Insulin-like Growth Factor 2 Protein Research Articles

OR25-3 Sperm-carried IGF2 Downregulates Mitogens Released by Sertoli Cells: A Paracrine Mechanism of Spermatogenetic Regulation?

Abstract The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p<0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p<0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p<0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p<0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Insulin-like growth factor 2 and autophagy gene expression alteration arise as potential biomarkers in Parkinson\u2019s disease

Insulin-like growth factor 2 (IGF2) and autophagy-related genes have been proposed as biomolecules of interest related to idiopathic Parkinson’s disease (PD). The objective of this study was to determine the IGF2 and IGF1 levels in plasma and peripheral blood mononuclear cells (PBMCs) from patients with moderately advanced PD and explore the potential correlation with autophagy-related genes in the same blood samples. IGF1 and IGF2 levels in patients' plasma were measured by ELISA, and the IGF2 expression levels were determined by real-time PCR and Western blot in PBMCs. The expression of autophagy-related genes was evaluated by real-time PCR. The results show a significant decrease in IGF2 plasma levels in PD patients compared with a healthy control group. We also report a dramatic decrease in IGF2 mRNA and protein levels in PBMCs from PD patients. In addition, we observed a downregulation of key components of the initial stages of the autophagy process. Although IGF2 levels were not directly correlated with disease severity, we found a correlation between its levels and autophagy gene profile expression in a sex-dependent pattern from the same samples. To further explore this correlation, we treated mice macrophages cell culture with α-synuclein and IGF2. While α-synuclein treatment decreased levels Atg5, IGF2 treatment reverted these effects, increasing Atg5 and Beclin1 levels. Our results suggest a relationship between IGF2 levels and the autophagy process in PD and their potential application as multi-biomarkers to determine PD patients' stages of the disease.

Intrathecal IGF2 siRNA injection provides long-lasting anti-allodynic effect in a spared nerve injury rat model of neuropathic pain.

Previous studies have shown an increase of insulin-like growth factor-2 (IGF2) in animal models of neuropathic pain. We aimed to examine the hypothesis that reducing the expression of IGF2 using intrathecal IGF2 small-interfering RNA (siRNA) would attenuate the development of neuropathic pain in rats after spared nerve injury (SNI). Male Wistar rats were divided into three groups: sham-operated group, in which surgery was performed to cut the muscles without injuring the nerves; SNI group, in which SNI surgery was performed to sever the nerves; and SNI + siRNA IGF2 group, in which SNI surgery was performed, and IGF2-siRNA was administered intrathecally 1 day after SNI. The rats were assessed for mechanical allodynia and cold allodynia 1 day before surgery (baseline), and at 2, 4, 6, 8, and 10 days after siRNA treatment. The rat spinal cord was collected for quantitative polymerase chain reaction and western blot analysis. Compared with the SNI group, rats that received IGF2 siRNA showed a significantly increased SNI-induced paw-withdrawal threshold to metal filament stimulation from Day 4 to Day 10 after SNI surgery. IGF2 siRNA significantly decreased the response duration from the acetone test from Day 2 to Day 10 following SNI surgery. SNI increased IGF2 mRNA expression on Day 2 and increased IGF2 protein expression on Day 8 and Day 10 in the spinal cord of the SNI rats. However, the above-mentioned effects of IGF2 mRNA and protein expression were significantly inhibited in the SNI + IGF2 siRNA group. We demonstrated that intrathecal administration of IGF2 siRNA provided significant inhibition of SNI-induced neuropathic pain via inhibition of IGF2 expression in the spinal cord. The analgesic effect lasted for 10 days. Further exploration of intrathecal IGF2 siRNA administration as a potential therapeutic strategy for neuropathic pain is warranted.

Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets.

