Human Leukocyte antigen-G mRNA Research Articles

Evaluation of the relationships between HLA-G 14 bp polymorphism and two acute leukemia in a Saudi population

The non-classical Human Leukocyte Antigen G (HLA-G) is an immunomodulatory molecule, with low polymorphism frequency and restricted tissue distribution. Its higher expression was associated with immunoinhibitory properties causing tolerance for cancer progression. The rs371194629 is a 14 bp insertion/deletion polymorphism (Ins/Del) in exon 8 of the 3′ untranslated region (3′-UTR) of the HLA-G gene, has been associated with the stability and the expression level of HLA-G mRNA. In this study, we evaluated the relationship between the HLA-G 14-bp Ins/Del polymorphism and two common blood cancers including acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) in a population from Riyadh in Saudi Arabia. The study groups include 145 patients diagnosed for ALL, 98 patients diagnosed for AML and 115 healthy individuals. For all these individuals, the 14 bp Ins/Del polymorphism was genotyped using PCR methodology. mRNA gene expression of the HLA-G was assessed using quantitative RT-PCR. Logistic regression model analysis for HLA-G 14 bp Ins/Del polymorphism shows no statistically significant correlation with AML and ALL. For the HLA-G mRNA expression, while slightly increased level among healthy individuals compared to patients was observed, no statistically significant differences were obtained. Although our results did not show association between HLA-G with the two leukemia cancer diseases, the role of HLA-G gene awaits further investigations including large number of subjects with more clinical data combinations.

Expression of Human Leukocyte Antigen-G in Placental Tissues from Pregnant Women with Preeclampsia

Objective: To study the gene and protein expressions of human leukocyte antigen-G (HLA-G) in the placental tissues from healthy women in late pregnancy and pregnant women with preeclampsia (PE), and to explore the relationship between HLA-G and PE. Methods: Immunohistochemistry, Western blot and RT-PCR were used to detect the expressions of HLA-G protein and mRNA in the maternal placenta of 60 pregnant women with PE (including 30 cases of mild PE and 30 cases of severe PE) and 30 healthy full-term pregnant women (control group) respectively. Results: HLA-G had its expression mainly on the surface of trophoblast cells, and its response range and reactivity of its immunologic tissue were further reduced and weakened as the patient's condition was aggravated. The protein expression of placental HLA-G in each PE group was significantly lower than control group, with the difference statistically significant (all P<0.05). Compared with mild PE group, the protein expression level of HLA-G was lower in severe PE group (P<0.05). The Spearman's correlation analysis for HLA-G and PE severity showed that the correlation coefficient was -0.892 (rs = -0.892) (P<0.05), indicating that the expression of HLA-G protein was significantly negatively correlated to the severity of PE. The mRNA expression level of placental HLA-G of each PE group was significantly lower than that of control group, and the difference was statistically significant (all P<0.05). But the mRNA expression level of HLA-G was significantly lower in severe PE group than in mild PE group (P<0.05). Conclusion: The decreased expression of HLA-G in placenta may be involved in the pathogenesis of PE, and can also reflect the severity of this disease.

Promoter methylation and mRNA expression of HLA-G in relation to HLA-G protein expression in colorectal cancer

Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues.RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G.

Identification of novel microRNAs regulating HLA-G expression and investigating their clinical relevance in renal cell carcinoma.

The non-classical human leukocyte antigen G (HLA-G) is expressed at a high frequency in renal cell carcinoma (RCC) and is associated with a higher tumor grade and a poor clinical outcome. This might be caused by the HLA-G-mediated inhibition of the cytotoxicity of T and NK cells. Therefore a selective targeting of HLA-G might represent a powerful strategy to enhance the immunogenicity of RCC lesions. Recent studies identified a number of HLA-G-regulating microRNAs (miRs) and demonstrated an inverse expression of some of these miRs with HLA-G in RCC in vitro and in vivo. However, it was postulated that further miRs might exist contributing to the tightly controlled selective HLA-G expression.By application of a miR enrichment assay (miTRAP) in combination with in silico profiling two novel HLA-G-regulatory miRs, miR-548q and miR-628-5p, were identified. Direct interactions of both miRs with the 3′ untranslated region of HLA-G were confirmed with luciferase reporter gene assays. In addition, qPCR analyses and immunohistochemical staining revealed an inverse, expression of miR-628-5p, but not of miR-548q to the HLA-G protein in primary RCC lesions and cell lines. Stable overexpression of miR-548q and miR-628-5p caused a downregulation of HLA-G mRNA and protein. This leads in case of miR-548q to an enhanced NK cell-mediated HLA-G-dependent cytotoxicity, which could be reverted by ILT2 blockade suggesting a control of the immune effector cell activity at least by this miR. The identification of two novel HLA-G-regulatory miRs extends the number of HLA-G-relevant miRs tuning the HLA-G expression and might serve as future therapeutic targets.

