Recombinant Mouse Transmembrane protein 235 (Tmem235)

What is Mouse Transmembrane protein 235 (Tmem235)?

Tmem235 refers to a long noncoding RNA (Lnc) located on chromosome 10 in mice. It consists of seven exons with a full transcript length of 2853 nucleotides and shares the same transcriptional direction as BIRC5 (Baculoviral IAP Repeat Containing 5), being located downstream of this gene . Despite its name suggesting a transmembrane protein, research has confirmed that Lnc Tmem235 does not encode a protein, as demonstrated through immunoblotting experiments with myc fusion protein analysis . The transcript primarily functions as a regulatory RNA molecule involved in cellular processes related to cell survival, particularly under hypoxic conditions. Lnc Tmem235 has gained significant research interest due to its role in preventing hypoxia-induced apoptosis in bone marrow mesenchymal stem cells.

What is the subcellular localization of Tmem235 and why is it significant?

Lnc Tmem235 is predominantly localized in the cytoplasm of bone marrow mesenchymal stem cells (BMSCs), as demonstrated through RNA fluorescence in situ hybridization (RNA-FISH) techniques . This cytoplasmic localization is crucial for understanding its function, as it positions Tmem235 to interact with cytoplasmic molecules including microRNAs and messenger RNAs. The cytoplasmic distribution allows Lnc Tmem235 to function as a competing endogenous RNA (ceRNA), specifically interacting with miR-34a-3p to regulate gene expression post-transcriptionally . This localization pattern distinguishes it from nuclear lncRNAs that typically function through chromatin remodeling or transcriptional regulation. The significance of this cytoplasmic localization lies in enabling Tmem235 to competitively bind miR-34a-3p, preventing this microRNA from silencing BIRC5 mRNA and thereby promoting BIRC5 expression, which ultimately protects BMSCs from hypoxia-induced apoptosis.

How does Tmem235 expression change under hypoxic conditions?

Under hypoxic conditions (0% O₂, 95% N₂, and 5% CO₂), the expression of Lnc Tmem235 is significantly downregulated in BMSCs . This downregulation coincides with altered expression of apoptosis-related proteins, specifically upregulation of pro-apoptotic Bcl-2-associated X protein (Bax) and downregulation of anti-apoptotic B-cell lymphoma-2 protein (Bcl-2) . The reduced expression of Lnc Tmem235 during hypoxia appears to be part of the cellular stress response that ultimately leads to increased BMSC apoptosis, with studies showing apoptotic rates exceeding 70% under these conditions . The mechanistic relationship between hypoxia and Tmem235 downregulation suggests that this lncRNA may serve as an oxygen-sensitive regulator of cell survival pathways. Understanding this hypoxia-induced change in Tmem235 expression provides insights into how BMSCs respond to the hypoxic microenvironment present in osteonecrotic areas, which is critical for developing strategies to enhance BMSC survival in therapeutic applications.

How does Lnc Tmem235 inhibit hypoxia-induced apoptosis in BMSCs?

Lnc Tmem235 inhibits hypoxia-induced apoptosis in BMSCs through a sophisticated molecular mechanism involving the regulation of BIRC5 (also known as Survivin), a potent inhibitor of apoptosis . When overexpressed in BMSCs, Lnc Tmem235 significantly reduces the apoptotic rate of these cells under hypoxic conditions (0% O₂, 95% N₂, and 5% CO₂) . Mechanistically, Lnc Tmem235 acts as a competitive endogenous RNA (ceRNA) that binds to miR-34a-3p, preventing this microRNA from targeting and silencing BIRC5 mRNA . This competitive binding ultimately promotes BIRC5 expression, which directly inhibits the activities of caspase-3 (CASP-3) and caspase-9 (CASP-9), two critical executioners of apoptosis . The anti-apoptotic effect of Lnc Tmem235 has been confirmed through multiple experimental approaches, including overexpression and silencing experiments, which demonstrated corresponding changes in apoptotic rates and expression levels of apoptosis-related proteins like Bax and Bcl-2 .

What is the miR-34a-3p/BIRC5 axis and how does Tmem235 regulate it?

