Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos

NADH dehydrogenase (complex I) couples the oxidation of NADH for the reduction of ubiquinone with the generation of a proton gradient that can be used for the synthesis of ATP. We have found a missense mutation in the MT-ND5 subunit of NADH dehydrogenase in the Tibet chicken breed. In the present study, the mitochondrial respiratory control ratio (RCR) and NADH dehydrogenase activity in Tibet chicken embryonic brain with different genotypes were measured. Significant differences between animals carrying mitochondria with the EF493865.1:m.1627A vs. EF493865.1:m.1627C alleles were observed for RCR and enzyme activity.     

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.     

Serum antibody responses against human papillomavirus in relation to tumor characteristics,response to treatment,and survival in carcinoma of the uterine cervix

To investigate whether the serum antibody responses to human papillomavirus (HPV) in cervical carcinoma were related to the clinical and histopathological features of the tumors and how the antibody responses were affected by treatment, pretreatment serum samples from 66 patients with carcinoma of the cervix were studied for the presence of IgA or IgG responses against six defined HPV epitopes. Posttreatment serum samples were drawn from the same patients 2–24 months after initiation of treatment. There was no significant correlation between pretreatment level of any of the investigated antibodies and clinical stage or differentiation of tumor. For the IgA responses to the epitopes 24516 and 24518 in the E2 protein there was a significant correlation between an increased pretreatment antibody level and a shortened survival. A high pretreatment value of IgA against 24516 was also associated with the absence of any complete response after therapy. The antibody levels declined dramatically after therapy for most of the antigens studied. However, this decline was seen both among the 53 patients with complete remission and among the 13 patients with remaining or progressive disease. Thus, the investigated serological responses were not useful as tumor markers, since patients with progressive, latestage disease may fail to mount an antibody response to these proteins. However, pretreatment levels of the serological responses to the HPV epitopes 24516 and 24518 were associated with prognosis in cervical cancer.     

高危型人乳头瘤病毒感染与宫颈癌中miR-218表达的关系

目的观察高危型人乳头瘤病毒(HPV)感染与宫颈癌中miR-218表达的关系。方法收集2015年6月至2018年12月我院手术切除的宫颈癌组织并检测高危型HPV感染情况和miR-218表达量。培养HPV16阳性的SiHa细胞株并进行分组,阴性对照(NC)组转染NC模拟物、miR-218组转染miR-218模拟物,检测两组细胞凋亡率、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Bcl-2相互作用细胞死亡介导因子(Bim)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-9、Caspase-3的mRNA表达量及凋亡通路分子c-Jun氨基末端激酶(JNK)、c-Jun基因(c-Jun)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)的蛋白表达量。结果高危型HPV阳性的宫颈癌组织中miR-218表达量减少。转染24 h后,miR-218组细胞凋亡率、细胞中Bax、Bim、Caspase-9、Caspase-3的mRNA表达量及JNK、c-Jun的蛋白表达量均明显高于NC组,而细胞中Bcl-2的mRNA表达量及PI3K、AKT、mTOR的蛋白表达量均低于NC组,差异均有统计学意义(均P<0.05)。结论miR-218在高危型HPV感染的宫颈癌组织中表达减少。增加miR-218的表达能够促进HPV感染宫颈癌细胞的凋亡。该调控作用与JNK/c-Jun通路的激活及PI3K/AKT/mTOR通路的抑制有关。     

Regulation of 5-phosphoribosyl-1-pyrophosphate synthesis in human fibroblasts by the concentration of inorganic phosphate