ZBED6 (zinc finger BED domain containing protein 6) is a transcription factor unique to placental mammals and its interaction with the IGF2 (insulin-like growth factor 2) locus plays a prominent role in the regulation of postnatal skeletal muscle growth. Here, we generated lean Bama miniature pigs by generating ZBED6-knockout (ZBED6−/−) and investigated the mechanism underlying ZBED6 in growth of muscle and internal organs of placental mammals. ZBED6−/− pigs show markedly higher lean mass, lean mass rate, larger muscle fiber area and heavier internal organs (heart and liver) than wild-type (WT) pigs. The striking phenotypic changes of ZBED6-/- pigs coincided with remarkable upregulation of IGF2 mRNA and protein expression across three tissues (gastrocnemius muscle, longissimus dorsi, heart). Despite a significant increase in liver weight, ZBED6-/- pigs show comparable levels of IGF2 expression to those of WT controls. A mechanistic study revealed that elevated methylation in the liver abrogates ZBED6 binding at the IGF2 locus, explaining the unaltered hepatic IGF2 expression in ZBED6-/- pigs. These results indicate that a ZBED6-IGF2-independent regulatory pathway exists in the liver. Transcriptome analysis and ChIP-PCR revealed new ZBED6 target genes other than IGF2, including cyclin dependent kinase inhibitor 1A (CDKN1A) and tsukushi, small leucine rich proteoglycan (TSKU), that regulates growth of muscle and liver, respectively.

Alterations of mRNA and lncRNA profiles associated with the extracellular matrix and spermatogenesis in goats.

ObjectiveSpermatozoa are produced within the seminiferous tubules after sexual maturity. The expression levels of mRNAs and lncRNAs in testicular tissues are different at each stage of testicular development and are closely related to formation of the extracellular matrix (ECM) and spermatogenesis. Therefore, we set out to study the expression of lncRNAs and mRNAs during the different developmental stages of the goat testis.MethodsWe constructed 12 RNA libraries using testicular tissues from goats aged 3, 6, and 12 months, and studied the functions of mRNAs and lncRNAs using the gene ontogeny (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. Relationships between differentially expressed genes (DEGs) were analyzed by lncRNA-mRNA co-expression network and protein-protein interaction network (PPI). Finally, the protein expression levels of matrix metalloproteinase 2 (MMP2), insulin-like growth factor 2 (IGF2), and insulin-like growth factor-binding protein 6 (IGFBP6) were detected by western blotting.ResultsWe found 23, 8, and 135 differentially expressed lncRNAs and 161, 12, and 665 differentially expressed mRNAs that were identified between 3 vs 6, 6 vs 12, and 3 vs 12 months, respectively. GO, KEGG, and PPI analyses showed that the differential genes were mainly related to the ECM. Moreover, MMP2 was a hub gene and co-expressed with the lncRNA TCONS-0002139 and TCONS-00093342. The results of quantitative reverse-transcription polymerase chain reaction verification were consistent with those of RNA-seq sequencing. The expression trends of MMP2, IGF2, and IGFBP6 protein were the same as that of mRNA, which all decreased with age. IGF2 and MMP2 were significantly different in the 3 vs 6-month-old group (p<0.05).ConclusionThese results improve our understanding of the molecular mechanisms involved in sexual maturation of the goat testis.

Effect of Coxsackievirus B4 Infection on the Thymus: Elucidating Its Role in the Pathogenesis of Type 1 Diabetes.

The thymus gland is a primary lymphoid organ for T-cell development. Various viral infections can result in disturbance of thymic functions. Medullary thymic epithelial cells (mTECs) are important for the negative selection of self-reactive T-cells to ensure central tolerance. Insulin-like growth factor 2 (IGF2) is the dominant self-peptide of the insulin family expressed in mTECs and plays a crucial role in the intra-thymic programing of central tolerance to insulin-secreting islet β-cells. Coxsackievirus B4 (CVB4) can infect and persist in the thymus of humans and mice, thus hampering the T-cell maturation and differentiation process. The modulation of IGF2 expression and protein synthesis during a CVB4 infection has been observed in vitro and in vivo in mouse models. The effect of CVB4 infections on human and mouse fetal thymus has been studied in vitro. Moreover, following the inoculation of CVB4 in pregnant mice, the thymic function in the fetus and offspring was disturbed. A defect in the intra-thymic expression of self-peptides by mTECs may be triggered by CVB4. The effects of viral infections, especially CVB4 infection, on thymic cells and functions and their possible role in the pathogenesis of type 1 diabetes (T1D) are presented.

Knockdown of insulin-like growth factor 2 gene disrupts mitochondrial functions in the liver.

