Overexpression Of Lin28a Research Articles

The role of Lin28A and Lin28B in cancer beyond Let-7.

Lin28A and Lin28B are paralogous RNA-binding proteins that play fundamental roles in development and cancer by regulating the microRNA family of tumor suppressor Let-7. Although Lin28A and Lin28B share some functional similarities with Let-7 inhibitors, they also have distinct expression patterns and biological functions. Increasing evidence indicates that Lin28A and Lin28B differentially impact cancer stem cell properties, epithelial-mesenchymal transition, metabolic reprogramming, and other hallmarks of cancer. Therefore, it is important to understand the overexpression of Lin28A and Lin28B paralogs in specific cancer contexts. In this review, we summarize the main similarities and differences between Lin28A and Lin28B, their implications in different cellular processes, and their role in different types of cancer. In addition, we provide evidence of other specific targets of each lin28 paralog, as well as the lncRNAs and miRNAs that promote or inhibit its expression, and how this impacts cancer development and progression.

Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells.

Thymic epithelial cells (TECs) are essential for T cell development in the thymus, yet the mechanisms governing their differentiation are not well understood. Lin28, known for its roles in embryonic development, stem cell pluripotency, and regulating cell proliferation and differentiation, is expressed in endodermal epithelial cells during embryogenesis and persists in adult epithelia, implying postnatal functions. However, the detailed expression and function of Lin28 in TECs remain unknown. In this study, we examined the expression patterns of Lin28 and its target Let-7g in fetal and postnatal TECs and discovered opposing expression patterns during postnatal thymic growth, which correlated with FOXN1 and MHCII expression. Specifically, Lin28b showed high expression in MHCIIhi TECs, whereas Let-7g was expressed in MHCIIlo TECs. Deletion of Lin28a and Lin28b specifically in TECs resulted in reduced MHCII expression and overall TEC numbers. Conversely, overexpression of Lin28a increased total TEC and thymocyte numbers by promoting the proliferation of MHCIIlo TECs. Additionally, our data strongly suggest that Lin28 and Let-7g expression is reliant on FOXN1 to some extent. These findings suggest a critical role for Lin28 in regulating the development and differentiation of TECs by modulating MHCII expression and TEC proliferation throughout thymic ontogeny and involution. Our study provides insights into the mechanisms underlying TEC differentiation and highlights the significance of Lin28 in orchestrating these processes.

Lin28a induced mitochondrial dysfunction in human granulosa cells via suppressing LARS2 expression

Granulosa cells surround the oocytes as a component of ovarian follicles and produce sex steroids and growth factors to support oocyte development. Mitochondria is one of the multiple factors regulating granulosa cell function by modulating bioenergetic pathways and maintaining cells' metabolic needs. Lin28a was reported to regulate the primordial germ cell development in the ovary and affect the fertility rate in females. However, whether Lin28a modulated mitochondria function in granulosa cells to regulate steroidogenesis remains a further exploration. In this study, we utilized immortal human granulosa cells (HGrC1) to overexpress or suppress the protein level of Lin28a. Results showed that overexpression of Lin28a could decrease the estrogen level, ATP content, mitochondrial membrane potential and Glutathione (GSH) level, while silencing Lin28a caused the opposite effect. Further, we found that overexpression of LARS2, a mitochondrial leucyl-tRNA synthetase, could increase the estrogen level, ATP content, mitochondrial membrane potential and GSH level while silencing LARS2 caused adverse results. Overexpression of LARS2 reversed Lin28a-induced estrogen downregulation and mitochondrial dysfunction. Moreover, overexpression of LARS2 increased the mRNA level of Pgc1α and Nrf2, which were involved in mitochondrial biogenesis. Besides, Lin28a could directly bind to the mRNA of Lars2 to suppress its translation. Taken together, overexpression of Lin28a caused mitochondrial dysfunction in granulosa cells via suppressing LARS2 expression. This study can provide new insights into how Lin28a regulates mitochondrial function in granulosa cells and influences female fertility.

Lin28A Reduced Sevoflurane-Induced Nerve Injury and Cognitive Dysfunction by Inhibiting Tau Acetylation and Phosphorylation via Activating SIRT1 in Elderly Rats.

