DNAH17-AS1 promotes pancreatic carcinoma by increasing PPME1 expression via inhibition of miR-432-5p

BACKGROUND The incidence and mortality rates of pancreatic carcinoma(PC)are rapidly increasing worldwide.Long noncoding RNAs(lncRNAs)play critical roles during PC initiation and progression.Since the lncRNA DNAH17-AS1 is highly expressed in PC,the regulation of DNAH17-AS1 in PC was investigated in this study.AIM To investigate the expression and molecular action of lncRNA DNAH17-AS1 in PC cells.METHODS The PC expression data for the lncRNA DNAH17-AS1 was downloaded from The Cancer Genome Atlas database and used to examine its profile.Western blot and reverse transcription-quantitative PCR were employed to assess protein and mRNA expression.A subcellular fractionation assay was used to determine the location of DNAH17-AS1 in cells.In addition,the regulatory effects of DNAH17-AS1 on miR-432-5p,PPME1,and tumor activity were investigated using luciferase reporter assay,MTT viability analysis,flow cytometry,and transwell migration analysis.RESULTS DNAH17-AS1 was upregulated in PC cells and was associated with aggressive tumor behavior and poor prognosis for patients.Silencing DNAH17-AS1 promoted the apoptosis and reduced the viability,invasion,and migration of PC cells.In addition,DNAH17-AS1 served as a PC oncogene by downregulating miR-432-5p which normally directly targeted PPME1 to downregulate its expression.CONLUSION DNAH17-AS1 functions in PC as a tumor promoter by regulating the miR-432-5p/PPME1 axis.This finding may provide new insights for PC prognosis and therapy.

DNAH1基因突变引起精子鞭毛多发形态异常的不育患者辅助生殖助孕结局分析

目的探讨DNAH1基因突变引起的精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella,MMAF)不育患者行卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)助孕后的临床结局。方法回顾性队列研究分析2018年2月至2020年1月期间在河南省人民医院生殖中心就诊的39例MMAF不育患者的临床资料和基因检测结果,12例由DNAH1突变引起的MMAF患者为DNAH1阳性组,27例未提示DNAH1突变的MMAF患者为DNAH1阴性组,选择同一时期男女双方年龄匹配进行ICSI助孕治疗的100例精子形态正常的男性不育症患者作为对照组,观察并分析3组不育夫妇进行辅助生殖助孕的治疗结局。结果39例MMAF患者均行全外显子组测序检测,其中12例患者检测到DNAH1基因突变,分别为10例复合杂合突变和2例纯合突变,另27例患者未检测到目前已知的引起MMAF的基因突变。3组患者夫妇均行ICSI助孕治疗,DNAH1阳性组、DNAH1阴性组和对照组在获卵数和MII卵子数上的差异均有统计学意义[(17.08±5.32)枚、(9.59±3.98)枚和(10.44±6.33)枚,P=0.001;(14.58±5.18)枚、(6.78±3.38)枚和(8.32±5.31)枚,P<0.001],在胚胎种植率、临床妊娠率、早期流产率和活产率上的差异均无统计学意义(均P>0.05)。12例由DNAH1突变引起的不育患者夫妇共接受12个取卵周期,形成第3天胚胎79枚,首次新鲜胚胎或复融胚胎移植共12次,获得10个亲生子代。结论对于由DNAH1基因突变引起的MMAF患者,ICSI助孕可以帮助其生育亲生子代,且有较高的临床妊娠率和活产率。

Novel compound heterozygous mutations in DNAH1 cause primary infertility in Han Chinese males with multiple morphological abnormalities of the sperm flagella

This study aimed to identify genetic causes responsible for multiple morphological abnormalities of the sperm flagella(MMAF)in the Han Chinese population.Three primary infertile males with completely immobile sperm and MMAF were enrolled.Whole-exome sequencing and Sanger sequencing were performed to identify disease-causing genes.Subsequently,morphological and ultrastructural analyses of sperm flagella were investigated.The probable impact of genetic variants on protein function was analyzed by online bioinformatic tools and immunofluorescence assay.Three patients with dynein axonemal heavy chain 1(DNAH1)gene compound heterozygous variations were identified.DNAH1 c.7435C>T,p.R2479X and c.10757T>C,p.F3586S were identified in the patient from Family 1,c.11726_11727delCT,p.P3909fs and c.12154delC,p.L4052fs were found in the patient from Family 2,and c.10627-3C>G and c.11726_11727delCT,p.P3909fs existed in the patient from Family 3.Four of these variations have not been reported,and all the mutations showed pathogenicity by functional effect predictions.The absence of the center pair and disorganization of the fibrous sheath were present in sperm flagella at the ultrastructural level.Moreover,the expression of DNAH1 was absent in spermatozoa from the participants,validating the pathogenicity of the variants.All three couples have undergone intracytoplasmic sperm injection(ICSI),and two couples of them became pregnant after the treatment.In conclusion,the newly identified DNAH1 mutations can expand the mutational and phenotypic spectrum of MMAF genes and provide a theoretical basis for genetic diagnosis in MMAF patients.It is recommended to conduct genetic screening in male infertility patients with MMAF and provide rational genetic counseling,and ICSI might be an optimal strategy to help with fertilization and conception for patients with DNAH1 mutations.