Maternal human leukocyte antigen-G (HLA-G) genetic variants associate with in utero mother-to-child transmission of HIV-1 in Black South Africans

A 14-bp insertion/deletion (indel) within the 3′ untranslated region (3′UTR) that affects HLA-G expression has been associated with HIV-1 mother-to-child transmission (MTCT). However, other 3′UTR single nucleotide polymorphisms (SNPs) that influence HLA-G mRNA stability have been described but not analysed in the context of MTCT, and little is known about the role of HLA-G alleles. We examined HLA-G alleles and 3′UTR SNPs, including the 14-bp indel, in 216 mother–infant pairs from Johannesburg, South Africa. Mother–infant pairs were classified as HIV-1 non-transmitting (NT, n=144) or HIV-1 transmitting (TR, n=72) with either intrapartum (IP, n=29) or in utero (IU, n=19) infected infants. We found HLA-G allele, G∗01:01:02 (in strong linkage disequilibrium with the 14-bp insertion) and +3187G SNP were significantly over-represented in IU-TR mothers compared to NT mothers (P=0.036, OR=2.26; P=0.011, OR=2.96, respectively). These findings suggest that maternal HLA-G alleles and/or SNPs that might alter expression of HLA-G potentially influence IU HIV-1 MTCT.

Expression and Potential Roles of HLA-G in Human Spermatogenesis and Early Embryonic Development

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-G's expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8–9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48–72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

The expression and functional activity of membrane-bound human leukocyte antigen-G1 are influenced by the 3′-untranslated region

Human Leukocyte Antigen (HLA)-G is an immunosuppressive molecule acting on both the innate and adaptive immune system. A 14 bp insertion/deletion polymorphism (rs66554220) in the 3'-untranslated region (3'UTR) of the HLA-G gene has been associated with a number of diseases, pregnancy complications, and graft rejection after organ transplantation. We have investigated the effect of HLA-G polymorphism in the 3'UTR on the processing and stability of the membrane-bound HLA-G1 (mHLA-G1) isoform, as well as its functional significance. Different HLA-G1 cDNA sequences were transduced into the human K562 cell line. Flow cytometry, immunohistochemistry, and ELISA were used to examine HLA-G1 protein expression. A quantitative RT-PCR assay was used to quantify transduced HLA-G1 DNA and mRNA transcript levels. Stability of mRNA and functional significance of HLA-G were investigated via Actinomycin D and NK cytotoxicity assays, respectively. Human leukocyte antigen-G mRNA from the 14 bp insertion K562-G1 cells showed a higher degree of stability than the other constructs, and increased mHLA-G1 expression relative to transductants lacking the 14 bp sequence. In line with this, transductants carrying the 14 bp insertion were the most efficient in inhibiting NK cytotoxicity but showed a lower soluble HLA-G1 per mHLA-G1 ratio than the HLA-G1 K562 cells lacking the 14 bp insertion. Our data suggest 3'UTR polymorphism may play an important role in HLA-G regulation with implications on a range of diseases.

Human Leukocyte Antigen-G Is Frequently Expressed in Glioblastoma and May Be Induced in Vitro by Combined 5-Aza-2′-Deoxycytidine and Interferon-γ Treatments: Results from a Multicentric Study

Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2'-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G-negative tumors.

A fast and easy real‐time PCR genotyping method for the HLA‐G 14‐bp insertion/deletion polymorphism in the 3′ untranslated region

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class Ib molecule shown to exhibit immunomodulatory function in a wide range of immune-based disorders. A number of functional HLA-G gene polymorphisms have been identified, including a 14-bp insertion/deletion polymorphism in exon 8 of the 3' untranslated region of the HLA-G gene, which has been associated with HLA-G mRNA stability. Moreover, studies show that homozygosity for the 14-bp insertion/deletion polymorphism is associated with lower HLA-G mRNA and protein levels and unique alternative splicing patterns. Here, we introduce a quick and reliable method to screen for the HLA-G 14-bp insertion/deletion polymorphism using an optimized real-time polymerase chain reaction protocol. The genotyping assay has been validated by comparison with conventional methods. As results can be obtained within a few hours, the assay will have a potential for clinical use.