Sevoflurane (Sev) might cause neurotoxicity in elderly rats. However, the role of Lin28A in Sev-induced neurotoxicity remains unclear in elderly rats. In this study, elderly rats were used to construct an Sev-induced nerve injury model. Learning and memory abilities were assessed by Morris water maze (MWM) trainings; pathological alterations in hippocampal region were assessed by HE staining; neuronal apoptosis was assessed by TUNEL; related protein expression was analyzed by immunofluorescence, immunohistochemistry, and Western blotting. Results of this study showed that Sev treatment caused nerve injury and cognitive dysfunction in elderly rats, with increased neuronal apoptosis and decreased Lin28A levels. Pathological impairment and learning and memory abilities of elderly rats were significantly improved after forced overexpression of Lin28A using AAV, accompanied by decreased expression of CD68, Iba-1, and GFAP. TUNEL analysis showed that Lin28A overexpression significantly reversed Sev-induced neuronal apoptosis. Further mechanistic analysis showed that Lin28A significantly promoted SIRT1 expression, which further reversed Sev-induced Tau acetylation at lysine 280 and 686 and Tau hyperphosphorylation, thereby alleviating nerve injury and cognitive dysfunction in elderly rats. The introduction of SIRT1 inhibitor EX527 further confirmed the involvement of SIRT1 in the regulation of Lin28A in elderly rats. In conclusion, our findings demonstrated that Lin28A reduced sevoflurane-induced nerve injury and cognitive dysfunction by inhibiting Tau acetylation and phosphorylation via activating SIRT1 in elderly rats.

Lin28A alleviates ovariectomy-induced osteoporosis through activation of the AMP-activated protein kinase pathway in rats.

To investigate the role of Lin28A in ovariectomy-induced osteoporosis and to elucidate the underlying molecular mechanism. Bilateral ovariectomy was conducted to generate an ovariectomy (OVX) rat model. Western blotting was performed to assess the relative expression levels of Lin28A, osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), adenosine monophosphate-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) proteins. Enzyme-linked immunosorbent assays were performed to detect the serum levels of calcium, E2, alkaline phosphatase (ALP) and interleukin (IL)-1β. Three-point bending test was used to assess biomechanical parameters of left femoral diaphysis. Hematoxylin and eosin (HE) staining was conducted to detect the trabecular structure of bone tissue. Dihydroethidium assay kit was used to measure the intracellular reactive oxygen species (ROS) level in osteoclasts. Alizarin red staining revealed the calcium deposit in bone marrow stromal cells (BMSC). The expression levels of Lin28A, OCN, RUNX2, AMPK and p-AMPK proteins were significantly decreased in OVX rats. The serum levels of calcium, E2, ALP and IL-1β were significantly declined in OVX rats. Biomechanical parameters of left femoral diaphysis were significantly decreased in OVX rats. OVX-induced trabecular abnormalities. ROS level was dramatically increased in the bone tissue of OVX rats, and calcium deposit was dramatically decreased in BMSC cells of OVX rats. These effects induced by OVX could be prevented by overexpression of Lin28A. Lin28A alleviates ovariectomy-induced osteoporosis through activation of AMPK pathway in rats.

LncRNA MALAT1 Aggravates Renal Tubular Injury via Activating LIN28A and the Nox4/AMPK/mTOR Signaling Axis in Diabetic Nephropathy.

