SPAG6 c.900 T>C affects boar semen quality and blood-testis barrier function by creating a new splice acceptor site.

Sperm associated antigen 6 (SPAG6) acts as a scaffolding protein in the center of the flagellar axoneme and has an impact on the maturation of the motility of mammalian sperm flagella and the maintenance of sperm structure. In our previous research, SPAG6 c.900 T>C in exon 7 and exon 7 skipped transcript was identified by analyzing RNA-seq data of testicular tissues from 60 day (sexually immature) and 180 day (sexually mature) Large White boars. Herein, we found porcine SPAG6 c.900 T>C to be associated with semen quality traits in Duroc, Large White and Landrace pigs. SPAG6 c.900 C can generate a new splice acceptor site, inhibit the occurrence of SPAG6 exon 7 skipping to a certain extent, thereby promote the growth of Sertoli cells and maintain the normal blood-testis barrier function. This study provides new insights into the molecular regulation of spermatogenesis and a new genetic marker for the improvement of semen quality in pigs.

Methyltransferase like 7B is a potential therapeutic target for reversing EGFR-TKIs resistance in lung adenocarcinoma.

BACKGROUND: Identification of potential novel targets for reversing resistance to Epidermal Growth Factor Receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) holds great promise for the treatment of relapsed lung adenocarcinoma (LUAD). In the present study, we aim to investigate the role of methyltransferase-like 7B (METTL7B) in inducing EGFR-TKIs resistance in LUAD and whether it could be a therapeutic target for reversing the resistance. METHODS: METTL7B-overexpressed LUAD cell lines, gefitinib and osimertinib-resistant Cell-Derived tumor Xenograft (CDX) and Patient-Derived tumor Xenograft (PDX) mouse models were employed to evaluate the role of METTL7B in TKIs resistance. Ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) was used to identify the metabolites regulated by METTL7B. Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis was performed to measure the N6-methyladenosine (m6A) status of mRNA of METTL7B targeted genes. Gold nanocluster-assisted delivery of siRNA targeting METTL7B (GNC-siMETTL7B) was applied to evaluate the effect of METTL7B in TKIs resistance. RESULTS: Increased expression of METTL7B was found in TKIs-resistant LUAD cells and overexpression of METTL7B in LUAD cells induced TKIs resistance both in vitro and in vivo. Activated ROS-metabolism was identified in METTL7B-overexpressed LUAD cells, accompanied with upregulated protein level of GPX4, HMOX1 and SOD1 and their enzymatic activities. Globally elevated m6A levels were found in METTL7B-overexpressed LUAD cells, which was reduced by knock-down of METTL7B. METTL7B induced m6A modification of GPX4, HMOX1 and SOD1 mRNA. Knock-down of METTL7B by siRNA re-sensitized LUAD cells to gefitinib and osimertinib both in vitro and in vivo. CONCLUSIONS: This study uncovered a new critical link in METTL7B, glutathione metabolism and drug resistance. Our findings demonstrated that METTL7B inhibitors could be used for reversing TKIs resistance in LUAD patients.

Oxidative stress in oocyte aging and female reproduction.

In a healthy body, reactive oxygen species (ROS) and antioxidants remain balanced. When the balance is broken toward an overabundance of ROS, oxidative stress appears and may lead to oocyte aging. Oocyte aging is mainly reflected as the gradual decrease of oocyte quantity and quality. Here, we aim to review the relationship between oxidative stress and oocyte aging. First, we introduced that the defective mitochondria, the age-related ovarian aging, the repeated ovulation, and the high-oxygen environment were the ovarian sources of ROS in vivo and in vitro. And we also introduced other sources of ROS accumulation in ovaries, such as overweight and unhealthy lifestyles. Then, we figured that oxidative stress may act as the "initiator" for oocyte aging and reproductive pathology, which specifically causes follicular abnormally atresia, abnormal meiosis, lower fertilization rate, delayed embryonic development, and reproductive disease, including polycystic ovary syndrome and ovary endometriosis cyst. Finally, we discussed current strategies for delaying oocyte aging. We introduced three autophagy antioxidant pathways like Beclin-VPS34-Atg14, adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR), and p62-Keap1-Nrf2. And we also describe the different antioxidants used to combat oocyte aging. In addition, the hypoxic (5% O2 ) culture environment for oocytes avoiding oxidative stress in vitro. So, this review not only contribute to our general understanding of oxidative stress and oocyte aging but also lay the foundations for the therapies to treat premature ovarian failure and oocyte aging in women.