Expression of Human Leukocyte Antigen-G during Normal Placentation and in Preeclamptic Pregnancies

Objectives. This study was undertaken to investigate the expression pattern of human leukocyte antigen-G (HLA-G) during normal placentation and determine whether altered expression of HLA-G is associated with severe preeclampsia. Methods. We investigated HLA-G protein levels in first (n = 27), second (n = 7), and third trimester placentas (n = 10) from normal pregnancies, and determined HLA-G levels in term placentas from normal (n = 15) and severe preeclamptic pregnancies (n = 14) using real-time RT-PCR and western blot analysis. Results. In normal placentas, HLA-G protein expression reached a peak level at gestational weeks 6 and 7, then gradually decreased from week 8 to third trimester (p < 0.05). In preeclamptic placentas, both HLA-G mRNA and protein levels were decreased significantly in comparison with normal term placentas (p < 0.05). Conclusion. HLA-G may contribute to placentation during early and mid-term pregnancy, and participate in maintaining gestation during term pregnancy. The reduced level of HLA-G may be associated with pathogenesis of preeclampsia.

Human Leukocyte Antigen G Expression in Breast Cancer: Role in Immunosuppression

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex Class Ib molecule. It is expressed by trophoblastic placental cells during pregnancy to protect the fetus from maternal alloreactivity. HLA-G is overexpressed in tumors and involved in cancer immune evasion. Reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were used to examine HLA-G expression in normal mammary and breast cancer cell lines and normal and human breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, whereas cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Twelve (12) normal and 38 breast cancer tissues were examined by IHC. Fifty-eight (58) percent (22/38) of cancers had medium to strong staining to HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was significant (p < 0.05). HLA-G staining was found in the membranes and cytoplasm of cancer cells. In conclusion, breast cancer cells overexpress HLA-G mRNA and protein, and this probably contributes to immune evasion.

Abstract 1488: Role of human leukocyte antigen-G (HLA-G) in cancer progression and susceptibility

Abstract Human Leukocyte antigen -G is an immunosuppressive protein with multiple functions. The 14bp insertion / deletion in exon-8 of HLA-G gene have been shown to play an important role in HLA-G mRNA stability as well as expression level; and can be correlated with its immunosuppressive function. The role of HLA-G in various cancers has already been demonstrated. Cancerous cells start expressing HLA-G which in turn provides a shield for various immune responses. In the present study we have tested the possibility of the role of 14bp insertion / deletion polymorphism of HLA-G in cancer progression and susceptibility. We have genotyped 59 cancer samples (26 benign and 33 malignant) and 79 matched controls for 14bp polymorphism of HLA-G. Data showed increase of +14bp/-14bp and −14bp/-14bp genotype with both benign and malignant cancer. We observed significantly higher frequency of +14bp/+14bp genotype in control (p=0.01). This is consistent with both benign and malignant tumors as well as cancer types showing possibility of protective effect of +14bp/+14bp genotype in various cancers. However, further studies will be required with large sample size to test this hypothesis. The +14bp/-14bp and −14bp/-14bp genotypes have already been shown to be associated with higher expression of HLA-G and its mRNA stability and might be providing favorable micro-environment for cancer progression. There is no published work till date showing the role of HLA-G 14bp polymorphism in various cancers. These preliminary data showed that HLA-G 14bp genotypes might be playing an important role in pathogenesis of various cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1488.

Correlation of Human Leukocyte Antigen-G (HLA-G) Expression and Disease Progression in Epithelial Ovarian Cancer

Human leukocyte antigen-G (HLA-G) expression has been reported to be relevant to cancer development and immune tolerance. The purpose of this study was to investigate the correlation between HLA-G expression and disease progression and to assess the use of HLA-G expression as a prognostic immunomarker in epithelial ovarian carcinoma. Human leukocyte antigen-G expression in 41 ovarian cancer tissues and 8 normal ovarian tissues was analyzed using immunohistochemistry and Western blot assay. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used for HLA-G messenger RNA (mRNA) expression. Human leukocyte antigen-G mRNA and protein levels were significantly greater in advanced ovarian cancer tissues than in normal or early stage ovarian cancer tissues (P < .05 and P < .05, respectively). Patients with HLA-G expression had a significantly worse prognosis. There is a significant correlation between HLA-G immunoreactivity and patient survival in univariate analysis (P = .04). Our data was consistent with the concept that HLA-G expression might play a pivotal role in the development and disease progression of epithelial ovarian cancer.