BackgroundDiabetic nephropathy (DN) is a serious complication among patients with diabetes. Elucidating its pathogenesis is crucial for identifying novel biomarkers and therapeutic targets for DN.MethodsDN tissues were harvested for examining MALAT1, LIN28A and Nox4. Human kidney-2 (HK-2) cells were treated with high glucose (HG) for establishing a cell model of DN. Cell viability was examined by MTT assay. HG-induced cell apoptosis and secretion of TNF-α and IL-6 were analyzed by TUNEL and ELISA assays, respectively. RIP and RNA pull-down assays were applied to analyze the interaction between MALAT1, LIN28A and Nox4 in HK-2 and human embryonic kidney 293T (HEK-293T) cells. A rat model of DN was established to determine the role of MALAT1 in DN in vivo.ResultsMALAT1, LIN28A and Nox4 were upregulated in DN tissues and HG-treated HK-2 cells. Overexpression of MALAT1, LIN28A or Nox4 reduced cell viability and enhanced cell apoptosis, ROS generation and secretion of inflammatory cytokines in HG-treated HK-2 cells, whereas knockdown of MALAT1, LIN28A or Nox4 exerted opposite effects. Furthermore, MALAT1 directly interacted with LIN28A. Moreover, MALAT1 facilitated the interaction between LIN28A and Nox4 to increase Nox4 stability. Knockdown of Nox4 relieved HG-induced injury by suppressing the AMPK/mTOR signaling in HK-2 cells. Knockdown of MALAT1 alleviated renal tubular epithelial injury by suppressing LIN28A and the Nox4/AMPK/TOR signaling in DN.ConclusionMALAT1 activates the AMPK/mTOR signaling via interacting with LIN28A to stabilize Nox4 mRNA, thereby aggravating high glucose-induced renal tubular epithelial injury. Our findings provide potential therapeutic targets for DN.

Involvement of LIN28A in Wnt-dependent regulation of hippocampal neurogenesis in the aging brain.

SummaryHippocampal neurogenesis declines with aging. Wnt ligands and antagonists within the hippocampal neurogenic niche regulate the proliferation of neural progenitor cells and the development of new neurons, and the changes of their levels in the niche mediate aging-associated decline of neurogenesis. We found that RNA-binding protein LIN28A remained existent in neural progenitor cells and granule neurons in the adult hippocampus and that it decreased with aging. Lin28a knockout inhibited the responsiveness of neural progenitor cells to niche Wnt agonists and reduced neurogenesis, thus impairing pattern separation. Overexpression of Lin28a increased the proliferation of neural progenitor cells, promoted the functional integration of newborn neurons, restored neurogenesis in Wnt-deficient dentate gyrus, and rescued the impaired pattern separation in aging mice. Our data suggest that LIN28A regulates adult hippocampal neurogenesis as an intracellular mechanism by responding to niche Wnt signals, and its decrease is involved in aging-associated decline of hippocampal neurogenesis and related cognitive functions.

ETMR-02. Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density

The RNA binding protein LIN28A is a stem- and progenitor marker and one of the factors necessary to induce pluripotent stem cells. An overexpression of LIN28A has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors. Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics within a crosstalk of Gsk3b/Rho-Rac/Map1b signaling. In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

OTHR-19. Disordered cell migration in the cerebral cortex caused by Lin28A overexpression and Wnt pathway activation in neural precursor cells

Abstract LIN28A overexpression and mutations of the Wnt pathway gene CTNNB1 have been described in rare malignant brain tumors of early childhood. In order to investigate the interplay of the oncogenic proteins Lin28A and Ctnnb1 during embryonal brain development, we overexpressed these components in neural progenitor cells in vivo. The sole overexpression of either Lin28A, stabilized Ctnnb1 (Ctnnb1Δex3) or the combination of both in hGFAP-positive forebrain precursor cells did not lead to brain tumor formation but resulted in distinct phenotypes in the cerebral cortex during embryonal development. The hGFAP-cre::IsI-Lin28A (GL) mouse model showed transiently increased proliferation in the cerebral ventricular zone and proper isocortical layering. hGFAP-cre::Ctnnb1Δex3fl/+ (GB) and hGFAP-cre::Ctnnb1Δex3fl/+::IsI-Lin28A (GBL) mice developed a hydrocephalus and showed disturbed cortical layering. GB mice displayed cerebral hypoplasia with a thinned cortex, while the GBL cortices showed variable thickness. Immunostainings with the pial marker Laminin and dendritic marker Map2c revealed a porous pia mater and aggregations of neurons above the pial border in the GBL model at embryonal day 14 (E14.5). At later embryonal stage (E18.5), the GBL model showed also large blood vessels located in deeper cortical layers. Proteome analyses of GB and GBL cortices revealed decreased abundance of the Lissencephaly associated component Reelin-receptor Lrp8 compared to hGFAP-cre control mice. Additionally, we found 92 proteins, which were altered specifically in the GBL mouse model. These results indicate that the co-expression of Lin28A and Ctnnb1Δex3 in neural precursor cells does not lead to brain tumor formation but results in neuronal migration disturbances with ectopic neurons in the subarachnoid area. Whereas the GB phenotype is reminiscent of human lissencephaly type I, GBL brain morphology showed similarities to neuronal overmigration observed in the migration disorder of human Cobblestone (Type II) Lissencephaly.