The miR-34a-3p/BIRC5 axis represents a regulatory pathway where microRNA-34a-3p (miR-34a-3p) targets and suppresses the expression of BIRC5 (Baculoviral IAP Repeat Containing 5), an important inhibitor of apoptosis . Lnc Tmem235 regulates this axis by functioning as a competing endogenous RNA (ceRNA) that shares the same binding site for miR-34a-3p as the BIRC5 mRNA 3'UTR . Through bioinformatic tools (miRDB and RNAhybrid), researchers identified that miR-34a-3p can simultaneously bind to both Lnc Tmem235 and the BIRC5 mRNA 3'UTR at the same binding site, creating competition for miR-34a-3p binding . RNA immunoprecipitation (RIP) experiments confirmed that overexpression of miR-34a-3p significantly increased the enrichment of both Lnc Tmem235 and BIRC5 mRNA in miRNA ribonucleoprotein complexes (miRNPs) . Luciferase reporter assays further validated the direct interaction between miR-34a-3p and both Lnc Tmem235 and BIRC5 mRNA, as demonstrated by decreased luciferase activity when miR-34a-3p was upregulated . By competitively binding to miR-34a-3p, Lnc Tmem235 reduces the suppressive effect of this microRNA on BIRC5, ultimately promoting BIRC5 expression and enhancing BMSC survival under hypoxic conditions.

How can the interaction between Lnc Tmem235 and miR-34a-3p be experimentally verified?

The interaction between Lnc Tmem235 and miR-34a-3p can be experimentally verified through multiple complementary approaches. RNA immunoprecipitation (RIP) assays provide direct evidence of physical interaction by detecting the enrichment of Lnc Tmem235 in miRNA ribonucleoprotein complexes (miRNPs) when miR-34a-3p is overexpressed . This technique involves immunoprecipitating miRNPs followed by quantification of associated Lnc Tmem235 using quantitative PCR . Luciferase reporter assays offer functional validation by inserting the cDNA of Lnc Tmem235 downstream of a luciferase reporter gene (Lv-Luc-Lnc Tmem235) and measuring luciferase activity when miR-34a-3p is overexpressed . A significant decrease in luciferase activity indicates direct binding between miR-34a-3p and Lnc Tmem235 . Mutation analysis provides specificity confirmation by introducing mutations in the predicted binding sites of Lnc Tmem235 (Lnc Tmem235 MUT) and assessing whether these mutations abolish the effect of miR-34a-3p on luciferase activity . Additionally, competitive binding experiments can demonstrate the ceRNA mechanism by showing that overexpression of Lnc Tmem235 reduces the enrichment of BIRC5 mRNA in miRNPs while increasing BIRC5 expression levels .

What methods can be used to overexpress or silence Tmem235 in BMSCs?

Overexpression or silencing of Tmem235 in BMSCs can be achieved through several molecular techniques. For overexpression, lentiviral vectors carrying the Lnc Tmem235 sequence (Lv-Lnc Tmem235) provide an efficient method for stable integration and expression in BMSCs . These vectors typically include fluorescent reporters like enhanced green fluorescent protein (EGFP) to track transduction efficiency . To silence Tmem235 expression, short hairpin RNA (shRNA) technology delivered via lentiviral vectors (Lv-Sh-Lnc Tmem235) effectively reduces endogenous Tmem235 levels . The efficacy of both overexpression and silencing can be verified through quantitative PCR (qPCR) to measure Tmem235 transcript levels before proceeding with functional studies . For more transient modulation, synthetic RNA oligonucleotides or plasmid-based expression systems represent alternative approaches. When designing these genetic tools, researchers should carefully consider the full-length transcript (2853 nucleotides) and ensure proper controls are included, such as empty vectors or non-targeting sequences, to distinguish specific effects from potential artifacts of the delivery method .

How can researchers evaluate the effect of Tmem235 on BMSC apoptosis?