Even though insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in nonalcoholic fatty liver disease (NAFLD), its role in the progression of NAFLD and the potential mechanism remain largely unclear. Using in vitro models, we found that IGF2 was the key overexpressed gene in steatosis, suggesting a possible association between IGF2 and NAFLD. Interestingly, loss-of-function experiments revealed that inhibition of IGF2 protein impaired mitochondrial biogenesis and respiration. It additionally disrupted the expression changes of mitochondrial fusion and fission-related proteins necessary in maintaining mitochondrial homeostasis. Consistently, IGF2 knockdown reduced the mitochondrial membrane potential and increased the production of reactive oxygen species. Mechanistically, IGF2 regulates mitochondrial functions by modulating the expression of SIRT1 and its downstream gene PGC1α. This research opens a new frontier on the role of IGF2 in energy metabolism, which potentially participates in the development of NAFLD. As such, IGF2 is a potential therapeutic target against NAFLD.

Adipocyte precursor cells from first degree relatives of type 2 diabetic patients feature changes in hsa-mir-23a-5p, -193a-5p, and -193b-5p and insulin-like growth factor 2 expression.

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.

The mechanism of enriched environment repairing the learning and memory impairment in offspring of prenatal stress by regulating the expression of activity-regulated cytoskeletal-associated and insulin-like growth factor-2 in hippocampus

BackgroundPrenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring.MethodsRats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting.ResultsThere was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress’s offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment.ConclusionsThe learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.

Insulin-like growth factor 2 (IGF2) expression in adrenocortical disease due to PRKAR1A mutations compared to other benign adrenal tumors.

Insulin-like growth factor-II (IGF2), a key regulator of cell growth and development, is tightly regulated in its expression by epigenetic control that maintains its monoallelic expression in most tissues. Biallelic expression of IGF2 resulting from loss of imprinting (LOI) has been reported in adrenocortical tumors. In this study, we wanted to check whether adrenocortical lesions due to PRKAR1A mutations lead to increased IGF2 expression from LOI and compare these findings to those in other benign adrenal lesions. We compared the expression of IGF2 by RNA and protein studies in primary pigmented nodular adrenocortical disease (PPNAD) caused by PRKAR1A gene mutations to that in primary macronodular adrenocortical hyperplasia (PMAH) and cortisol-producing adenomas (CPA) that did not have any mutations in known genes. We also checked LOI in all lesions by DNA allelic studies and the expression of other components of IGF2 signaling at the RNA and protein level. We identified cell clusters overexpressing IGF2 in PPNAD; although immunostaining was patchy, overall, by RNA and immunoblotting PPNAD expressed high IGF2 message and protein. However, this was not due to LOI, as there was no correlation between IGF2 expression and the presence of LOI. Our data pointed to over-expression of IGF2 protein in PPNAD compared to other benign adrenocortical lesions, such as PMAH and CPA. However, there was no correlation of IGF2 mRNA levels with LOI of IGF2/H19. The discrepancy between mRNA and protein levels with regards to LOI points, perhaps, to different control of IGF2 gene expression in PPNAD.

Structure of insulin-like growth factor 2 (IGF2) gene from striped catfish (Pangasianodon hypophthalmus)

Gene coding Insulin-like growth factor 2 (IGF2) is one of genes relating the growth of many fish species, including striped catfish. In this study, cDNA coding to protein IGF2 was isolated from total RNA extracted from liver tissue of catfish, then was amplified through reverse transcription PCR and standard PCR with IGF2 specific primer pair to generate the 642bp-in-size PCR product. The sequence of this cDNA-IGF2 is similar up to 95% to the cDNA-IGF2 gene sequence of catfish included in family Ictalurus. Based on that sequence, 3 primer pairs were designed to amplify 3 regions of IGF2 gene from genomic DNA extracted from fins of catfish, which have 900bp, 1500bp and 1200bp in length. That 3 regions were sequenced and assembled to give the final sequence with 3387bp in length of IGF2 gene, which includes 4 exons coding to IGF2 protein, and is similar up to 96% to sequence of mRNA coding to IGF2 protein of channel catfish Ictalurus punctatus and that of blue catfish Ictalurus furcatus. Determining the structure of IGF2 gene of striped catfish Pangasianodon hypophthalmus enable us to further examine its function and genetic variation.