447-P: C/EBPß-Lin28a Positive Feedback Loop Mediated by C/EBPß Hypomethylation Promotes the Proliferation and Migration of Vascular Smooth Muscle Cells in Restenosis

Background: The main reason for restenosis (RS) after percutaneous transluminal angioplasty is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs) . C/EBPβ plays an important role in the process of vascular remodeling. However, the role of C/EBPβ in RS has not been clarified. Methods: The type 2 diabetes rat models of RS and atherosclerosis (AS) were established to detect the difference in DNA methylation and expression levels of C/EBPβ. Simultaneously, the effect of C/EBPβ on the proliferation and migration of VSMCs was clarified by regulating C/EBPβ and Lin28a in vitro. The effect of decitabine, a demethylating agent, on the expression of C/EBPβ and Lin28a, and the further regulation of VSMCs proliferation and migration was confirmed. Results: C/EBPβ was highly expressed and had a lower methylation level in RS compared with AS. In vitro C/EBPβ overexpression facilitated the proliferation and migration of VSMCs with increased expression of Lin28a. Conversely, C/EBPβ knockdown resulted in opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to the promoter of Lin28a. Furthermore, increased expression of C/EBPβ and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. And mechanical stretch promoted the expression of C/EBPβ and Lin28a. Additionally, C/EBPβ expression increased following the overexpression of Lin28a. The reversed result was found in the Lin28a knockdown cells. Conclusion: Our results have demonstrated that C/EBPβ expression is regulated partly by DNA methylation. Meanwhile, as a transcription factor, C/EBPβ promotes the transcription of Lin28a and enhances the proliferation and migration of VSMCs through a positive feedback loop between the two. Our study suggests C/EBPβ-Lin28a axis is a driver of the RS progression, providing a promising therapeutic strategy for RS. Disclosure S.Jiang: n/a. L.Liao: None. J.Dong: None. X.Zhou: n/a. Q.Zhang: None. S.Guo: None. Q.Sheng: None. X.Li: None. R.Zhang: None. C.Xu: None. P.Gong: None. Funding National Natural Science Foundation of China Grants (81800732,81670757,82170824) , Shandong Provincial Medicine and Health Science and Technology Development Program (No.202103060766) and Shandong Provincial Hospital Research incubation Fund (No.2021FY059)

Overexpression of Lin28a blocks mitochondrial respiration and promotes Glycolysis to induce chondrocyte reprogramming

Overexpression of Lin28a blocks mitochondrial respiration and promotes Glycolysis to induce chondrocyte reprogramming

The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse.

Cortical expansion and folding are key processes in human brain development and evolution and are considered to be principal elements of intellectual ability. How cortical folding has evolved and is induced during embryo development is not well understood. Here, we show that the expression of human FOXM1 promotes basal progenitor cell proliferation and induces cortical thickening and folding in mice. Human-specific protein sequences further promote the generation of basal progenitor cells. Human FOXM1 increases the proliferation of neural progenitors by binding to the Lin28a promoter and increasing Lin28a expression. Furthermore, overexpression of LIN28A rescues the proliferation of human FOXM1 knockout neural progenitor cells. Together, our findings demonstrate that a human gene can increase the number of basal progenitor cells in mice, leading to brain size increase and gyrification, and may thus contribute to evolutionary brain development and cortical expansion.

Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density

LIN28A overexpression has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors.Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics.In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

Lin28A/CENPE Promoting the Proliferation and Chemoresistance of Acute Myeloid Leukemia.