Researchers can employ multiple complementary techniques to comprehensively evaluate the effect of Tmem235 on BMSC apoptosis. Flow cytometry with Annexin V/PI (propidium iodide) staining offers quantitative assessment of early and late apoptotic populations, enabling calculation of apoptotic rates exceeding 70% under hypoxic conditions and demonstrating reduction to approximately 20% with Tmem235 overexpression . Western blotting for apoptosis-related proteins provides mechanistic insights through measurement of Bax (pro-apoptotic), Bcl-2 (anti-apoptotic), and caspase-3 (CASP-3) expression levels, revealing that Tmem235 overexpression reverses hypoxia-induced changes in these proteins . Caspase activity assays specifically quantify the enzymatic activities of caspase-3 and caspase-9, key executioners of apoptosis, showing significant reduction in activity with Tmem235 overexpression . TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) staining visualizes DNA fragmentation in apoptotic cells both in vitro and in vivo, demonstrating decreased TUNEL-positive cells in Tmem235-overexpressing BMSCs transplanted into osteonecrotic areas . For in vivo tracking, fluorescent dyes like DiR combined with GFP expression allow monitoring of BMSC survival and apoptosis in animal models, with higher fluorescence intensity indicating greater cell survival .

How can tissue-engineered bone with BMSCs overexpressing Tmem235 be constructed for in vivo studies?

Construction of tissue-engineered bone with BMSCs overexpressing Tmem235 for in vivo studies involves a multi-step protocol combining cellular engineering with scaffold preparation. First, BMSCs should be isolated from bone marrow and cultured under standard conditions prior to genetic modification . Lentiviral transduction with Lv-Lnc Tmem235 vectors enables stable overexpression of Tmem235, with verification of successful transduction through qPCR and fluorescent marker expression . For cell tracking purposes, BMSCs can be labeled with near-infrared fluorescent dye DiR, allowing subsequent in vivo visualization . Xenogeneic antigen-extracted cancellous bone (XACB) serves as an appropriate scaffold material, providing both structural support and osteoinductive properties . BMSCs overexpressing Tmem235 should be thoroughly mixed with the XACB scaffold and cultured for a short period to ensure cell attachment and distribution throughout the scaffold . The resulting tissue-engineered bone construct can then be surgically implanted into animal models of early steroid-induced osteonecrosis of the femoral head (SONFH), with careful preparation of the defect site to ensure proper integration . Post-surgical evaluation includes fluorescence imaging to track cell survival, micro-CT examination to assess bone formation, histological analysis through H&E and Masson staining, and molecular analysis of osteogenic markers to comprehensively evaluate therapeutic efficacy .

How does Tmem235 overexpression improve the therapeutic effect of BMSCs in early SONFH?

Tmem235 overexpression substantially improves the therapeutic effect of BMSCs in early steroid-induced osteonecrosis of the femoral head (SONFH) through multiple cellular and molecular mechanisms. By inhibiting hypoxia-induced apoptosis, Tmem235 overexpression significantly enhances the survival rate of transplanted BMSCs in the hypoxic microenvironment of osteonecrotic areas, as evidenced by higher DiR fluorescence intensity and GFP expression, alongside decreased TUNEL-positive cells compared to control BMSCs . This improved survival directly translates to enhanced therapeutic efficacy, with micro-CT examination at 12 weeks post-surgery revealing complete repair of the defect area in the Lnc Tmem235 overexpression group compared to incomplete repair in control groups . Histological evaluation through H&E and Masson staining demonstrated more mature bone tissue formation with Tmem235 overexpression, indicating superior quality of regenerated bone . Quantitative bone parameters further supported these findings, with significant increases in trabecular number (Tb.N), trabecular thickness (Tb.Th), bone volume (BV), and bone volume fraction (BVF) in the Tmem235 overexpression group . Additionally, molecular analysis showed upregulation of osteogenic markers including Runx2, osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG), suggesting enhanced osteogenic differentiation of the surviving BMSCs .

What are the potential limitations and challenges in translating Tmem235-based therapy to clinical applications?