BET proteins inhibitor JQ-1 impaired the extinction of remote auditory fear memory: An effect mediated by insulin like growth factor 2

Fear extinction is an important preclinical model for behavior therapy in human anxiety disorders, such as post-traumatic stress disorder (PTSD). Histone acetylation is involved in the extinction of fear memory. As the “readers” of histone acetylation markers, the role of the bromodomain and extraterminal domain (BET) proteins in fear extinction is still unclear. In the present study, we found that suppression of BET proteins using small molecule JQ-1 had no effects on the acquisition of auditory fear or on the extinction of recent auditory fear, but it impaired the extinction of remote auditory fear. We found that insulin like growth factor 2 (IGF-2) mRNA and protein were up-regulated in the anterior cingulate cortex (ACC) after the extinction training of remote fear memory, and that this effect was inhibited by JQ-1 administration. Further, the local delivery of IGF-2 protein to the ACC region rescued the impaired extinction of remote memory caused by JQ-1 administration, which suggesting IGF-2 mediates the effects of JQ-1 on remote memory extinction. Gene expression profiling analysis demonstrated that JQ-1 treatment inhibited the up-regulated expression of a key set of neuroplasticity-related genes following remote memory extinction. Together, these findings establish BET proteins as epigenetic mediator for the extinction of remote fear memory. In particular, the findings of this study imply that as a prospective preclinical cancer drug, JQ-1 (or other BET bromodomain inhibitors) should be modified to prevent it from crossing the blood brain barrier and causing neurological side effects.

LncRNA TUG1 regulates proliferation and apoptosis by regulating miR-148b/IGF2 axis in ox-LDL-stimulated VSMC and HUVEC

Vascular smooth muscle cell (VSMC) accumulation and endothelial cell dysfunction are associated with pathogenesis of atherosclerosis. Long noncoding RNA taurine up-regulated gene 1 (TUG1) has been reported to play an important role in cardiovascular diseases, including atherosclerosis. However, the regulatory mechanism underlying TUG1 in atherosclerosis is far from understood. VSMC and human umbilical vein endothelial cells (HUVEC) stimulated by oxidized low-density lipoprotein (ox-LDL) were used as cellular model of atherosclerosis. Cell proliferation and apoptosis were detected by CCK-8, flow cytometry and Western blot. The expression levels of TUG1, microRNA (miR)-148b and insulin-like growth factor 2 (IGF2) were measured by quantitative real-time polymerase chain reaction or Western blot. The target association among TUG1, miR-148b and IGF2 was determined by luciferase reporter assay and RNA immunoprecipitation. The expression of TUG1 was increased in ox-LDL-treated VSMC and HUVEC. Silence of TUG1 inhibited proliferation and promoted apoptosis in ox-LDL-treated VSMC but induced proliferation promotion and apoptosis inhibition in HUVEC stimulated by ox-LDL. miR-148b was a target of TUG1 and its knockdown reversed the effect of TUG1 silence on proliferation and apoptosis of VSMC and HUVEC challenged by ox-LDL. IGF2 was a target of miR-148b and miR-148b regulated proliferation and apoptosis in ox-LDL-treated VSMC and HUVEC by targeting IGF2. TUG1 promoted IGF2 protein expression by sponging miR-148b. TUG1 knockdown attenuated ox-LDL-induced injury through regulating proliferation and apoptosis of VSMC and HUVEC by miR-148b/IGF2 axis, providing a novel mechanism for pathogenesis of atherosclerosis.

Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. Primary human cumulus GCs in culture. University infertility center. GCs of women undergoing in vitro fertilization. Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. Quantification of mRNA, protein, promoter activity, and DNA methylation. FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Insulin-Like Growth Factor 2 (IGF2) Signaling in Colorectal Cancer-From Basic Research to Potential Clinical Applications.

