Study On The Role Of HIF-1? In The Development Of Pelvic Organ Prolapse And Its Related Molecular Mechanism

BackgroundPelvic organ prolapse(POP),the fall of the female pelvic organs(vagina,uterus,bladder,and/or rectum)into or through the vagina,is common and costly condition in elderly women.Pelvic organ prolapse(POP)occurs in approximately 50%of women,and the risk of POP increases with advancing age.Over 200,000 surgeries are performed annually in the United States for POP,costing more than 1 billion dollars a year.Although several risk factors for POP such as advancing age,vaginal childbirth,increasing body-mass index,family history of POP and chronic intra-abdominal pressure are well established,the underlying molecular mechanism remains unknown.Hypoxia triggers the expression of hypoxia-inducible factor-1?(HIF-1?)protein and increases its nuclear translocation.HIF-1?,the master regulator of cellular response to hypoxia microenvironments,has various of downstream target genes,including cell proliferation,angiogenesis,energy metabolism and cell death.HIF-1? upregulates the expression of matrix metalloproteinases,and these proteins are associated with collagen metastasis.HIF-1? also regulates some pro-apoptotic genes,such as BCL2/adenovirus E1B 19 kDa protein-interacting protein 3(BNIP3)and Bcl-2 interacting protein 3-like(NIX),and stabilizes the tumor suppressor p53 to induce apoptosis.Thus,hypoxia may contribute to the pathological process of POP by increasing apoptosis via activating HIF-1?.Thus,we hypothesized that hypoxia may contribute to the pathological process of POP via HIF-1? pathway.Long noncoding RNA(LncRNA)are defined as RNA molecules with over 200 nucleotides in length but lack of protein coding capacity.In recent years,many studies have shown that many lincRNAs may participate in multiple biological and physiological processes,such as cell proliferation,apoptosis,autophagy,immune response and cancer metastasis,by binding to DNA,transcription factors and miRNAs.LncRNA is also widely involved in the physiological and pathological processes of collagen metabolism and degeneration.For example,lncRNA H19 binding with p53 reduces the level of its downstream target gene Bax and play an important role in the hypertrophic scar.The changes in the expression of RP11-296A18.3 may lead to the up-regulation of FAF11 and eventually lead to the excessive apoptosis of the cells in the degenerative pulposus.Therefore,the change of IncRNA expression profiles should be regarded as an important factor affecting the incidence of various diseases.However,the role of lncRNAs in POP are still largely unknown at present.This study is the first to explore the role of HIF-1? and its relationship with fibroblast apoptosis in the development of POP.The mechanism of HIF-1? in inducing apoptosis is also studied.The expression profile of lncRNA in POP is further clarified by second generation sequencing of lncRNA,which lays a foundation for further clarifying the etiology of POP.Part ? Expression and clinical significance of HIF-la in the uterosacral ligament of patients with POPObjective:1.To test the expression of HIF-1? and BNIP3 in the uterosacral ligament of POP patients,and to clarify the correlation between HIF-v and cell apoptosis..2.To explore the correlation between HIF-1? and clinical features,and to study the role of HIF-1? in the occurrence and development of POP.Methods:Immunohistochemical and Western Blot were used to explore HIF-1? and BNIP3 expression,and HIF-1? expression level was analyzed in terms of clinical characteristics.QRT-PCR was used to explore HIF-1? and BNIP3 mRNA expression.TUNEL was used to explore cell apoptosis and the relationship with HIF-1? was analyzed.PFIQ-7 questionnaire was used to follow up the patients after operation,and the relationship between HIF-1? and prognosis was analyzed.Results:1.Clinical characteristics of patients with POP and control groups.The clinical characteristics of patients with POP and control groups were matched in terms of demographic and clinical characteristics.No significant differences were observed in age,body-mass index,parity,or menopausal status between the two groups2.The expression of HIF-la and BNIP3 in USL tissues of POP and control groups.Using immunohistochemical assay,we found that the immunoreactivity of HIF-1? and BNIP3 in USL tissues of the POP group was significantly higher than the control group(P<0.05).Correlation analyses revealed a significant positive correlation between HIF-1? and BNIP3(P<0.001).Similarly,qRT-PCR and Western Blot analysis revealed the expression of HIF-1? and BNIP3 was increased in the level of mRNA and protein,respectively.3.Relationship between the expression of HIF-1? in POP USL tissues and clinical characteristics.We found that the immunoreactivity of HIF-1? in stage ?group was significantly higher than stage ? group(P<0.05),and no significant differences were found between stage ? and stage ? group(P>0.05).In addition,the expression level of HIF-1 a in USL tissues was not correlated with age and menopausal status(P>0.05).4.The level of apoptosis cells in USLs of POP patients and controls.The percentage of apoptosis cells was