Despite promising preclinical results, several limitations and challenges exist in translating Tmem235-based therapy to clinical applications. Safety concerns regarding lentiviral vectors used for Tmem235 overexpression present significant regulatory hurdles, as viral integration could potentially lead to insertional mutagenesis or oncogene activation, necessitating development of non-viral delivery methods or safer integration-free vectors . The long-term stability of Tmem235 overexpression in transplanted BMSCs remains uncertain, as gradual silencing of transgene expression could diminish therapeutic efficacy over time, requiring strategies to maintain stable expression . Species differences between mouse and human Tmem235 may affect functional conservation, potentially limiting direct translation of murine findings to human applications without additional validation studies in human cells and tissues . The complex hypoxic microenvironment in human SONFH likely differs from animal models, possibly affecting the anti-apoptotic efficacy of Tmem235 overexpression . Furthermore, patient heterogeneity in SONFH etiology, progression stage, and individual responses to cell therapy presents challenges for standardized treatment protocols . The competition between therapeutic mechanisms and ongoing pathological processes in more advanced SONFH cases may reduce efficacy, suggesting potential limitation to early-stage disease . Finally, scalability issues in generating sufficient quantities of autologous Tmem235-modified BMSCs for clinical use present practical challenges, potentially requiring exploration of allogeneic approaches with associated immunological considerations .

How can contradictory results in Tmem235 research be reconciled?

Reconciling contradictory results in Tmem235 research requires systematic analysis of methodological differences and experimental contexts. Variation in hypoxia protocols, including oxygen concentration, exposure duration, and gradual versus sudden onset, can significantly influence cellular responses and Tmem235 function, necessitating standardized hypoxia conditions to ensure comparability between studies . Differences in BMSC sources (donor age, species, isolation methods) and passage number affect cellular phenotype and response to Tmem235 modulation, suggesting the need for detailed reporting of cell characteristics . The extent of Tmem235 overexpression or knockdown achieved in different studies directly impacts functional outcomes, with insufficient modulation potentially yielding inconsistent results, highlighting the importance of quantitative verification of expression changes . Context-dependent functions of the miR-34a-3p/BIRC5 axis may exist across different cell types or disease models, as miR-34a-3p likely regulates multiple targets beyond BIRC5, creating complex regulatory networks that vary by cellular context . In vivo discrepancies could stem from differences in SONFH animal models, including induction methods, severity, and timing of intervention . Additionally, variations in scaffold properties when constructing tissue-engineered bone (porosity, mechanical strength, biodegradation rate) influence BMSC behavior and therapeutic outcomes . To reconcile these contradictions, researchers should implement comprehensive reporting of methodological details, perform dose-response studies of Tmem235 modulation, validate findings across multiple experimental systems, and directly address inconsistencies through targeted comparative experiments.

How can researchers accurately quantify the expression and activity of Tmem235 in experimental settings?

Accurate quantification of Tmem235 expression and activity in experimental settings requires a multi-faceted approach combining various molecular techniques. Quantitative real-time PCR (qRT-PCR) serves as the primary method for measuring Tmem235 transcript levels, requiring carefully designed primers specific to the 2853-nucleotide transcript and appropriate housekeeping genes for normalization . Northern blotting provides visualization of the full-length transcript and potential isoforms, offering information about transcript integrity that qPCR alone cannot provide . RNA fluorescence in situ hybridization (RNA-FISH) enables subcellular localization analysis and single-cell expression heterogeneity assessment, as demonstrated in studies confirming the cytoplasmic distribution of Tmem235 . For functional activity measurement, RNA immunoprecipitation (RIP) quantifies Tmem235 enrichment in miRNA ribonucleoprotein complexes (miRNPs), directly reflecting its interaction with miR-34a-3p . Luciferase reporter assays with wild-type and mutated Tmem235 binding sites provide functional readouts of Tmem235's ability to competitively bind miR-34a-3p . Measurement of downstream effectors including BIRC5 expression, caspase-3/9, activities, and apoptotic rates serves as indirect indicators of Tmem235 activity . RNA stability assays using transcription inhibitors like actinomycin D can evaluate whether interventions affect Tmem235 stability rather than transcription . Additionally, developing Tmem235-specific molecular beacons or biosensors would enable real-time monitoring of expression dynamics in living cells, though these advanced techniques require further development .