratory efficiency, which is abrogated by AGK overexpression or SLC25A11 knockdown. This study functionally characterizes the LINC00493-encoded microprotein SMIM26 and establishes its anti-metastatic role in ccRCC, and therefore illuminates the importance of hidden proteins in human cancers.

A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is of poor clinical outcomes, and currently lacks reliable prognostic biomarkers. By analyzing the datasets of the Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we established a five-protein prognostic signature containing GBP2, HLA-DRA, ISG15, ISG20 and ITGAX. Our data indicate that this signature was closely correlated with advanced stage, higher pathological grade, and unfavorable survivals in patients with ccRCC. We further functionally characterized GBP2. Overexpression of GBP2 enhanced the phosphorylation of STAT2 and STAT3 to trigger JAK-STAT signaling and promote cell migration and invasion in ccRCC. Treatment of Ruxolitinib, a specific inhibitor of JAK/STAT, attenuated the GBP2-mediated phenotypes. Patients with high GBP2 expression were accompanied with more infiltration of immune cells positively stained with CD3, CD8, CD68, and immune checkpoint markers PD-1 and CTLA4, which was validated by Opal multiplex immunohistochemistry in ccRCC tissues. More CD8 + T cells and CD68 + macrophages were observed in patients expressing high GBP2. Taken together, a five-protein prognostic signature was constructed in our study. GBP2 has an oncogenic role via modulating JAK-STAT signaling and tumor immune infiltration, and thus may serve as a potential therapeutic target in ccRCC.

Smilax china L. rhizome extract inhibits nuclear factor-κB and induces apoptosis in ovarian cancer cells.

OBJECTIVE: To study the antitumor effects and associated mechanisms of extract of the Smilax china L. rhizome (SCR) on ovarian cancer cells. METHODS: Ovarian cancer cells A2780 were treated with different concentrations of SCR extract (SCRE), and compared with controls. Effects on cell growth were evaluated by cell counting kit-8 (CCK-8) assay; proliferation effects by EdU incorporation assay; cell cycle by propidium iodide staining; apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide; cellular distribution of nuclear factor-κB (NF-κB) by immunofluorescence; protein levels of NF-κB, caspase-3, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP), Bcl-2-associated X protein (Bax), cellular inhibitor of apoptosis (cIAP)-1, anti-X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-extra large (Bcl-XL), B-cell lymphoma-2 (Bcl-2) and AKT by Western blotting; and effects of SCRE combined with cisplatin or adriamycin on A2780 cells by CCK-8 assay. RESULTS: SCRE suppressed A2780 cell proliferation in a dose-dependent manner (P<0.05,P<0.01), arrested cells in G2/M phase and induced apoptosis by activating caspase-3, PARP and Bax. SCRE treatment also correlated with inhibition of NF-κB and downregulation of Bcl-2, Bcl-XL, cIAP-1, XIAP and AKT. SCRE can promote chemosensitivity to cisplatin and adriamycin in A2780 cells (P<0.01). CONCLUSION: SCR effectively inhibits NF-κB, induces apoptosis and reduces chemoresistance to cisplatin and adriamycin in ovarian cancer cells, which might be its molecular basis for treating ovarian cancer.

mRNA expression of IGF-1 and IGF-1R in patients with colorectal adenocarcinoma and type 2 diabetes.

BACKGROUND AND AIMS: Increasing studies show that messenger RNA (mRNA) levels of local IGF-system are overexpressed in cancer tissue of patients with colorectal cancer (CRC). However, the influence of type 2 diabetes (T2DM) on the expression of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) mRNA in colorectal cancer tissue and adjacent non-cancerous tissue (ANCT) is unknown. The aim of this study was to assess mRNA expression of IGF-1 and IGF-1R in paired samples of cancer tissue and ANCT between colorectal adenocarcinoma (CA) patients with and without T2DM. METHODS: To quantify the levels of IGF-1 and IGF-1R mRNA in CA, we analyzed the expression of IGF-1 and IGF-1R mRNA levels in paired samples of cancer tissue and ANCT in CA patients with and without T2DM using real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: mRNA levels of IGF-1 and IGF-1R were significantly higher in cancer tissue compared with its ANCT in CA patients with and without T2DM. Compared with the CA group, significantly higher levels of IGF-1 and IGF-1R mRNA were observed in cancer tissue in CA with T2DM group. No significant differences were observed in the role of cancer locations, Dukes stages and diabetes duration on mRNA expression of IGF-1. After adjusting for age, gender and Dukes stages, multivariate analysis indicated IGF-1 mRNA level was a risk factor for prognosis (p <0.05). CONCLUSIONS: Our results support the hypothesis that IGF system plays an important role in CRC. Further larger studies are needed.

[The cloning, expression and the binding ability with TRß1 of retinoid X receptor-α gene].

Retinoid X receptor-α (RXR-α), a member of nuclear receptor family, is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription. To further study the function of RXR-α, abundant of recombinant RXR-α protein in hand is necessary. In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α. Thereafter, competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium. Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses. The results showed that a protein, with the molecular mass around 50 kDa, could be selectively recognized by anti-RXR-α antibody. Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRß1 to form a heterodimer, which could specifically bind the target DNA fragment. This was confirmed by EMSA. In conclusion, the recombinant human retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.

Cloning and analysis of rat osteoclast inhibitory lectin gene promoter.

Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.