During the growth cycle of normal fibroblasts and of fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the concentration of 5-phosphoribosyl-1-pyrophosphate and of P_i, as well as the activity of 5-phosphoribosyl-1-pyrophosphate synthetase, decreased to stable values in confluent cultures. A high degree of correlation (0.89 and 0.91 for two normal and 0.69 for one glucose-6-phosphate dehydrogenase-deficient cell strain, respectively) was shown between intracellular P_i and 5-phosphoribosyl-1-pyrophosphate concentrations under varying culture and incubation conditions. 5-phosphoribosyl-1-pyrophosphate concentrations were elevated in normal fibroblasts incubated with methylene blue only if intracellular P_i levels were high. Neither methylene blue nor 6-aminonicotinamide, singly, affected intracellular P_i concentrations. However, when normal cells were pretreated with 6-aminonicotinamide and then with methylene blue, intracellular P_i decreased, 5-phosphoribosyl-1-pyrophosphate was depleted, and its rate of generation decreased. Under similar conditions, glucose-6-phosphate dehydrogenase-deficient fibroblasts maintained unaltered P_i levels, and 5-phosphoribosyl-1-pyrophosphate concentration and generation were slightly increased. The decrease in intracellular P_i in normal cells after the combined treatment was commensurate with an accumulation of 6-phosphogluconate, which did not take place in mutant cells. The changes in 5-phosphoribosyl-1-pyrophosphate synthesis, whether due to the stage of growth or various experimental manipulations, were always concordant with changes in intracellular P_i level. The regulatory role of P_i is consistent with the known enzymic properties of 5-phosphoribosyl-1-pyrophosphate synthetase.     

Relation between HPV-16 serology and clinico-pathological data in cervical carcinoma patients: prognostic value of anti-E6 and/or anti-E7 antibodies

 To investigate the clinical significance of the enhanced sensitivity of antibody detection by radio immunoprecipitation assays (RIPA), using in vitro translated HPV-16 E6 and E7 proteins, over synthetic-peptide enzyme-linked immunosorbent assay (ELISA), RIPA for HPV-16 E6 and E7 were performed. The results obtained with E6 and E7 RIPA were related to clinico-pathological data from cervical carcinoma patients positive for HPV type 16 DNA in their primary tumour. The data obtained with E6 and E7 RIPA were then compared to the results obtained using the E7/6-35 synthetic-peptide ELISA. The prevalence of antibodies to E6, E7, E6 and/or E7 and E6 and E7, as determined by RIPA, was significantly higher in cervical cancer patients than in both controls and cervical intraepithelial neoplasia patients. Odds ratios, calculated for cervical carcinoma patients versus controls, ranged from 7.4 to 15.4. Antibodies to E6 and/or E7 were largely restricted to patients with HPV DNA in their primary tumour. Analysis of the relation between prevalence of antibodies to E6 and E7 and clinico-pathological parameters was limited to 85 patients positive for HPV-16 DNA. The strongest relation with clinico-pathological data, such as lesion size, lymph node involvement, and prognosis, was found for E7 synthetic-peptide ELISA, whereas E6 and E7 RIPA did not reach significance. The significance of these findings is discussed. Received: 17 February 1997 / Accepted: 13 March 1997     

Discovery of human lactate dehydrogenase 5 inhibitors (hLDH5) with anti-lung cancer activity through an in silico method and biological validation

Human lactate dehydrogenase 5 (hLDH5) is an important metabolic enzyme playing critical roles in the anaerobic glycolysis. Herein, we employed an in silico method and biological validation to identify a novel hLDH5 inhibitor with a promising cellular activity under hypoxia condition. The identified compound 9 bound to hLDH5 with a K_d value of 1.02 µM, and inhibited the enzyme with an EC₅₀ value of 0.7 µM. Compound 9 exhibited a weak potency against NCI-H1975 cell proliferation under normal condition (IC₅₀ = 36.5 µM), while dramatically increased to 5.7 µM under hypoxia condition. In line with the observation, hLDH5 expression in NCI-H1975 cell under hypoxia condition is much higher as compared to the normal oxygenated condition, indicating the hLDH5 inhibition may contribute to the cancer cell death.     

Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

SRSF1和BIRC5在胃癌组织中的表达及临床意义

目的通过对丝氨酸/精氨酸富有剪接因子1(SRSF1)和凋亡抑制因子(BIRC5)在胃癌组织中表达的检测及两者之间相关性的研究,来探讨两者与胃癌的发生、发展之间的关系;为胃癌的转移及复发预警,以及预后评估提供新靶点。方法应用逆转录-聚合酶链式反应(RT-PCR)和蛋白质印迹(Western blotting)检测60例术中切除的胃癌肿组织以及对应的癌旁组织中SRSF1与BIRC5的表达水平,分析这两种蛋白的表达水平与临床病理学特征的关系。结果 (1)胃癌肿组织中SRSF1 mRNA和BIRC5mRNA表达情况:SRSF1阳性表达率(75.0%,45/60)高于癌旁组织中(8.3%,5/60);BIRC5阳性表达率(81.7%,49/60)高于癌旁组织中(11.7%,7/60)。胃癌肿组织中SRSF1mRNA与BIRC5mRNA的表达与患者的年龄、性别、肿瘤大小等因素无关,与胃癌肿组织的分化程度、淋巴结转移以及TNM分期有关;SRSF1和BIRC5 mRNA在胃癌肿组织中的阳性表达呈正相关(r=0.3496,P=0.0062)。(2)胃癌肿组织中SRSF1蛋白与BIRC5蛋白表达情况:胃癌肿组织中SRSF1蛋白表达量相较于其在癌旁组织中的表达量明显增多(t=44.87,P0.01),BIRC5蛋白表达量较癌旁组织同样异常增高(t=69.84,P0.01)。结论在胃癌肿组织中SRSF1和BIRC5表达均存在异常增高,与胃癌的发生发展存在正相关,这可能与SRSF1选择性剪接BIRC5片段,促进凋亡抑制基因BIRC5的高表达有关,具体机制还需进一步探讨。     

Clinical implication of elevated human cervical cancer oncogene-1 expression in esophageal squamous cell carcinoma

The human cervical cancer oncogene 1 (HCCR-1), a novel human oncoprotein, has been shown to be upregulated in various human tumors and plays a critical role in tumorigenesis and tumor progression. Here, the authors investigated HCCR-1 level in esophageal squamous cell carcinoma (ESCC) tissues and assessed the correlation between HCCR-1 level and prognosis of the patients with ESCC. HCCR-1 levels were investigated by immunohistochemistry, in situ hybridization, real-time quantitative RT-PCR and Western blotting methods; Kaplan-Meier curve was used to evaluate the prognostic value of HCCR-1 level in patients with ESCC using log-rank test. HCCR-1 displayed high levels in ESCC tissues compared to squamous dysplasia tissues and normal esophageal epithelial tissues. No significant correlation was observed between the levels of HCCR-1 mRNA and protein and gender and age (all p>0.05) but obviously related to histological grade, clinical stage, and lymph node metastasis (all p<0.001). Moreover, the survival rate of the patients with low HCCR-1 levels was higher than that of the patients with high HCCR-1 levels (both p<0.05). These data demonstrate that HCCR-1 may be used as a novel predictor for the prognosis of the patients with ESCC.     

宫颈癌患者人乳头瘤病毒感染情况及STING、ZEB1蛋白水平分析

分析宫颈癌患者人乳头瘤病毒（HPV）感染情况及癌灶组织内干扰素基因刺激因子（STING）、E盒锌指结合蛋白1（ZEB1）表达情况,为该类患者的治疗提供参考。

收集我院病理检验科2019年1月至2022年1月63例宫颈癌标本（宫颈癌组）、55例宫颈上皮内瘤变（CIN）标本（CIN组）及50例正常宫颈标本（正常组）,采用免疫组化法检测各标本内STING及ZEB1表达情况,采用原位杂交法检测各标本内高危HPV感染情况,分析STING及ZEB1蛋白表达情况与宫颈癌病理特征及高危HPV感染之间的关系。

宫颈癌组标本STING及ZEB1表达量均高于CIN组及正常组,CIN组标本ZEB1表达量高于正常组,差异均有统计学意义（均P＜0.05）;CIN组标本STING表达量与正常组比较差异无统计学意义（P＞0.05）。宫颈癌组标本HPV16、18检出率高于CIN组及正常组,CIN组标本HPV16、18检出率高于正常组,差异均有统计学意义（均P＜0.05）。宫颈癌患者FIGO分期及浸润深度与其组织内STING及ZEB1蛋白阳性情况均存在相关性;此外,宫颈癌淋巴结转移及肿瘤分化程度与组织内STING蛋白阳性情况存在相关性,差异均有统计学意义（均P＜0.05）。宫颈癌患者STING及ZEB1蛋白表达与其HPV16、18检出率间均呈正相关（均P＜0.05）。