两例精子鞭毛多发形态异常男性不育症患者的DNAH1基因变异分析

目的对2例由严重弱精子症导致原发性男性不育患者进行临床和遗传学分析,明确其可能的致病原因。方法提取患者及父母外周血基因组DNA,采用全外显子组测序技术对患者进行基因变异分析,并对疑似致病变异进行Sanger测序验证和致病性分析。结果全外显子测序显示例1 DNAH1基因存在c.2016T>G(p.Y672X)和c.6017T>G(p.V2006G)复合杂合变异;例2 DNAH1基因存在c.2610G>A(p.W870X)纯合变异,分别遗传自父亲和母亲。按照美国医学遗传学会与基因组学学会遗传变异分类标准与指南,DNAH1基因c.2016T>G(p.Y672X)和c.2610G>A(p.W870X)变异均为致病(PVS1+PM2+PM3+PP3)。结论2例患者可能均为DNAH1基因变异导致的精子鞭毛多发形态异常,进而引起原发性男性不育。

圆头精子症患者精子形态观察及全外显子测序分析

目的:观察1例圆头精子症患者的精子形态,并进行全外显子测序,探索其发病的遗传学基础。方法:取1例圆头精子症患者和1名健康对照精液各2μL,滴于GoldCyto Spermblue精子形态染色分析玻片上,用光学显微镜观察精子形态。抽取患者静脉血3 mL,提取基因组DNA,进行全外显子测序,并采用实时荧光定量PCR和Sanger测序方法对全外显子测序检测到的基因变异进行验证。结果:患者所有精子均为圆形头,仅见深染的细胞核,无顶体;该患者DPY19L2基因纯合缺失,同时携带DNAH1基因c.871+3G>A杂合突变以及ZMYND10基因c.833G>C杂合突变。结论:DPY19L2基因纯合缺失可能是本例圆头精子症的发病原因。

沉默lncRNA DNAH8-AS1靶向miR-186-5p/YY1调控前列腺癌细胞的增殖和侵袭

目的探讨lncRNA DNAH8-AS1与miR-186-5p之间的靶向关系及沉默lncRNA DNAH8-AS1对前列腺癌细胞增殖和侵袭的影响。方法采用GEPIA数据库分析lncRNA DNAH8-AS1在前列腺癌和癌旁组织中的表达及其与前列腺癌临床分期的关系。应用原位杂交实验检测lncRNA DNAH8-AS1在前列腺癌和癌旁组织中的表达差异。采用qRT-PCR检测lncRNA DNAH8-AS1在前列腺癌细胞系中的表达水平。采用MTT法和Transwell实验分别检测各组LNCaP细胞增殖和侵袭情况。应用生物信息学技术预测lncRNA DNAH8-AS1与miR-186-5p/YY1之间的靶向关系,并经双荧光素酶报告基因实验验证。结果与癌旁组织相比,前列腺癌组织中lncRNA DNAH8-AS1呈高表达(P<0.01),其与前列腺癌临床分期呈正相关(P<0.05)。与RWPE-1细胞相比,前列腺癌细胞中lncRNA DNAH8-AS1呈高表达(P<0.05)。Control组和si-DNAH8-AS1组LNCaP细胞中lncRNA DNAH8-AS1表达分别为6.44±0.56和1.08±0.37,si-DNAH8-AS1组lncRNA DNAH8-AS1表达比Control组显著降低(P<0.01)。si-DNAH8-AS1组LNCaP细胞的增殖能力,比Control组显著降低(P<0.05)。Control组和si-DNAH8-AS1组侵袭细胞数分别是(63.39±8.03)个和(23.94±3.86)个,si-DNAH8-AS1组LNCaP细胞的侵袭能力比Control组显著降低(P<0.01)。lncRNA DNAH8-AS1可靶向调控miR-186-5p的表达(P<0.01),miR-186-5p可靶向调控YY1的表达(P<0.01)。与Control组相比,si-DNAH8-AS1组LNCaP细胞中miR-186-5p的表达显著减少(P<0.01),YY1基因表达显著增加(P<0.01),Hippo信号通路蛋白表达显著减少(P<0.01)。结论lncRNA DNAH8-AS1在前列腺癌组织和细胞中高表达,与前列腺癌临床分期相关;沉默lncRNA DNAH8-AS1通过靶向miR-186-5p/YY1,有抑制前列腺癌细胞的增殖和侵袭能力。