The HLA‐G 14 bp insertion/deletion polymorphism is a putative susceptible factor for active human cytomegalovirus infection in children

Human leukocyte antigen-G (HLA-G) expression is a potential factor for the pathogenesis of virus infection. A 14 bp insertion/deletion polymorphism (rs16375) in the 3'-untranslated region of the HLA-G gene is involved in the stability of HLA-G mRNA and HLA-G protein expression. Therefore, the HLA-G 14 bp polymorphism might be involved in human cytomegalovirus (hCMV) infection. To test a possible association between the HLA-G 14 bp deletion/insertion polymorphism and the active hCMV infection, in this study, a total of 54 patients with active hCMV infection and 165 age- and sex-matched, unrelated, normal Chinese Han population were genotyped for the 14 bp insertion/deletion polymorphism. Association of 14 bp polymorphism with hCMV urine DNA copies and the odds ratio (OR) of the polymorphism as a risk factor for active hCMV infection were analyzed. Our results showed that the prevalence of -14 bp/ -14 bp genotype in active hCMV patients was markedly increased [P(c) = 0.00034, OR = 3.31, 95% confidence interval (CI): 1.77-6.18], and similar significance was also observed for the frequency of -14 bp allele (P c = 0.0023, OR = 2.24, 95% CI: 1.38-3.64) when compared with that of healthy controls. Furthermore, urine hCMV DNA copies in patients with the -14 bp/ -14 bp genotype were significantly higher than those in patients with the +14 bp/ +14 bp genotype (P = 0.041). Our findings support a potential role of HLA-G 14 bp insertion/deletion polymorphism as a susceptible factor for the active hCMV infection.

Immunohistochemical Study of HLA-G Expression in Lung Transplant Recipients

Human leukocyte antigen-G (HLA-G), a nonclassical HLA class I protein, promotes immune tolerance of solid-organ allografts, yet its role in lung transplantation (LTx) is unknown. We examined the expression of HLA-G in lung allografts through immunohistochemistry by a cross-sectional study of 64 LTx recipients, classified into four groups (stable patients, acute rejection [AR], bronchiolitis obliterans syndrome [BOS] and symptomatic viral shedders). A marked expression of HLA-G in bronchial epithelial cells (BEC) was frequently observed in stable recipients (n = 18/35 [51%]), but not in patients with AR (n = 14) or with BOS (n = 8). HLA-G was also expressed by 4 of 7 symptomatic viral shedders. In addition, HLA-G-positive patients from the stable group (n = 35) experienced lower incidence of resistant AR and/or BOS during long-term follow-up, as compared with their HLA-G-negative counterparts. Finally, in vitro data showed that interferon-gamma, a cytokine present in lung allograft microenvironment, upregulated HLA-G mRNA and protein expression in primary cultured human BEC. We conclude that HLA-G expression in the bronchial epithelium of lung allograft is elevated in some LTx recipients in association with their functional stability, suggesting a potential role of HLA-G as a tolerance marker.

HLA‐G polymorphism and in vitro fertilization failure in a Polish population

To investigate whether human leukocyte antigen-G (HLA-G) gene polymorphism is associated with in vitro fertilization (IVF) failure, we sequenced exons 2-4 of the HLA-G gene in 50 couples with three or more IVFs (including 10 couples with five or more IVFs) and 58 control fertile couples from a Polish population. Of the 10 different HLA-G alleles identified in our study subjects, neither allele was found to be associated with IVF. We also genotyped 50 couples with IVF and 71 control couples for the -725C>G variant in the promoter region and the 14 bp insertion or deletion polymorphism in the 3' untranslated region of the HLA-G gene. The frequency of -725GG or GC genotype in women with IVF and in control fertile women was similar [26% vs 25.3%; odds ratio (OR) = 1.0; P = 1.0]. The 14 bp ins/ins or ins/del genotype was more common in women with IVF than in control women (76.9% vs 59.1%; OR 2.4; P = 0.03), but the difference was not significant after Bonferroni correction for multiple comparisons. The frequency of the ins/ins or ins/del genotype was particularly high (90%) in women who experienced five or more IVFs (OR = 6.2; P = 0.08), but again, the excess was not statistically significant, possibly because of small sample sizes. These results are in line with functional studies that show lower levels of HLA-G mRNA and protein related to the HLA-G allele including the 14 bp sequence and suggest that the insertion allele may be associated with an increased risk of IVF.

A single base-pair mutation in the 3'-untranslated region of HLA-G mRNA is associated with pre-eclampsia

Human leukocyte antigen-G (HLA-G) is a non-classical class I HLA molecule that is expressed by extravillous cytotrophoblast cells. This protein may play a critical role in the protection of cytotrophoblasts from maternal immune response, allowing these semi-allogeneic cells to invade the uterus unimpeded. We have demonstrated that diminished placental HLA-G expression is associated with pre-eclampsia. In order to explore fundamental mechanisms underlying this reduced HLA-G expression in pre-eclampsia, we looked for, and found by sequences analysis, a single base-pair mutation in the HLA-G gene 3'-untranslated region (3'UTR) adjacent to an AUUUA motif. This mutation is significantly associated with pre-eclampsia, the severe form being more strongly associated with homozygosity for this mutation than the mild form. Since the null allele was discovered in the HLA-G mRNA 3'UTR adjacent to an AUUUA motif, we also examined the effect of this mutation on HLA-G mRNA stability, and found that half-lives of HLA-G mRNA with the mutation were significantly shorter than without the mutation. These data provide evidence that this mutation could be one of the fundamental mechanisms for lower levels of placental HLA-G protein expression in patients with pre-eclampsia.

Shift in expression of HLA-G mRNA spliceforms in pregnancies complicated by preeclampsia.

Despite emerging data on the in vitro modulatory effects of trophoblast-associated human leukocyte antigen G (HLA-G), its in vivo function needs to be determined. Immunohistochemical studies show a decrease in protein expression of trophoblast HLA-G in preeclampsia. Such a decrease in protein might be the consequence of a shift in HLA-G mRNA spliceform patterns. In an exploratory pilot study we determined trophoblast HLA-G mRNA spliceform distribution in preeclampsia. Placental samples were collected immediately after cesarean delivery from pregnancies complicated by preeclampsia or the syndrome hemolysis, elevated liver enzymes, and low platelet count (HELLP) and uncomplicated normotensive pregnancies as controls. HLA-G mRNA spliceform distribution was analyzed using a semiquantitative reverse transcriptase polymerase chain reaction procedure. Analysis of HLA-G spliceform distribution showed a significant increase in frequency of the G5 form encoding for a soluble HLA-G molecule in preeclampsia. This increase in G5 form was not found in pregnancies complicated by HELLP. The increased frequency in the expression of the HLA-G G5 spliceform may play a role in the pathophysiology of preeclampsia, in particular through a recently suggested effect of this soluble HLA-G molecule on remodeling of the spiral arteries.

Inhibition of HLA-G production in JEG-3 choriocarcinoma cells by RNA interference.

Human leukocyte antigen-G (HLA-G) plays a major role in escape of trophoblast cells from maternal cytotoxicity. Unless its malignant transformation, Jeg-3 choriocarcinoma cell line maintained the capacity of HLA-G production. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G would be very helpful. RNA interference (RNAi) is a very elegant method for this approach, but the design of appropriate oligonucleotide sequences may provide difficulties. We designed oligonucleotides to interfere exclusively with HLA-G mRNA, which were applied at different concentrations to 50% confluent Jeg-3 cells. After 36 hr, the HLA-G content in Jeg-3 cells was analyzed by Western blots. Applying the described RNAi method and oligonucleotides the cellular content of HLA-G was dose-dependently reduced as assessed in several independent Western blots. This method provides a tool for extensive investigation of HLA-G functions in vitro and if vivo.

Detection of alternatively spliced transcripts of human leukocyte antigen-g in single human preimplantation blastocysts using nested RT-PCR

Objective: Human leukocyte antigen G (HLA-G) is a virtually nonpolymorphic HLA class I gene expressed by extravillous cytotrophoblast, which is believed to play a key role in maternal immune tolerance of the fetus. HLA-G mRNA can be alternatively spliced into 6 transcripts, which encode 4 membrane isoforms (G1, G2, G3, G4) and 2 soluble isoforms (G5 and G6). HLA-G is expressed by some human pre-implantation embryos and the levels of soluble HLA-G secreted may relate to implantation success (1). However, which of the mRNA isoforms are expressed and in what proportion of embryos they are found is unknown.