Methyltransferase-like protein 11A promotes migration of cervical cancer cells via up-regulating ELK3.

Cervical cancer is one of the common malignancies in women, which is characterized with high invasion and metastatic tendency in its advanced stage. Increasing evidence indicates that methyltransferase-like (METTL) gene family is involved in the progression of various cancers. However, the functional role of methyltransferase-like gene family in cervical cancer remains unclear. In the present study, we found that METTL11A, a member of the methyltransferase-like gene family, was significantly over-expressed in cervical carcinoma by analyzing TCGA database. This finding was further validated in clinical tissue samples. Moreover, ectopic expression of METTL11A in cervical cancer cell lines promoted cell proliferation and migration both in vitro and in vivo. Differential gene expression analysis in the transcriptomic sequencing data indicated that ELK3 was down-regulated in METTL11A-silenced cervical cancer cells, which was further verified at both protein and mRNA levels. Functional experiments identified that METTL11A promoted migration of cervical cancer cells in an ELK3-dependent manner. This study will promote understanding the mechanism of cervical cancer progression and the functional role of methyltransferase-like gene families in cancers.

H2AFZ, RNF4 and NR4A1 loci are associated with boar semen quality by population association studies.

Spermatogenesis is a complex process regulated by many genes. In this study, H2AFZ, RNF4 and NR4A1 genes were selected as candidate genes for boar semen quality traits based on their functions during spermatogenesis, and the associations of three loci (H2AFZ c.192 + 210-192 + 213delCGAT, RNF4 c.374 + 358 T > C and NR4A1 c.956 + 796 A > G) with sperm quality traits were analyzed in Duroc (n = 185), Large White (n = 87) and Landrace (n = 49) pig populations. The results showed H2AFZ c.192 + 210-192 + 213delCGAT AA boars produced 1.52% lower abnormal sperm rate (ASR) than AB boars in Landrace pigs (p < 0.05); RNF4 c.374 + 358 TC boars produced 0.31 × 108/ml higher sperm concentration (SCON) than CC boars (p < 0.05) in Large White pigs; NR4A1 c.956 + 796 A > G was associated with ASR in Duroc and Large White pigs and was associated with sperm motility (MOT) in Large White and Landrace pigs. This study indicated the H2AFZ, RNF4 and NR4A1 loci were the potential molecular markers for improving the semen quality traits in boars.

A novel biomarker NIFK-AS1 promotes hepatocellular carcinoma cell cycle progression through interaction with SRSF10.

Background: Hepatocellular carcinoma (HCC) is one of the most common carcinomas all over the world, with high mortality and low survival rate. Notably, many studies have showed that a variety of molecules play vital roles in the progression of HCC. Therefore, it is urgent to find reliable biomarkers to diagnose HCC and developing novel strategies are required for the effective treatment of patients with HCC. Methods: This study obtained an HCC cohort from The Cancer Genome Atlas (TCGA). For prognostic analysis, the TCGA cohort was grouped according to different median boundaries. The key module associated with HCC was adopted by Weighted Gene Co-expression Network analysis (WGCNA). We also analyzed the survival ability, functional enrichment, and potential binding proteins of key lncRNAs. The expression of hub lncRNAs in HCC tissues and cell lines was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8) and flow cytometry were applied to detect the cell proliferation, apoptosis, and cell cycle. The interaction between NIFK-AS1 and SRSF1 was examined using an RNA pull-down assay. Results: The green module is the key module in HCC. NIFK-AS1 was highly expressed in HCC tissues and correlated with a poor prognosis in HCC patients (P=0.008). NIFK-AS1 was also significantly associated with cell mitosis, the cell cycle, and other biological processes. NIFK-AS1 deletion prevented cell proliferation, induced apoptosis, caused G2/M arrest, and affected cell cycle progression. RNA pull-down validated the NIFK-AS1/SRSF10 interaction. The overexpression of NIFK-AS1 was sufficient to rescue the growth of SRSF10 knockdown HepG2 cells. Conclusions: This study suggested that NIFK-AS1 promotes HCC cell cycle progression through interaction with SRSF10 and its findings provide new insights into therapeutic targets for HCC.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

OBJECTIVES: BCL2-associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24-skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis. MATERIALS AND METHODS: CRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high-testosterone mice model. H&E staining, qRT-PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis. RESULTS: Bag6 exon24 knockout mice show sub-fertility along with partially impaired blood-testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24-deficient testes and sterol regulatory element-binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up-regulated, which causes a high serum testosterone level. Additionally, serine/arginine-rich splicing factor 1 down-regulates BAG6 exon24-skipped transcripts in porcine Sertoli cells by binding to 35-51 nt on BAG6 exon24 via its N-terminal RNA-recognition domain. CONCLUSIONS: Our findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

The Function of Pre-mRNA Alternative Splicing in Mammal Spermatogenesis.

Alternative pre-mRNA splicing plays important roles in co-transcriptional and post-transcriptional regulation of gene expression functioned during many developmental processes, such as spermatogenesis. The studies focusing on alternative splicing on spermatogenesis supported the notion that the development of testis is regulated by a higher level of alternative splicing than other tissues. Here, we aim to review the mechanisms underlying alternative splicing, particularly the splicing variants functioned in the process of spermatogenesis and the male infertility. There are five points regarding the alternative splicing including ⅰ) a brief introduction of alternative pre-mRNA splicing; ⅱ) the alternative splicing events in spermatogenesis-associated genes enriched in different stages of spermatogenesis; ⅲ) the mechanisms of alternative splicing regulation, such as splicing factors and m6A demethylation; ⅳ) the splice site recognition and alternative splicing, including the production and degradation of abnormal transcripts caused by gene variations and nonsense-mediated mRNA decay, respectively; ⅴ) abnormal alternative splicing correlated with male infertility. Taking together, this review highlights the impacts of alternative splicing and splicing variants in mammal spermatogenesis and provides new insights of the potential application of the alternative splicing into the therapy of male infertility.

Epitranscriptomics and epiproteomics in cancer drug resistance: therapeutic implications.

Drug resistance is a major hurdle in cancer treatment and a key cause of poor prognosis. Epitranscriptomics and epiproteomics are crucial in cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In recent years, epitranscriptomic and epiproteomic modification has been investigated on their roles in overcoming drug resistance. In this review article, we summarized the recent progress in overcoming cancer drug resistance in three novel aspects: (i) mRNA modification, which includes alternative splicing, A-to-I modification and mRNA methylation; (ii) noncoding RNAs modification, which involves miRNAs, lncRNAs, and circRNAs; and (iii) posttranslational modification on molecules encompasses drug inactivation/efflux, drug target modifications, DNA damage repair, cell death resistance, EMT, and metastasis. In addition, we discussed the therapeutic implications of targeting some classical chemotherapeutic drugs such as cisplatin, 5-fluorouridine, and gefitinib via these modifications. Taken together, this review highlights the importance of epitranscriptomic and epiproteomic modification in cancer drug resistance and provides new insights on potential therapeutic targets to reverse cancer drug resistance.

Possible involvement of HSP70 in pancreatic cancer cell proliferation after heat exposure and impact on RFA postoperative patient prognosis.

PURPOSE: As an alleviative treatment measured in patients with unresectable advanced pancreatic cancer, radiofrequency ablation (RFA) needed more clinical data to prove its advantages and to explore limitations in its utilization. This study was determined to observe the efficacy of RFA, and to explore its impact on perioperative periphery carcinoma as well as the normal pancreatic tissues. METHODS: Clinical data of 32 patients with pancreatic cancer accepted RFA surgery were collected. Followed up patients' pain degree and the changes in serum tumor markers CA19-9 and CA 242 before and after surgery. Ex vivo, gave human pancreatic cancer cell line PANC-1 heat treatment to simulate the heat exposure condition periphery carcinoma was experienced during RFA surgery, and to observe the proliferation rate and HSP70 expression change compared with control group. RESULTS: Of the 32 patients, 1 died of upper gastrointestinal hemorrhage, and 29 survived for more than 5 months, 2 of which for more than 16 months. The average CA19-9 and CA 242 levels of the patients were significantly decreased in 3 months after surgery (t = 9.873, 5.978, P < 0.001). During in vitro experiments, the proliferation rate of PANC-1 cells after heating was significantly increased, accompanied with the increased HSP70 expression. The addition of HSP70 inhibitor can inhibit the rise of proliferation after heat therapy. CONCLUSION: Utilizing RFA treat patients with unresectable advanced pancreatic cancer, could effectively relieve the pain, decline jaundice, and deduce tumor marker levels significantly. However, it failed to extend the long-term survival rate of the patients significantly. This study found that a higher proliferative rate accompanied with a higher HSP70 expression level were observed on in vitro cultured pancreatic carcinoma cells after heat treatment, which could be altered by HSP70 inhibitor. And these findings indicated that the heat exposure might impact periphery carcinoma during RFA surgery and HSP70 might play an important role in patients' prognosis.

miR-638 Inhibits immature Sertoli cell growth by indirectly inactivating PI3K/AKT pathway via SPAG1 gene.

Numerous studies have demonstrated that microRNAs (miRNAs) play important roles in cell growth, apoptosis and spermatogenesis. Our previous study showed that miR-638 was differentially expressed in sexually immature and mature testes of Large White boars. Here we reported that sperm-associated antigen 1 (SPAG1) was a direct target gene of miR-638. Moreover, miR-638 inhibited cell proliferation and cell cycle, and promoted apoptosis of porcine immature Sertoli cells. Key genes including phosphorylated phosphatidylinositide 3-kinases (p-PI3K) and phosphorylated serine/ threonine kinase (p-AKT) in PI3K/AKT pathway as well as cell cycle factors including c-MYC, cyclin-D1 (CCND1), cyclin-E1 (CCNE1) and cyclin-dependent kinase 4 (CDK4) were all significantly down-regulated after overexpression of miR-638 or RNAi of SPAG1. Notably, mRNA levels of SRY-related HMG-box 2 (SOX2) and POU domain, class 5, transcription factor 1 (POU5F1) essential for spermatogonia proliferation were significantly suppressed in SPAG1 siRNA- transfected ST cells. This study suggests that miR-638 regulates immature Sertoli cell growth and apoptosis by targeting SPAG1 gene which can indirectly inactivate PI3K/AKT pathway, and plays roles in pig spermatogenesis.

Characterization of swine testicular cell line as immature porcine Sertoli cell line.

Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions.

miR-762 promotes porcine immature Sertoli cell growth via the ring finger protein 4 (RNF4) gene.

A growing number of reports have revealed that microRNAs (miRNAs) play critical roles in spermatogenesis. Our previous study showed that miR-762 is differentially expressed in immature and mature testes of Large White boars. Our present data shows that miR-762 directly binds the 3' untranslated region (3'UTR) of ring finger protein 4 (RNF4) and down-regulates RNF4 expression. A single nucleotide polymorphism (SNP) in the RNF4 3'UTR that is significantly associated with porcine sperm quality traits leads to a change in the miR-762 binding ability. Moreover, miR-762 promotes the proliferation of and inhibits apoptosis in porcine immature Sertoli cells, partly by accelerating DNA damage repair and by reducing androgen receptor (AR) expression. Taken together, these findings suggest that miR-762 may play a role in pig spermatogenesis by regulating immature Sertoli cell growth.

Exploiting RNA-sequencing data from the porcine testes to identify the key genes involved in spermatogenesis in Large White pigs.

Mammalian testis development and spermatogenesis play critical roles in male fertility. However, little genomic information is available for porcine sexually mature and immature testis. Presently, we detected approximately 76% of previously annotated genes that were expressed in the porcine testes by RNA sequencing. Taking an FDR of 0.001 and a |log2Ratio| of 1 as cutoffs, 10,095 genes were significantly differentially expressed between two stages, including 242 spermatogenesis-associated genes. These genes were significantly enriched to GO BP terms concerning spermatogenesis, male gamete generation, developmental process and sexual reproduction; to the KEEG pathways, including focal adhesion, ECM-receptor interaction, and phagosome. 186 extended transcripts, 1273 alternative splicing events and 2846 SNPs were detected in spermatogenesis-associated DEGs. Two PIWIL4 SNPs were successfully validated and suggested to be the potential molecular markers for semen quality. This study will help identify the specific genes and isoforms that are active in porcine spermatogenesis and sexual maturity.