Colorectal cancer (CRC) is one of the most common cancers in men and women worldwide as well as is the leading cause of death in the western world. Almost a third of the patients has or will develop liver metastases. While genetic as well as epigenetic mechanisms are important in CRC pathogenesis, the basis of the most cases of cancer is unknown. High spatial and inter-patient variability of the molecular alterations qualifies this cancer in the group of highly heterogeneous tumors, which makes it harder to elucidate the mechanisms underlying CRC progression. Determination of highly sensitive and specific early diagnosis markers and understanding the cellular and molecular mechanism(s) of cancer progression are still a challenge of the current era in oncology of solid tumors. One of the accepted risk factors for CRC development is overexpression of insulin-like growth factor 2 (IGF2), a 7.5-kDa peptide produced by liver and many other tissues. IGF2 is the first gene discovered to be parentally imprinted. Loss of imprinting (LOI) or aberrant imprinting of IGF2 could lead to IGF2 overexpression, increased cell proliferation, and CRC development. IGF2 as a mitogen is associated with increased risk of developing colorectal neoplasia. Higher serum IGF2 concentration as well as its tissue overexpression in CRC compared to control are associated with metastasis. IGF2 protein was one of the three candidates for a selective marker of CRC progression and staging. Recent research indicates dysregulation of different micro- and long non-coding RNAs (miRNAs and lncRNAs, respectively) embedded within the IGF2 gene in CRC carcinogenesis, with some of them indicated as potential diagnostic and prognostic CRC biomarkers. This review systematises the knowledge on the role of genetic and epigenetic instabilities of IGF2 gene, free (active form of IGF2) and IGF-binding protein (IGFBP) bound (inactive form), paracrine/autocrine secretion of IGF2, as well as mechanisms of inducing dysplasia in vitro and tumorigenicity in vivo. We have tried to answer which molecular changes of the IGF2 gene and its regulatory mechanisms have the most significance in initiation, progression (including liver metastasis), prognosis, and potential anti-IGF2 therapy in CRC patients.

Effects of down-regulation of IGF2 gene on the biological characteristics of HCT116 colon cancer stem cells

Objective: To investigate the effect of down-regulation of insulin-like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs). Methods: Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133(+) cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT-qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs. Results: CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum-free stem cell culture medium. We found that the morphology of sphere-forming-like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non-CSCs group and CSCs group were (28.10±2.66)% and (43.73±2.30)% respectively, with significant difference (P<0.01). The RT-qPCR results showed that the transcriptional level IGF2 gene in non-CSCs group and CSCs group were (1.06±0.24) and (2.17±0.51) respectively, with significant difference (P<0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non-CSCs group were (1.10±0.55) and (2.14±0.23) respectively, with significant difference (P<0.05). Knockdown of IGF2 significantly decreased the percentage of CD133(+) cells in CSCs and cell proliferation (P<0.01). Knockdown of IGF2 increased the percentage of G(2)/M phase (23.46% of siNC group vs 60.14% of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G(0)/G(1) phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P<0.01). The rate of sphere-forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)% and (21.27±2.35)% respectively, with significant difference (P<0.01). The clone formation rate of siNC group and siIGF2 group were (37.20±3.87)% and (18.23±2.25)% respectively, with significant difference (P<0.01). Conclusion: IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self-renewal and stemness of colon CSCs.

The insulin-like growth factor 2 gene in mammals: Organizational complexity within a conserved locus.

The secreted protein, insulin-like growth factor 2 (IGF2), plays a central role in fetal and prenatal growth and development, and is regulated at the genetic level by parental imprinting, being expressed predominantly from the paternally derived chromosome in mice and humans. Here, IGF2/Igf2 and its locus has been examined in 19 mammals from 13 orders spanning ~166 million years of evolutionary development. By using human or mouse DNA segments as queries in genome analyses, and by assessing gene expression using RNA-sequencing libraries, more complexity was identified within IGF2/Igf2 than was annotated previously. Multiple potential 5’ non-coding exons were mapped in most mammals and are presumably linked to distinct IGF2/Igf2 promoters, as shown for several species by interrogating RNA-sequencing libraries. DNA similarity was highest in IGF2/Igf2 coding exons; yet, even though the mature IGF2 protein was conserved, versions of 67 or 70 residues are produced secondary to species-specific maintenance of alternative RNA splicing at a variable intron-exon junction. Adjacent H19 was more divergent than IGF2/Igf2, as expected in a gene for a noncoding RNA, and was identified in only 10/19 species. These results show that common features, including those defining IGF2/Igf2 coding and several non-coding exons, were likely present at the onset of the mammalian radiation, but that others, such as a putative imprinting control region 5’ to H19 and potential enhancer elements 3’ to H19, diversified with speciation. This study also demonstrates that careful analysis of genomic and gene expression repositories can provide new insights into gene structure and regulation.