The prognosis of chemoresistant acute myeloid leukemia (AML) is still poor, mainly owing to the sustained proliferation ability of leukemic cells, while the microtubules have a major role in sustaining the continuity of cell cycle. In the present study, we have identified CENPE, a microtubular kinesin-like motor protein that is highly expressed in the peripheral blood of patients with chemoresistant AML. In our in vitro studies, knockdown of CENPE expression resulted in the suppression of proliferation of myeloid leukemia cells and reversal of cytarabine (Ara-C) chemoresistance. Furthermore, Lin28A, one of the RNA-binding oncogene proteins that increase cell proliferation and invasion and contribute to unfavorable treatment responses in certain malignancies, was found to be remarkably correlated with CENPE expression in chemoresistance AML. Overexpression of LIN28A promoted the proliferation and Ara-C chemoresistance of leukemic cells. RIP assay, RNA pull-down, and dual luciferase reporter analyses indicated that LIN28A bound specifically to the promoter region GGAGA of CENPE. In addition, the impacts of LIN28A on cell growth, apoptosis, cell cycle progression, and Ara-C chemoresistance were reverted by the knockdown of CENPE. Hence, Lin28A/CENPE has enhanced the proliferation and chemoresistance of AML, and therefore, it could be a prospective candidate for AML treatment.

Overexpression of Lin28a induces a primary ovarian insufficiency phenotype via facilitation of primordial follicle activation in mice

Lin28a is an RNA binding protein and increasing evidence has indicated its role in regulating female fertility. Lin28a has been reported to be involved in ovarian follicle activation. However, its role and mechanisms in regulating primordial follicle activation have not yet been explored. To test whether overexpression of Lin28a activates ovarian primordial follicles, studies were conducted in wild type (WT) and Lin28a Tg mice. Female Lin28a Tg mice at 4-month old exhibited significantly smaller litter size and fewer ovulated oocytes when compared with the WT mice. By 6-month of age, these parameters in Lin28a Tg mice were less than 20% of the WT mice. At postnatal day (PD) 14, the number of primordial follicles was significantly decreased but the number of primary follicles was significantly increased in the transgenic mice. The number of primordial follicles, secondary and antral follicles in these mice were drastically reduced at PD21. In the ovary of Lin28a Tg mice, there were activation of Wnt/β-catenin signaling and its downstream mTOR pathway. Interestingly, overexpression of Lin28a, which can also act as transcriptional activator, activated Wnt signaling through enhancing the transcription of Wnt co-receptor LRP5. In conclusion, overexpression of Lin28a induced a primary ovarian insufficiency phenotype in long term via facilitating Wnt/β-catenin signaling leading to activation of primordial follicles.

Overexpression of Lin28a Aggravates Psoriasis-Like Phenotype by Regulating the Proliferation and Differentiation of Keratinocytes.

PurposePsoriasis is a common and well-studied autoimmune skin disease, which is characterized by plaques. The formation of psoriasis plaques occurs through the hyperproliferation and abnormal differentiation of keratinocytes, infiltration of numerous immune cells into the dermis, increased subepidermal angiogenesis, and various autoimmune-associated cytokines and chemokines. According to previous research, Lin28 regulates the let-7 family, and let-7b is associated with psoriasis. However, the link between Lin28 and psoriasis is unclear. In this study, an association was identified between Lin28a and psoriasis progression, which promoted the pathological characteristic of psoriasis in epidermal keratinocytes.Patients and MethodsThis study aims to investigate the role of Lin28a and its underlying mechanism in psoriasis through in vivo and in vitro models, which include the Lin28a-overexpressing transgenic (TG) mice and Lin28a-overexpressing human keratinocyte (HaCaT) cell lines, respectively.ResultsIn vivo and in vitro results revealed that overexpression of Lin28a downregulated microRNA let-7 expression levels and caused hyperproliferation and abnormal differentiation in keratinocytes. In imiquimod (IMQ)-induced psoriasis-like inflammation, Lin28a overexpressing transgenic (TG) mice exhibited more severe symptoms of psoriasis.ConclusionMechanistically, Lin28a exacerbated psoriasis-like inflammation through the activation of the extracellular-signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 signaling (STAT 3) by targeting proinflammatory cytokine interleukin-6 (IL-6).