significantly higher in POP group than in controls(1 6.42%±7.94%vs.7.91%±5.56%,P<0.01),and significant positive correlation was detected between the immunoreactivity of HIF-1 a and the percentage of apoptosis cells(r=0.60,P<0.01).5.The level of apoptosis proteins in USLs of POP patients and controls.The expression of Bax and Bad in USL tissues from the POP group was higher than that in the control group(P<0.05).The Bcl-2/Bax ratio(P<0.01)and Bcl-xl/Bax ratio(P<0.05)were significantly different between two groups.Similarly,Western Blot analysis showed the expression of procaspase-3 and procaspase-9 decreased,and the expression of Cyto-c,cleaved caspase-3,and caspase-9 increased in patients with POP(P<0.05).6.PFIQ-7 scores of postoperative patients and the relationship with HIF-la.The PFIQ-7 presents seven questions with urinary Impact,colorectal-anal Impact and pelvic organ prolapse Impact.The total scores were significantly different between groups,with higher scores in HIF-la positive group(38.49±19.63 vs.17.26± 10.78,P<0.05).Conclusions:1.HIF-1? may induce apoptosis of hUSL cells by up-regulating the expression of BNIP3,which may be involved in the occurrence and development of POP.2.HIF-1? may be associated with POP-Q of patients and life quality.Part II The mechanisms of HIF-1? in the development of POPObjective:To investigate the effects of HIF-1? on human uterosacral ligament fibroblasts(hUSLFs)following treatment with the chemical inducer of hypoxia,cobalt chloride(CoCl2),and to explore the underlying mechanisms.Methods:Ten women who underwent hysterectomy for benign disease provided uterosacral ligament tissue for cell extraction.HUSLFs were exposed to Cobalt Chloride to mimic hypoxic conditions.Following CoCl2 treatment,cell viability of isolated and cultured hUSLFs was evaluated by the MTT assay.Flow cytometry and TUNEL was used to test the effect of different times of Cobalt Chloride on the HUSLFs apoptosis.Western Blot was used to detect the variation of HIF-la and COL1A1 after 48 h of CoCl2 treatment.JC-1 fluorescence mitochondrial imaging was used to study the change in mitochondrial membrane potential.Results:1.HUSLF viability was reduced by treatment with the chemical inducer of hypoxia,cobalt chloride(CoCl2).The growth of human uterosacral ligament fibroblasts(hUSLFs)was observed using an inverted microscope after between 3-5 days following inoculation of the tissue explants and isolated cells in the culture flask.The fibroblasts were seen to become elongated and formed long,spindle,stellate,bipolar,or polygonal shapes.Following passage to the third generation,cell immunofluorescence indicated that more than 95.6±2.4%of the cells were vimentin-positive and cytokeratin-negative,which indicated most of the cells were fibroblasts.To evaluate whether hypoxic conditions were cytotoxic to primary hUSLFs,the fibroblasts were treated with 0,50,100,150,200 and 300 ?M of CoCl2 for 24,48,60,and 72 h,respectively,following which cell viability was examined using the MTT assay.The hUSLF cell viability decreased following CoCl2 treatment in a time-dependent and dose-dependent manner2.Effect of CoCl2 on apoptosis and expression of HIF-la and COL1A1 expression in hUSLFs.Apoptosis of hUSLFs of 24,48,60,and 72h following treatment with CoCl2 was observed,and when hUSLFs were stimulated with CoCl2 at 24 h,no significant difference in the percentage of apoptotic cells was detected using either the TUNEL method or flow cytometry analysis(P>0.05).However,48 h later,a gradual increase in the number of apoptotic cells were found after CoCl2 treatment.A significant increase in the HIF-1? protein level was detected after 48 h of CoCl2 treatment(P<0.01).However,the expression of COL1A1 was significantly decreased by treatment with CoCl2 by 48 h(P<0.05).3.Involvement of the death receptor-associated pathway in CoCl2-treated hUSLFs.When hUSLFs were incubated with 100 ?M of CoCl2for 48 h,increased expression of Fas,death receptor 5(DR5),tumor necrosis factor related apoptosis-inducing ligand(TRAIL),and cleaved caspase-8 were detected by Western Blot(P<0.05).However,cellular FLICE inhibitory protein(c-FLIP)and decoy receptor 2(DcR2)protein expression levels were significantly decreased(P<0.05).The activity of caspase-8 was increased in the hypoxia group and levels of lactate dehydrogenase(LDH)were also increased.Also,when the caspase-8 inhibitor Z-IETD-FMK was added before treatment with CoCl2,the cell viability significantly increased compared to the hUSLFs under the same hypoxic conditions(P<0.05).4.The mitochondrial apoptotic pathway in hUSLFs treated with CoCl2.When hUSLFs were incubated with 100 ?M of CoCl2 for 48 h,increased expression of cytochrome C,Bcl-2 interacting protein 3(BNIP3),cleaved caspase-3,and cleaved caspase-9 were detected by Western Blot(P<0.05).However,Bcl-2/Bax protein expression levels were significantly decreased(P<0.05).The activity of caspase-3 and caspase-9 was increased in the hypoxia group(P<0.05).Also,the cell viability rate was significantly increased when hUSLFs were seeded with CoCl2 and the caspase-9 inhibitor,Z-LEHD-FMK,compared to treatment with Z-LEHD-FMK alone(P<0.05).However,Z-IETD-FMK further increased the cell viability rate when added to each cell group.JC-1 fluorescence mitochondrial imaging was show the rate of breakdown of red to green fluorescence(P<0.05).5.Inhibition of HIF-la down-regulated the apoptosis rate of hUSLFs and normalized the apoptotic related proteins.Following transfection with small-interfering RNA(siRNA),apoptosis of hUSLFs was reduced following HIF-1a silencing by staining with Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI).The expression of apoptotic proteins was evaluated by Western Blot.The protein levels of c-FLIP and DcR2 were increased,whereas the expressions of cleaved caspase-8,DR5,TRAIL,and Fas were reduced(P<0.05).Also,the ratio of Bcl-2/Bax was upregulated,while cytochrome C,BNIP3,and cleaved caspase-3,and cleaved caspase-9 were downregulated in hUSLFs following HIF-1? silencing(P<0.05).Therefore,apoptosis was induced by hypoxia of hUSLFs through the death receptor-associated pathway and the mitochondrial apoptosis-associated pathway.Conclusions:1.Hypoxia lead to apoptosis of hUSLFs increased and expression of COL1A1 decreased.2.Apoptosis was induced by HIF-la through the death receptor-associated pathway and the mitochondrial apoptosis-associated pathway in POP.Part III The screening and verification of differentially expressed long non-coding RNAs in pelvic organ prolapse based on RNA-SeqObjective:1.To screen the differentially expressed lncRNAs in the uterosacral ligaments based on RNA-Seq.2.To verify the results of RNA-Seq by qRT-PCR..Methods:5 pairs of the uterosacral ligament tissues from POP and control groups were collected.We surveyed the lncRNAs and mRNAs differentially expressed by RNA-Seq and bioinformatics methods.LncRNA-mRNA co-expression network based on differentially expressed lncRNAs and mRNAs was built to identify interactions between genes and lncRNAs with critical role in POP.The functional annotation and enrichment analysis using GO and KEGG showed that the differentially expressed lncRNAs were enriched in the biological processes and signaling pathways involved in POP.5 lncRNAs were further identified and validated by real-time PCR in 48 uterosacral ligament tissues except the 5 pairs for RNA-Seq.Results:1.LncRNAs identification.Excluding the low-quality and adaptor sequences,there are 1,006,596,942 clean reads accounting for 151.01 Gb to be analyzed.Subsequently,five steps were used to screen the transcripts for identify the confidently expressed lncRNAs.Based on the specific structure characteristics of lncRNAs,the final results yielded 289 novel lncRNAs(Fig.1 A)including 256(88.6%)lncRNAs and 33(11.4%)antisense lncRNAs.2.Differential expression and clustering analyse of lncRNAs and mRNAs.In patients with POP,a total of 41 lncRNAs(21 up-regulated and 20 down-regulated)and 808 mRNAs(548 up-regulated and 260 down-regulated)were aberrantly expressed compared with the controls.3.The results of lncRNA biological information analysis.GO enrichment analysis showed that many important items were obtained,including:embryonic skeletal system development,DNA polymerase complex,molecular function,central nervous system neuron differentiation,embryo development,biosynthetic process,cellular biosynthetic process,protein binding,cell cycle and so on.KEGG pathway analysis showed that many important pathways were obtained,including:base excision repair,Galactose metabolism,Purine metabolism,DNA replication,TGF-beta signaling pathway,Metabolic pathways and so on.4.The results of mRNA biological information analysis.GO enrichment analysis showed that many important items were obtained,including:developmental process,extracellular matrix,organ development,tissue development,extracellular space,cell differentiation,cell proliferation,cell adhesion,vasculature development,cardiovascular system development and so on.KEGG pathway analysis showed that many important pathways were obtained,including:Cell adhesion molecules(CAMs),Protein digestion and absorption,ECM-receptor interaction,Protein export,p53 signaling pathway,TGF-beta signaling pathway,Wnt signaling pathway and so on.5.Verification test of sequencing results.To confirm the sequencing and bioinformatics results,3 up-regulated and 2 down-regulated lncRNA transcripts(LINC01291,LINC01605,LINC00968,MIR4458HG.,RP11-219A1 5.4)were chosen for qRT-PCR.Consistent with sequencing results,all 5 lncRNA transcripts were found to be differentially expressed in the hUSL samples with the same trends.6.The network of lncRNAs and targeted mRNAs.To further indicate the interaction and correlation between lncRNAs and mRNAs in hUSLs,a network based on differentially expressed IncRNAs and mRNAs was constructed.The network consisted of dysregulated lncRNAs and their targeted mRNAs.A total of 8 lncRNAs related to HIF-1? were found in the co-expression network,including 3 down-regulated and 5 up-regulated.Conclusions:1.There were a large number of differentially expressed lncRNA and mRNA in the uterosacral ligament tissues of POP patients through RNA-Seq.2.To construct a co-expression network of lncRNA-mRNA and explore the relationship between the expression of lncRNA in POP and the occurrence,development and prognosis of POP.