宫颈癌组标本STING及ZEB1蛋白表达量均高于正常组及CIN组,且其表达量还与宫颈癌浸润、FIGO分期相关。STING和ZEB1可能与HPV16、18感染共同参与宫颈癌的发生及进展。

     

Hepatitis B virus x gene-downregulated growth-arrest specific 5 inhibits the cell viability and invasion of hepatocellular carcinoma cell lines by activating Y-box-binding protein 1/p21 signaling

The long noncoding RNA growth-arrest specific 5 (GAS5) is a suppressor of many cancers. However, the role and mechanism of action of GAS5 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain unclear. Here, the expression of hepatitis B virus x gene (HBx) mRNA and GAS5 was assessed by qRT–PCR, and western blot analysis was performed to determine the protein expression levels. In addition, the cell viability and invasion of cells were confirmed using  MTT assay and Transwell assay, respectively. The DNA methylation level of GAS5 was measured by methylation-specific PCR. Moreover, RIP assay and RNA pull down assay were carried out to examine the combination of Y-box-binding protein 1 (YBX1) and GAS5. First, our data proved that HBx is increased, while GAS5 is decreased in HCC cell lines. Subsequently, we found that HBx facilitates HCC cell viability and invasion by inhibiting GAS5 expression. Then, we further clarified that HBx induces the DNA methylation of GAS5 by promoting methyltransferase expression, thereby suppressing GAS5 expression. Furthermore, GAS5 binds YBX1 and promotes YBX1 and p21 expression. Finally, the functional analysis revealed that the upregulation of GAS5 could attenuate cell viability and invasion by boosting p21 expression via binding YBX1. Overall, our results demonstrated that HBx promotes HCC progression by inducing GAS5 methylation to reduce its expression. The upregulation of GAS5 suppressed HBV-related HCC by activating YBX1/p21 signaling. Our data provide novel evidence supporting the potential of GAS5 as a treatment target in HBV-related HCC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00645-z.     

宫颈癌组织RACK-1、Lgr5表达与临床病理特征及预后的关系

目的:探讨宫颈癌(CC)组织激活的蛋白激酶C受体1(RACK-1)、富含亮氨酸重复序列的G蛋白偶联受体5(Lgr5)表达与临床病理特征及预后的关系。方法:选取2012年1月-2016年1月于三峡大学附属仁和医院接受手术治疗的134例CC患者作为研究对象,选取CC组织以及对应的癌旁正常组织,同时选取该院因宫颈良性病变行肿物剥除或附件切除的134例患者的正常宫颈组织作为对照,采用免疫组化链霉素抗生物素蛋白-过氧化物酶连(SP)法对各组织进行免疫组化染色,依据阳性细胞率和染色强度分析CC组织中RACK-1、Lgr5的表达情况,并分析RACK-1、Lgr5表达与CC患者临床病理特征的相关性,分析Lgr5及RACK-1不同阳性表达强度的CC患者的预后。结果:CC组织中RACK-1、Lgr5阳性表达率高于癌旁组织和正常宫颈组织,差异有统计学意义(P0.05);RACK-1、Lgr5阳性表达与TNM分期、肿瘤分化程度、淋巴结转移、脉管内癌栓相关(P0.05),与患者的年龄、肿瘤大小、病理类型、肿瘤浸润深度、宫旁侵犯、手术切缘无关(P0.05);Lgr5、RACK-1阳性高表达组患者的3年生存率、生存时间低于Lgr5、RACK-1阳性低表达组患者,差异有统计学意义(P0.05)。结论:RACK-1、Lgr5在CC组织中表达上调,并与部分临床病理特征及预后有关,检测RACK-1、Lgr5有助于CC患者的诊断及预后评估。     

Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR

Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC.