lncRNA and mRNA sequencing of the left testis in experimental varicocele rats treated with Morinda officinalis polysaccharide
- Authors:
- Published online on: August 6, 2021 https://doi.org/10.3892/etm.2021.10570
- Article Number: 1136
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Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Varicocele, an abnormal varix of the pampiniform plexus vein, is the most common cause of male infertility. The global incidence of varicocele is 15-20% in the general population and ~40% in patients who are infertile (1-4); this may have a negative impact on human evolution. For spermatogenesis to occur, the testis must descend into the scrotum during the embryonic period to provide proper the conditions of a temperature ~2˚C lower than the central body temperature (5). The upright human posture requires spermatic veins to work against gravity to return deoxygenated blood back to the heart (6). If the valves inside these veins fail, gravity can make the blood pool inside the testicle, eventually leading to enlargement of the veins and formation of a varicocele (7). In ~90% of cases, the disease occurs on the left side, due to the longer left testicular vein, hemodynamics and higher incidence of abnormal venous valves (8,9). Effective therapies for varicocele have yet to be determined (8,10-12). The experimental rat varicocele model, first established in 1981(13) by partly ligating the left renal vein, is widely used to investigate the pathophysiology, diagnosis and treatment of the disease. Researchers have shown that the following mechanisms contribute to the pathogenesis of varicocele: i) Neuroendocrine system dysfunction; ii) hypoxia; iii) accumulation of metabolites and toxicants; iv) oxidative stress; v) disruption of the blood-testis barrier (BTB); and vi) cell damage resulting from increased testicular temperature (14,15).
In China, Morinda officinalis F.C.How grows in the Guangdong, Guangxi and Fujiang provinces. The roots of M. officinalis are used in a Chinese herbal medicine known as Bajitian; it has a long history of use for the improvement of male sexual function and the treatment of male reproductive system defects in China (16). M. officinalis polysaccharide (MOP) is one of the main active components of M. officinalis. Our previous research found that MOP can repair varicocele-induced damage to the male rat reproductive system by promoting spermatogenesis, reconstructing the BTB, increasing the expression of tight junction (TJ) proteins and restoring hormonal balance, with 300 mg/kg being the most effective dosage (17). However, the molecular mechanisms underlying the pathophysiology of varicocele and the physiological functions and therapeutic effects of MOP in varicocele are yet to be explored in detail.
Advances in high-throughput RNA sequencing (RNA-Seq) technology and bioinformatics analysis methods have enabled researchers to gain insight into the nature of diseases at the RNA level by studying the dynamics of mRNA, microRNA and long non-coding RNA (lncRNA) (18) expression levels. According to previous research, lncRNAs, which are >200 nucleotides long and found in multiple organisms (19), play notable roles at almost every step of gene expression and take part in various disease processes (20); for example, in Parkinson's disease (21), leukemia (22), diabetes (23), cardiovascular disease (24), colon cancer (25,26), lung cancer (27) and prostate cancer (28). Due to their diverse bioactivities, lncRNAs are regarded as potential targets for the diagnosis and treatment of varicocele.
In the present study, rats with surgically induced varicocele were treated with either saline or 300 mg/kg MOP by gavage. Identification of differentially expressed (DE) mRNAs and lncRNAs in rat left testicular tissue was conducted by RNA-Seq, and the results were verified by reverse transcription-quantitative PCR (RT-qPCR). Bioinformatics resources, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and co-expression network analysis, were utilized to explore the mechanisms underlying varicocele pathophysiology and the therapeutic effect of MOP, in addition to the interactions between DE mRNAs and lncRNAs in varicocele. The aim of the current study was to identify novel targets and methods for varicocele diagnosis and therapy, and establish a theoretical basis for the use of MOP in clinical practice.
Materials and methods
Extraction of MOP
The plant M. officinalis used in the present study was grown (via artificial cultivation) in the Nanjing county of Fujian province in China. MOP was extracted from dried root according to a previously described protocol (17,29,30). In brief, after being ground, the dried root was boiled in ethyl alcohol to remove oligosaccharides and lipids, extracted using deionized water and precipitated with 60% alcohol; thereafter, Sevag reagent (Sinopharm Chemical Reagent Co., Ltd) was added to remove the protein.
Experimental design
Male, 6- to 7-week-old Sprague-Dawley rats, weighing 200±10 g, were purchased from and kept in The Laboratory Animal Center of Fujian Medical University (Fuzhou, China). All rats were housed in normal atmosphere (N2, 78%; O2, 21%; CO2, 0.03%) and specific pathogen-free controlled environmental conditions, with a temperature of ~23˚C, a 12-h day/night cycle, a humidity of 40-70% and free access to standard rat food and water. All procedures conformed to the Guidelines for the Care and Use of Laboratory Animals established by Fujian Medical University. The study was approved by The Animal Approval Committee of Fujian Medical University (approval no. SYXK-2012-0001).
A total of 24 rats were randomly divided into three equal groups: Control group, varicocele non-treatment group (VC) and varicocele treatment group (VC + MOP). Induction of varicocele was attempted by partial ligation of the left renal vein under anesthesia using 30 mg/kg sodium pentobarbital by intraperitoneal injection (31). Control rats underwent the same operation without the partial ligation, and were fed a conventional diet for 12 weeks. Subsequently, 8 weeks post-surgery, rats in the VC and VC+MOP groups were given a daily oral gavage of 2 ml normal saline or 300 mg/kg MOP, respectively, for 4 weeks; rats in the Control group were given nothing except conventional diet. The rats (weight, 500±25 g) were sacrificed by cervical dislocation after being anesthetized using 30 mg/kg sodium pentobarbital by intraperitoneal injection to collect left testicular tissue, which was immediately placed in liquid nitrogen. Varicocele rats in which vascular dilation did not occur or left kidney atrophy occurred were excluded from the follow-up experiment.
mRNA and lncRNA sequencing analysis
RNA-Seq analysis was performed by Kangchen BioTech Co., Ltd. The process used was divided into three parts: i) Extraction of total RNA from rat left testicular tissue; ii) construction of a cDNA library; and iii) RNA sequencing.
In brief, total RNAs were extracted using TRIzol® Reagent (Takara Bio Europe SAS) according to the manufacturer's instructions, followed by purification by Ribo-Zero™ Magnetic Gold Kit (Human/Mouse/Rat) (cat. no. MRZG12324; Epicentre, Illumina, Inc.). The quantity, integrity and concentration of the extracted RNAs were verified by 1% agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometry (Thermo Fisher Scientific, Inc.). For library preparation, the KAPA Stranded RNA-Seq Library Prep kit (cat. no. KK8401; Illumina, Inc.) was used (paired-ended sequencing; 500 bp), with 1-2 µg of sample RNA. The established libraries were examined using an Agilent 2100 Bioanalyzer G2938C (Agilent Technologies, Inc.) and quantified by RT-qPCR. The samples were denatured to single-stranded DNA with 0.1 M NaOH (final density, 8 pM; concentrations measured by RT-qPCR) and amplified using TruSeqSR Cluster Kit v3-cBot-HS (cat. no. GD-401-3001; Illumina, Inc.). The libraries were pooled and sequenced on the Illumina HiSeq 4000 system (Illumina, Inc.) using 150 cycles of paired-end sequencing.
The quality of the raw sequencing data was assessed using FastQC software v0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), before the adapter sequences and poor quality bases were trimmed from the reads using the Cutadapt software v1.17 (http://dx.doi.org/10.14806/ej.17.1.200) (32). The Hisat2 software v2.0.4 (http://ccb.jhu.edu/software/hisat2) and StringTie software v1.2.2 (http://ccb.jhu.edu/software/stringtie) (33) were used to calculate fragments per kilobase of transcript per million mapped reads (FPKM) values, which represented the final expression levels of the genes. Differential gene expression level analyses were performed using the Ballgown package in R v2.10.0 (https://www.r-project.org) (32). An average FPKM value >0.5 was chosen as the cut-off for gene expression in the samples.
RT-qPCR
To confirm the results of RNA-Seq, specific DE patterns were validated by RT-qPCR, with three biological replicates for each group. TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract the total RNAs from rat testicular tissue, according to the manufacturer's instructions. cDNA was synthesized using 3 µg total RNAs, SuperScript III reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.), 5X reverse transcription buffer solution (Invitrogen; Thermo Fisher Scientific, Inc.), 2.5 mM dNTPs (HyTest, Ltd), primers (Invitrogen; Thermo Fisher Scientific, Inc.), and the GeneAmp PCR 9700 System (50˚C for 60 min, 70˚C for 15 min and stored at 4˚C for infinite time; Applied Biosystems; Thermo Fisher Scientific, Inc.). The RT-qPCR analysis was performed using the SYBR®-Green Real-time PCR Master mix (Arraystar, Inc.) and ViiA™ 7 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), with three technical replicates set for each sample. The thermocycling conditions were as follows: 95˚C for 10 min; 95˚C for 10 sec, then 60˚C for 1 min (40 cycles); 95˚C for 10 sec; 60˚C for 1 min; and 95˚C for 15 sec. The primers were designed and purchased from Kangchen BioTech Co., Ltd. The primer sequences are shown in Table I. The relative expression of each gene was quantified by comparing its Cq value (2-ΔΔCq method) with that of the housekeeping gene, β-actin (34).
Statistical analyses
RNA-Seq and RT-qPCR were performed in three independent biological replicates for each group. Only genes with an absolute fold-change >1.2 and P-value ≤0.05 were considered to be differentially expressed between the groups. The data were analyzed in SPSS v21.0 (IBM Corp.), and the results are presented as the mean ± standard deviation. Pearson's correlation was used in scatter plot of DE genes. One-way ANOVA was used to compare gene expression levels between two groups, and P≤0.05 was considered to indicate a statistically significant difference.
Bioinformatic analyses
GO classification and KEGG pathway enrichment analyses were performed using the website of Gene Ontology Resource (http://www.geneontology.org) and KEGG pathway database (https://www.kegg.jp/kegg/pathway.html) respectively. The co-expression of DE mRNA (coding genes) and lncRNA (non-coding genes) networks were visualized using Cytoscape v2.8.3 (http://cytoscape.org). The present study data were uploaded to the Gene Expression Omnibus (GEO) website (https://www.ncbi.nlm.nih.gov/geo/; accession no. GSE139447; secure token: sfaxocskhpoxnix).
Results
Identification of DE mRNAs and lncRNAs
The present study data were uploaded to the Gene Expression Omnibus website. The top 10 upregulated and downregulated DE genes (including mRNAs and lncRNAs) are listed in Tables II and III, respectively. Hierarchical cluster (Fig. 1), scatter plot (Fig. 2) and volcano plot (Fig. 3) analyses of the DE genes are presented. Overall, there were 144 DE mRNAs and 63 DE lncRNAs in the VC group vs. the Control group, 63 DE mRNAs and 148 DE lncRNAs in the VC + MOP group vs. the VC group and 173 DE mRNAs and 54 DE lncRNAs in the VC + MOP group vs. the Control group. The most significantly upregulated DE genes in VC group vs. the Control group, VC + MOP group vs. the VC group, and VC + MOP group vs. the Control group were TMEM255B, actin g 2 smooth muscle and calsyntenin 2, respectively. The most significantly downregulated DE genes in VC group vs. the Control group, VC + MOP group vs. the VC group, and VC + MOP group vs. the Control group were AABR07050146.1, YIPF7 and ATP-dependent helicase ATRX chromatin remodeler, respectively.
 | Table IITop 10 upregulated differentially expressed genes between groups by RNA-sequencing analysis. |
| Table IIITotal of 10 downregulated differentially expressed genes between groups by RNA-sequencing analysis. |
GO and KEGG analyses of DE genes
To elucidate the functions of the DE genes, GO term enrichment and KEGG pathway analyses were performed. The GO functional annotations were classified into three categories: Molecular function, cellular components and biological processes. The top 10 enriched GO terms of DE genes, both upregulated and downregulated (Figs. 4 and 5) and the top 10 enriched KEGG pathways of DE genes, both upregulated and downregulated, (Fig. 6) are shown.
Co-expression network of the chosen DE mRNAs and lncRNAs
Correlation coefficients of selected DE mRNAs (protein-coding genes) and lncRNAs (non-protein-coding genes) were calculated to build a co-expression network (Pearson correlation coefficient ≥0.8; P≤0.05; false discovery rate, ≤1; Fig. 7). These DE mRNAs and lncRNAs presented in Fig. 7 were selected according to their function based on the GO term enrichment result, but not the total DE genes. The enriched DE genes were selected based on the GO terms the present study were interested in, such as the ‘response to steroid hormone’, ‘oxidation-reduction process’, ‘positive regulation of MAPK cascade’ and ‘DNA repair’ terms.
Validation of DE mRNAs and lncRNAs through RT-qPCR
The DE mRNAs and lncRNAs identified by RNA-Seq were validated using RT-qPCR. The results are presented in Table IV and Fig. 8. There were six DE mRNAs and four DE lncRNAs in the VC group vs. the Control group, one DE mRNA and four DE lncRNAs in the VC + MOP group vs. the VC group and four DE mRNAs and two DE lncRNAs in the VC + MOP group vs. the Control group. Varicocele and MOP treatment appeared to have opposing effects on the expression levels of YIPF7, AABR07007833.1, AABR07014649.1 and AABR07069067.1. To be specific, the varicocele increased the expression levels of YIPF7, AABR07007833.1 and AABR07014649.1, and decreased the expression level of AABR07069067.1, while MOP treatment reversed these changes.
Discussion
In our previous study, varicocele induced reproductive dysfunction in male rats via mechanisms such as destruction of the seminiferous epithelium and TJ structure, downregulation of TJ proteins (occludin, claudin-11 and zona occludens protein 1), deregulation of hormone levels and an increase in cytokine (TGF-β3 and TNF-α) levels. MOP repaired varicocele-induced damage and promoted spermatogenesis (the most effective dose was found to be 300 mg/kg) (17). However, the molecular mechanisms underlying the pathophysiology of varicocele and the therapeutic effect of MOP are still unknown. In the present study, mRNA and lncRNA sequencing analyses, combined with validation of DE genes through RT-qPCR, were performed to bridge these data gaps.
According to GO results, the pathophysiology of varicocele may be associated with ‘epidermal growth factor receptor binding’, ‘ligand-gated calcium channel activity’, ‘growth factor receptor binding’, ‘histone kinase activity’ and ‘microtubule motor activity’. Notably, numerous DE genes between the VC and control groups were enriched in cancer-related pathways, such as the ‘p53 signaling pathway’, which is downregulated in breast cancer and melanoma; this warrants further research and validation.
According to the KEGG pathway analysis, ‘cytokine-cytokine receptor interaction’, ‘Wnt signaling pathway’ and ‘p53 signaling pathway’ were all implicated in the varicocele-therapeutic effect of MOP. In our previous study, the levels of TGF-β3 and TNF-α were upregulated in experimental rat left testicular tissue and downregulated following MOP treatment, potentially through the cytokine-cytokine receptor interaction pathway (17). The Wnt signaling pathway plays a notable role in the normal physiology and pathology of the male reproductive system. The Wnt/β-catenin pathway is involved in the annexin 5-mediated stimulation of testosterone synthesis (35), and its dysregulation is linked to the development of granulosa cell tumors in the testis (36). Additionally, the activation of Wnt/β-catenin signaling in Sertoli cells results in germ cell loss and seminiferous tubule degeneration (37). Moreover, the Wnt/β-catenin signaling pathway facilitates the proliferation of spermatogonial stem cells in the testis (38). Thus, proper regulation of Wnt/β-catenin signaling is necessary for adult spermatogenesis, and its disruption may result in infertility (39). The results of the present study provide compelling evidence for the involvement of Wnt/β-catenin signaling in varicocele progression and the therapeutic effect of MOP in varicocele; this evidence is worthy of further research.
The RT-qPCR analysis revealed that one coding gene, YIPF7, and three non-coding genes, AABR07007833.1, AABR07014649.1 and AABR07069067.1, were DE between the VC and Control groups, and between the VC + MOP and VC groups. Varicocele and MOP treatment appeared to have opposing effects on the expression of these genes, implying that these genes may play a role in varicocele pathophysiology and the repair effect of MOP. Specifically, varicocele increased the expression levels of YIPF7, AABR07007833.1 and AABR07014649.1, and decreased the expression level of AABR07069067.1, while MOP treatment reversed these effects. The YIP1 family of proteins are involved in protein transport between the endoplasmic reticulum and Golgi apparatus (40), as well as the regulation of membrane dynamics (41). Decreasing the expression of YIPF7 enhances the intestinal inflammatory response and upregulates the TNF mRNA level (42). The change in YIPF7 expression may be associated with the testicular inflammatory response and the fluctuation of the TNF-α expression level induced by varicocele and MOP. This finding is in accordance with the results of our previous study; thus, we hypothesize that YIPF7 is involved in the process of varicocele development by increasing the TNF-α level in the left testicular tissue, which MOP treatment is able to decrease (17). Due to the lack of extensive research on lncRNA, the functions of AABR07007833.1, AABR07014649.1 and AABR07069067.1 remain elusive. In the present study, according to the co-expression network analysis, these non-coding genes were positively associated with phosphatidylinositol glycan anchor biosynthesis class C insulin-like growth factor 1 (IGF1), P2RX4, TMEM255B and WNT9B, as well as others.
Aldo-keto reductase family 1, member B8 (AKR1B8), leukocyte immunoglobulin-like receptor B2 (NILR1), P2RX4, TMEM255B and WNT9B were also DE between the VC and control groups. TMEM255B, also known as FAM70B, encodes the transmembrane protein 255b, which has been proposed as a prognostic marker for muscle-invasive bladder cancer (43). The expression level of TMEM255B in the VC group was ~63-fold higher compared with that of the control group. Such a large difference in expression levels suggests that TMEM255B could be used as a novel diagnostic maker for varicocele; however, further clinical research is required to confirm this hypothesis. WNT9B takes part in the Wnt/β-catenin signaling pathway, described as aforementioned. P2RX4, also known as P2X4 or P2X4R, encodes the ATP-gated P2RX4 ion channel. According to a previous study, extracellular ATP is a danger molecule for peritubular cells and aggravates inflammatory responses in the testis (44). P2RX4 is involved in pain processing (45-47), and its downregulation has been shown to moderate allergen-induced airway inflammation (48). Although the function of P2RX4 in the male reproductive system is unclear, according to its diverse biological activities under physiological and pathological conditions (44-49), it is hypothesized that changes in P2RX4 expression levels are involved in varicocele pathogenesis via the stimulation of inflammatory responses and disruption of transmembrane ion channel function.
In the present study, RNA sequencing and RT-qPCR were performed on rat testicular tissue to reveal the molecular mechanisms underlying varicocele pathophysiology and the restorative effect of MOP on varicocele-induced damage in male reproductive systems. The results of GO and KEGG pathway enrichment analyses showed that ‘ligand-gated calcium channel activity’, ‘cytokine-cytokine receptor interaction’ and the ‘Wnt signaling pathway’ may all be implicated to underlie this effect. RT-qPCR confirmed that YIPF7 is upregulated in varicocele and downregulated by MOP. Based on its diverse biological activities and intimate connection to TNF-α, YIPF7 is considered a key mediator of varicocele pathogenesis and therapeutic effects of MOP. Furthermore, differential expression of AKR1B8, NILR1, P2RX4, TMEM255B and WNT9B was detected between the VC group and the control group. TMEM255B may be a potential novel diagnostic marker for varicocele; the role of WNT9B and P2RX4 in varicocele is possibly mediated by the activation of Wnt signaling and the regulation of transmembrane ion channels and the inflammatory response, respectively.
In summary, the present study provides a foundation for understanding the molecular basis of varicocele pathophysiology and the varicocele-therapeutic effect of MOP, offers insights into novel strategies for varicocele diagnosis and treatment, and suggests directions for further study.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by The Training Program Foundation for the Talents of Health and Family Planning Commission in Fujian Province (grant no. 2018-1-74), The Start-up Foundation of Fujian Medical University (grant no. 2017XQ1005), The Research Foundation for High-Level Talents of Fujian Medical University (grant. no. XRCZX2017033), The Natural Science Foundation of Fujian Province (grant no. 2017J01819) and The Co-construction Science Foundation of National Health and Family Planning Commission-Joint Program for Tackling Key Problems of Health and Education in Fujian Province (grant no. WKJ2016-2-35).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request. The datasets generated and analyzed during the current study are available in the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi (accession no. GSE139447; secure token: sfaxocskhpoxnix).
Authors' contributions
LZ performed the experiments and the statistical analysis, and drafted the manuscript. WW and XZ conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by The Animal Approval Committee of Fujian Medical University (approval no. SYXK-2012-0001).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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