Recombinant Human Uncharacterized protein C7orf13 (C7orf13)

What is C7orf13 and how is it classified in current genomic databases?

C7orf13 was initially designated as an uncharacterized protein, but current research classifies it as LINC01006, a long intergenic non-protein coding RNA . Located on chromosome 7, it is part of the approximately 1,000 genes encoded by this chromosome, which makes up about 5% of the human genome . Despite being initially annotated as a protein-coding gene, its function as a long non-coding RNA is now better established through transcriptomic analyses. The gene product has been provisionally designated C7orf13 pending further characterization .

How should recombinant C7orf13 protein be stored and handled for optimal stability?

For optimal stability, recombinant C7orf13 protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use. Repeated freeze-thaw cycles should be avoided . The lyophilized powder can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add 5-50% glycerol (with 50% being the default final concentration) and aliquot before storing at -20°C/-80°C . Working aliquots can be stored at 4°C for up to one week .

What expression systems are most effective for producing recombinant C7orf13 protein?

Based on commercially available recombinant C7orf13 proteins, E. coli has been successfully used as an expression system . The recombinant full-length human C7orf13 protein (Q8NI28) spanning amino acids 1-216 has been produced with an N-terminal His tag in E. coli . This bacterial expression system appears effective for generating functional protein for research applications. Researchers should consider testing mammalian expression systems when studying post-translational modifications or when proper protein folding is critical for functional studies.

What antibodies are available for C7orf13 detection, and what are their applications?

Anti-C7ORF13 rabbit polyclonal antibodies are commercially available, including those conjugated with fluorescent markers such as ALEXA FLUOR® 750 . These antibodies are generally raised against an immunogen range covering amino acids 1-100/216 of the protein . Applications include immunofluorescence microscopy, flow cytometry, and potentially Western blotting, though specific validation for each application should be verified before experimental use. When selecting antibodies, researchers should consider the host species (rabbit in this case), clonality (polyclonal), and the specific epitope targeted to ensure compatibility with their experimental design.

How can researchers effectively validate the expression and function of C7orf13 in cellular models?

For validating C7orf13 expression and function:

• Expression validation: Real-time quantitative PCR (RT-qPCR) has been effectively used to determine C7orf13 expression levels in nasopharyngeal carcinoma tissues and cell lines .

• Functional validation: Knockdown experiments using siRNA or shRNA targeting C7orf13 have been employed to assess its role in cell proliferation, migration, and invasion . These functional assays should include appropriate controls and multiple cell lines to ensure reproducibility.

• Molecular interaction studies: RNA immunoprecipitation and dual-luciferase reporter assays have been successfully used to investigate the interaction between C7orf13 and its target microRNAs (miR-449c and miR-28-5p) .

• Pathway analysis: Researchers should consider examining downstream effectors such as FMNL2, which has been shown to have a positive relationship with C7orf13 in nasopharyngeal carcinoma tissues .

What is the association between C7orf13 expression and nasopharyngeal carcinoma progression?

Research has demonstrated that C7orf13 is overexpressed in nasopharyngeal carcinoma (NPC) tissues compared to normal tissues, and this overexpression is associated with malignant features of the cancer . Functional studies have shown that C7orf13 knockdown significantly suppresses proliferation, migration, and invasion of NPC cells, suggesting its oncogenic role in NPC progression . Furthermore, C7orf13 has been shown to sequester miR-449c and miR-28-5p in NPC cells, thereby positively regulating FMNL2 expression, which promotes cell invasion and migration . This indicates that C7orf13 contributes to NPC progression through a microRNA-mediated regulatory mechanism.

How does C7orf13 expression correlate with patient survival in glioblastoma?

Among the genes on chromosome 7, C7orf13 is one of the genes whose expression changes have shown the strongest association with patient survival in glioblastoma . While the specific mechanism of C7orf13 in glioblastoma hasn't been fully elucidated in the provided search results, it's noteworthy that gain of whole chromosome 7 is an early event in gliomagenesis that occurs in proneural-like precursor cells . Given that C7orf13 is located on chromosome 7, its increased expression due to chromosomal gain may contribute to the aggressive phenotype of glioblastoma and potentially affect patient outcomes. Further research specifically targeting C7orf13's role in glioblastoma is warranted to clarify its prognostic significance.

What methodologies have been validated for studying C7orf13's role in tumorigenesis?

Several methodologies have been validated for studying C7orf13's role in tumorigenesis:

• Gene expression analysis: RT-qPCR has been used to quantify C7orf13 expression levels in cancer tissues compared to normal tissues .

• Loss-of-function studies: RNA interference (siRNA/shRNA) targeting C7orf13 has been employed to investigate its function in cancer cell proliferation, migration, and invasion .

• Molecular interaction assays:

  • Dual-luciferase reporter assays have been used to confirm direct interactions between C7orf13 and microRNAs .

  • RNA immunoprecipitation analysis has validated the binding of C7orf13 to specific miRNAs .

• Cell-based functional assays: Migration and invasion assays have been utilized to assess the effect of C7orf13 manipulation on cancer cell behavior .

• Correlation studies: Analysis of the relationship between C7orf13 expression and downstream targets (e.g., FMNL2) in cancer tissues has provided insights into its molecular mechanisms .

How does C7orf13 interact with the miR-449c/miR-28-5p-FMNL2 axis in cancer?

C7orf13 functions as a competing endogenous RNA (ceRNA) that sequester miR-449c and miR-28-5p in nasopharyngeal carcinoma cells . Through dual-luciferase reporter assays and RNA immunoprecipitation analysis, research has confirmed that C7orf13 directly binds to these microRNAs . This interaction prevents these microRNAs from binding to their target gene, formin-like 2 (FMNL2), thereby upregulating FMNL2 expression . FMNL2, in turn, promotes cell invasion and migration in NPC . Experimental data has shown that C7orf13 has a positive relationship with FMNL2 in NPC tissues, and C7orf13 knockdown weakens FMNL2-mediated cell invasion and migration . This regulatory axis (C7orf13-miR-449c/miR-28-5p-FMNL2) represents a critical mechanism by which C7orf13 contributes to cancer progression.

How does chromosome 7 gain in cancer cells affect C7orf13 expression and function?

Gain of whole chromosome 7 is an early event in gliomagenesis that occurs in proneural-like precursor cells, which ultimately give rise to all isocitrate dehydrogenase (IDH) wild-type glioblastoma transcriptional subtypes . Among the genes on chromosome 7, C7orf13 is one of the genes whose expression changes have shown the strongest association with patient survival . This suggests that chromosome 7 gain leads to increased C7orf13 expression, potentially contributing to cancer progression. While platelet-derived growth factor A (PDGFA) on chromosome 7 is known to drive gliomagenesis, research indicates that there are likely several other genes on chromosome 7, potentially including C7orf13, that select for increased whole-chromosome copy number within glioblastoma cells . The specific mechanisms by which increased C7orf13 copy number and expression contribute to cancer cell fitness advantages require further investigation.

What are the key methodological challenges in researching C7orf13's function?

Several methodological challenges exist in researching C7orf13's function:

• Dual classification confusion: C7orf13 was initially annotated as an uncharacterized protein but is now recognized as a long non-coding RNA (LINC01006) . This dual classification may lead to confusion in experimental design and interpretation of results.

• Limited structural information: The lack of detailed structural information about C7orf13, particularly regarding its functional RNA domains, hampers structure-based functional studies.

• Complex regulatory networks: C7orf13 appears to be involved in complex regulatory networks involving microRNAs and downstream targets . Disentangling these interconnected pathways requires sophisticated experimental approaches and careful validation of results.

• Cancer-specific context: C7orf13's function may be highly context-dependent, varying across different cancer types. Research has primarily focused on nasopharyngeal carcinoma and potentially glioblastoma , but its role in other cancers remains unexplored.

• In vivo relevance: Translating in vitro findings to in vivo models and ultimately to clinical relevance presents significant challenges, particularly for molecular targets with complex regulatory functions.

What potential therapeutic applications might emerge from targeting C7orf13 in cancer?

Based on current research, several potential therapeutic applications might emerge from targeting C7orf13 in cancer:

• RNA interference therapy: The development of siRNA or antisense oligonucleotides targeting C7orf13 could potentially suppress its oncogenic function in cancers where it is overexpressed .

• MicroRNA mimics: Since C7orf13 functions by sequestering miR-449c and miR-28-5p , introducing mimics of these microRNAs might counteract C7orf13's effect and inhibit cancer progression.

• Diagnostic biomarker: C7orf13 overexpression in nasopharyngeal carcinoma and its association with survival in glioblastoma suggest its potential use as a diagnostic or prognostic biomarker.

• Combination therapy: Targeting C7orf13 in combination with conventional therapies might enhance treatment efficacy, particularly in cancers where C7orf13 contributes to therapy resistance.

• Downstream target inhibition: Targeting downstream effectors of C7orf13, such as FMNL2 , might provide an alternative therapeutic strategy when direct targeting of C7orf13 is challenging.

How can researchers address the contradictions in C7orf13 literature regarding its classification as a protein versus non-coding RNA?

To address contradictions regarding C7orf13's classification:

• Comprehensive transcriptomic analysis: Employing RNA-seq and ribosome profiling to definitively determine whether C7orf13 transcripts are translated into protein or function primarily as non-coding RNA.

• Proteomic verification: Using mass spectrometry to detect potential C7orf13 protein products in various tissues and cell types, confirming or refuting its protein-coding potential.

• Evolutionary conservation analysis: Examining the evolutionary conservation of C7orf13's open reading frame versus its RNA structure across species to infer functional significance.

• Functional validation: Designing experiments that specifically distinguish between protein-dependent and RNA-dependent functions, such as introducing frameshift mutations that disrupt the protein sequence without affecting the RNA structure.

• Dual-function hypothesis testing: Investigating whether C7orf13 might have dual functions, acting both as a non-coding RNA and producing a functional protein under specific cellular conditions.