Down Regulation of Let-7 Triggers a Critical Double-Negative Feedback Loop to Determinate the Malignant VSMCs Fate During Restenosis

Background: The excessive proliferation and migration of VSMCs is the main cause of restenosis (RS). Our previous results suggested that overexpression of Lin28a is responsible for this “malignant” behaver of VSMCs. However, the mechanisms driving the specific VSMCs behavior in RS are far less understood. Methods and Findings: Using an atherosclerosis followed by PTA treatment rats as RS model, we discovered a decreased expression of let-7c, let-7g, let-7i, miR98 and miR202, that associated with an increased expression of Lin28a in RS plaques compared to those of atherosclerosis. Combined with in vitro results, we demonstrated that let-7c, let-7g, and miR98 were Lin28a downstream targets in RS. More importantly, the decreased expression of let-7c, let-7g, in turn, will enhance the expression of Lin28a, indicating a double-negative feedback loop between Lin28a and let-7c/let-7g, which may be responsible for the vicious behavior of the VSMCs in RS. Transfection of let-7c and let-7g in vivo by AAV9 attenuated the aggregation of VSMCs and decreased the RS plaque size. In addition, unlike the other member of let-7, let-7d downregulates Lin28a and plays a restrictive regulator on the Lin28a/let-7 loop, that may bias VSMCs fate decision. Conclusion: In this study, by using a “two-step lesion” protocol to create RS models, we discovered that the double negative feedback system of Lin28a/let-7 axis act as a regulation center in vicious behavior of VSMCs in RS. These discoveries give us a better understanding of RS in lower extremity arterial disease and expected to present opportunities for discovering new therapeutic approaches. Funding: This work was supported by the National Natural Science Foundation of China Grants (No. 81670757, 81770822, 81800732, 81900685), Shandong Provincial Natural Science Foundation of China Grants (No. ZR2017LH025, ZR2020MH101), Shandong Provincial Medicine and Health Science and Technology Development Program (No. 2017WS461), China Postdoctoral Science Foundation (No. 2020M682193). Declaration of Interest: The authors confirm that there are no conflicts of interest. Ethical Approval: The protocol was authorized by the Ethics Committee of Qilu Hospital, Shandong University.

Lin28a ameliorates glucotoxicity-induced β-cell dysfunction and apoptosis

An excessive and prolonged increase in glucose levels causes β-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicity-induced β-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced β-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in β-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirus-mediated Lin28a overexpression in β-cells reversed the glucotoxicity-induced reduction of insulin secretion and insulin mRNA expression via regulation of β-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic β-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway.

Hypoxia-Induced LIN28A mRNA Promotes the Metastasis of Colon Cancer in a Protein-Coding-Independent Manner.

The hypoxic microenvironment is beneficial to the metastasis but not to the proliferation of cancer cells. However, the mechanisms regarding to hypoxia differentially regulating cancer metastasis and proliferation are largely unknown. In this study, we revealed that hypoxia induced the expression of LIN28A at mRNA level but segregated LIN28A mRNAs in the P-bodies and thus inhibits the production of LIN28A protein. This unexpected finding suggests that there may be non-coding role for LIN28A mRNA in the progression of colon cancer. We further showed that the non-coding LIN28A mRNA promotes the metastasis but not proliferation of colon cancer cells in vitro and in vivo. Mechanistically, we revealed that methionyl aminopeptidase 2 (METAP2) is one of the up-regulated metastasis regulators upon over-expression of non-coding LIN28A identified by mass spectrum, and confirmed that it is non-coding LIN28A mRNA instead of LIN28A protein promotes the expression of METAP2. Moreover, we demonstrated that knockdown of DICER abolished the promotional effects of non-coding LIN28A on the metastasis and METAP2 expression. Conclusively, we showed that hypoxia induces the production of LIN28A mRNAs but segregated them into the P-bodies together with miRNAs targeting both LIN28A and METAP2, and then promotes the metastasis by positively regulating the expression of METAP2. This study uncovered a distinctive role of hypoxia in manipulating the metastasis and proliferation by differently regulating the expression of LIN28A at mRNA and protein level.

Lin28a attenuates TGF-β-induced renal fibrosis

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-β-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-β-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-β-stimulated SMAD3 activity, via inhibition of SMAD3 phos-phorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease.