Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

Abstract

Background Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). Methods We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. Results miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. Conclusion miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

Evidence That Regulation of Pri-miRNA/miRNA Expression Is Not a General Rule of miPEPs Function in Humans

“…In summary, our results and the recent reports [ 11 , 15 , 16 ] reveal that among 32 potentially translatable MIRHGs according to ribosome profiling experiments, four out of four tested (MIR155HG, MIR497HG, MIR22HG, and pri-miR-34a) were experimentally validated.…”

“…In plants, it was initially shown for two miPEPs that they enhance the transcription of their own pri-miRNAs [ 7 ], a concept further extended to other miRNA genes and in various plant species [ 8 , 10 ]. So far in humans, only miPEP133 has been shown to promote such a positive autoregulatory loop [ 16 ]. To determine whether this is a general mechanism, we investigated the potential regulatory role of miPEP155 and miPEP497 on the expression of their pri-miRNAs.…”

“…However, recent studies have shown that a few lnc pri-miRNAs encode small peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants [ 7 ], miPEPs encoded by the lncRNAs MIR22HG (pri-miRNA-22) [ 11 ], MIR155HG (pri-miR-155) [ 15 ], and MIR34AHG (pri-miR34a) [ 16 ] have also been identified in humans. Analysis of the 48 lnc MIRHGs identified in UCSC Genome Browser with the GWIPS-viz genome browser [ 17 ] revealed that 32 of them contain sORFs exhibiting ribosome profiling marks ( Table S1 ).…”

“…Another miPEP, miPEP133, was recently identified as a 133 amino acid peptide encoded by pri-miRNA 34a. MiPEP133 is expressed in various normal tissues and downregulated in cancer cell lines and tumors, and was shown to function as a tumor suppressor in cellulo and in vivo when overexpressed [ 16 ]. Interestingly, miPEP133 localizes to mitochondria and enhances p53 transcriptional activity by disrupting mitochondrial function.…”

“…Interestingly, miPEP133 localizes to mitochondria and enhances p53 transcriptional activity by disrupting mitochondrial function. Since MIR34AHG, encoding pri-miR-34a/miR-34a, is a p53-target gene, this results in an increase of pri-miR-34a/miR-34a expression [ 16 ].…”