Short Communication: Genetic diversity of the IGF2 gene as a source of genetic marker for halal authentication

Hamdani AD, Madihah, Suryohastari RB, Virgianti DP, Putrie RFW. 2018. Genetic diversity of the IGF2 gene as a source of genetic marker for halal authentication. Nusantara Bioscience 10: 203-209. The main issue of halal authenticity is the availability of reliable and rapid analytical methods to identify animal species in raw and processed food. The two most popular procedures for identifying the source of the meat are protein-based and DNA-based methods, including mass spectroscopy (MS) using a peptide marker or PCR based DNA marker, respectively. This study aims to investigate the genetic diversity of insulin-like growth factor 2 (IGF2) gene, mainly from porcine (Sus scrofa) and bovine (Bos taurus) as a source of genetic marker for halal authentication by in-silico analysis using bioinformatic tools. Multiple sequence alignment and phylogenetic tree construction of IGF2 protein sequences from representative species of mammals, birds, and fishes showed the paraphyletic relationship between the IGF2 protein of S. scrofa and B. taurus. Protein structure analysis revealed differences in helical structures near the carboxyl end of the protein, while gene analysis showed different number of exon and motifs. By in-silico analysis, we have designed a peptide marker from amino acids at position of 93-107 (S. scrofa) and 93-112 (B. taurus) from peptide preptin region that resulted in different pattern of mass spectrum between the two species. We have also identified two DNA markers that can be detected by PCR using two primers sets designed from the IGF2 transcript sequences, to examine the presence of porcine in the sample. Thus, this study provides new genetic predictive markers, derived from the IGF2 gene, to identify the source of meat for halal authentication.

Mitotically Stable Modification of DNA Methylation in IGF2/H19 Imprinting Control Region Is Associated with Activated Hepatic IGF2 Expression in Offspring Rats from Betaine-Supplemented Dams.

The growth-promoting action of betaine involves activation of GH/IGF-1 signaling, yet it remains unclear whether insulin-like growth factor 2 (IGF2), an imprinting gene, is affected by maternal dietary betaine supplementation. In this study, F1 offspring rats derived from dams fed basal or betaine-supplemented diet were examined at D21 and D63. Maternal betaine significantly upregulated the hepatic expression of IGF2 mRNA and protein in offspring rats at both D21 and D63, which was accompanied by enhanced hepatic IGF2 immunoreactivity and elevated serum IGF-2 level. Higher protein expression of betaine-homocysteine methyltransferase and DNA methyltransferase 1 was detected in the betaine group at D21, but not D63. However, hypermethylation of the imprinting control region of the IGF2/H19 locus at D21 was maintained at D63. These results indicate that maternal betaine modifies DNA methylation of IGF2/H19 imprinting control region in a mitotically stable fasion, which was associated with the activation hepatic IGF2 expression in offspring rats.

Long Noncoding RNA Lnc-SEMT Modulates IGF2 Expression by Sponging miR-125b to Promote Sheep Muscle Development and Growth

Background/Aims: Long noncoding RNAs (lncRNAs) are RNA transcripts that are more than 200 nt long but have little protein-coding potential. Within the last few years, thousands of lncRNAs have been identified and their functions in biological processes have begun to be understood. Although many studies havebegun to examine the functions of many noncoding RNAs, very little is known about the functions of long noncoding (lncRNA) function of livestock production and molecular mechanisms of their functions are still lackingrelated to livestock production. Methods: Expression of sheep enhanced muscularityTranscript lncRNA (lnc-SEMT) and miR-125b were examined in sheep using quantitative reverse-transcription polymerase chain reaction. Expression of Myod (myogenic determination factor), Myog (myoglobin) and Insulin-like growth factor 2 (IGF2)were examined by Western Blot.Luciferase reporter assays were performedto confirm the relationship between lnc-SEMT and miR-125b. Results: Here, we identified a novel lnc-SEMT that promote sheep myoblast differentiation in vitro and enhanced sheep muscularity in vivo. Functional analyses showed that lnc-SEMT accelerates sheep myoblast differentiation in vitro. lnc-SEMT transgenic sheep exhibit a muscle hypertrophy phenotype characterized by increased body weight, and increased the number of muscle fibers indicating that lnc-SEMT play an important role in the regulation of skeletal muscle differentiation in vivo. Our results show that lnc-SEMT acts as a molecular sponge by antagonizing miR-125b to control IGF2 protein labundance in vitro and in vivo. Conclusion: In brief, lnc-SEMT is the first example of a lncRNA could be a useful candidate for improving biological growth traits such as skeletal muscle production in sheep.