Analysis of RNA Exosome Subunit Transcript Abundance Across Tissues: Implications for Neurological Disease Pathogenesis: 10.14800/rd.1247

Exosomopathies are a collection of rare diseases caused by mutations in genes that encode structural subunits of a ribonuclease complex termed the RNA exosome. The RNA exosome mediates both RNA processing and degradation of multiple classes of RNA. This complex is evolutionarily conserved and required for fundamental cellular functions, including rRNA processing. Recently, missense mutations in genes encoding structural subunits of the RNA exosome complex have been linked to a variety of distinct neurological diseases, many of them childhood neuronopathies with at least some cerebellar atrophy. Understanding how these missense mutations lead to the disparate clinical presentations that have been reported for this class of diseases necessitates investigation of how these specific changes alter cell-specific RNA exosome function. Although the RNA exosome complex is routinely referred to as ubiquitously expressed, little is known about the tissue- or cell-specific expression of the RNA exosome complex or any individual subunit. Here, we leverage publicly available RNA-sequencing data to analyze RNA exosome subunit transcript levels in healthy human tissues, focusing on those tissues that are impacted in exosomopathy patients described in clinical reports. This analysis provides evidence to support the characterization of the RNA exosome as ubiquitously expressed with transcript levels for the individual subunits that vary in different tissues. However, the cerebellar hemisphere and cerebellum have high levels of nearly all RNA exosome subunit transcripts. These findings could suggest that the cerebellum has a high requirement for RNA exosome function and potentially explain why cerebellar pathology is common in RNA exosomopathies

MiR-1294, mediated by KLF4, negatively regulates cell proliferation and apoptosis by targeting MYH9 in Non-Small Cell Lung Cancer: DOI: 10.14800/rd.1206

Lung cancer, especially non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related mortality worldwide. MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules. In recent years, many studies have confirmed that abnormal miRNAs expression in tumor can participate in many biological processes of NSCLC. However, whether miR-1294 is involved in the development of NSCLC remains unclear. In this study, miR-1294 was inhibited in NSCLC cell lines, and its expression was associated with tumor size and progression. MiR-1294 overexpression inhibited cell proliferation and cell cycle, conversely promoted cell apoptosis and senescence, and miR-1294 binding to MYH9 3’-UTR mediated suppression of it. Besides, three bioinformatics software were searched, and KLF4 was predicted as an upstream regulator of miR-1294. This study is the first to illuminate that miR-1294, mediated by KLF4, by targeting MYH9 to regulate NSCLC cell proliferation and apoptosis, and is a potential biomarker and therapeutic target for NSCLC

RNA binding activity of HSV-2 ICP27 protein: DOI: 10.14800/rd.1165

Herpes Simplex virus (HSV) protein ICP27 is a multifunctional gene expression regulator, which assumes various roles during the course of the viral infection. Previously we identified ICP27 switches RNA isoforms expression of particular genes such as PML (promyelocytic leukemia). Although ICP27 protein contains several RNA binding domains, it remains unclear what domain of ICP27 protein is critically important for RNA binding activity to both host and viral transcripts. Here we characterized the RNA binding activity of ICP27 protein in vitro. We found that R2 domain and three KH domains in the C terminal region of ICP27 protein bound to PML pre-mRNA and also suppressed retention of PML intron 7a, indicating that the C-terminal region contributes to such alternative splicing regulation of host gene. Notably C-terminal region of ICP27 protein efficiently binds to poly G, poly U and poly C, although the N-terminal region preferentially binds to poly G. In addition, the mutant proteins that lack either R2 or KH domains decreases RNA binding activity to poly U. We also employed Cross-Linking and Immuno-Precipitation (CLIP)-PCR approach in HSV-2 infected cells and identified 39 RNA binding targets of ICP27 protein in the viral genome that is G-rich. Finally, we showed that loss of either the N or C terminal regions of ICP27 significantly impaired RNA binding to such viral RNAs. Overall our results suggest that combination of ICP27 protein domains determines specificity of RNA binding to host and HSV-2 RNAs

Homologous recombination repair pathway alteration and its association with survival of breast cancer patients: DOI: 10.14800/rd.462

Breast cancer is a highly heterogeneous neoplasm with different response to chemotherapy. In this study, we investigated if homologous recombination repair (HRR), one of the important pathways of DNA damage repair, could serve as biomarkers for breast cancer. Breast cancer patients were selected from the Cancer Genome Atlas (TCGA) database.  Data of RNA-seq or mutation alteration of HRR pathway-related genes were extracted and analyzed. Correlations between HRR pathway mutation and clinicopathological features of breast cancer were analyzed using chi-square test. Based on the Kaplan-Meier method and log-rank test, survival analysis was done to identify the correlation between each HRR gene and survival rates. Using data retrieved from TCGA database, 1108 cases were identified of breast cancer with full data on RNA-seq and 986 cases with full data on mutation. We demonstrated that high expression of HRR gene RAD50, RAD51, RAD51C, RAD54L and XRCC2 were associated with favorable prognosis (Log-rank P=0.02686, 0.03734, 0.00664, 0.01147 and 0.01818, respectively). Moreover, mutation in the HRR pathway was present in 15.0% of cases. RIM1, PPP4R2, PPP4R4, RAD50 and RAD51D gene mutation were associated with unfavorable outcome (Log-rank P=0.0346, 0.0051, 0.0326, 0.0213 and 0.0007, respectively). The N stage and estrogen receptor (ER) status were significantly related to HRR pathway mutation (all factors P<0.05). Additionally, basal-like breast cancer subtype took up more percentage in HRR pathway mutation patients. Low expression or mutation in HRR pathway were associated with unfavorable prognosis in breast cancer. HRR pathway could serve as potential predictor, emphasizing the significance of more research on HRR pathway genes to facilitate more profound clinical implications in breast cancer molecular treatment.   DOI: 10.14800/rd.46

Apoptosis and autophagy inhibited by autophagy-related circular RNA in Schwann cells via miR-144-3p/PI3K/AKT/mTOR pathway: DOI: 10.14800/rd.461

Diabetic peripheral neuropathy is a common complications of Type 2 Diabetes and its main pathological feature is myelin sheath damage of peripheral nerve that was induced by Schwann cells (SCs) apoptosis. Increasing evidence suggested that noncoding RNAs might play a role in improving DPN because of its ability to prevent SCs apoptosis. In this study, we explore the effect of autophagy-related circular RNA on preventing SCs apoptosis and its underlying mechanism. Our study found that ACR could relieve HG-aroused RSC96 cell apoptosis, autophagy, and oxidative stress via reducing miR-144-3p and then promoting PI3K/AKT/ mTOR pathway activation, which provides some clues that ACR might be effective and feasible candidate for the treatment of DPN

Modeling Pathogenic Variants in the RNA Exosome: DOI: 10.14800/rd.1166

Exosomopathies are a collection of rare diseases caused by mutations in genes that encode structural subunits of the RNA exosome complex. The RNA exosome is critical for both processing and degrading many RNA targets. Mutations in individual RNA exosome subunit genes (termed EXOSC genes) are linked to a variety of distinct diseases. These exosomopathies do not arise from homozygous loss-of-function or large deletions in the EXOSC genes likely because some level of RNA exosome activity is essential for viability. Thus, all patients described so far have at least one allele with a missense mutation encoding an RNA exosome subunit with a single pathogenic amino acid change linked to disease. Understanding how these changes lead to the disparate clinical presentations that have been reported for this class of diseases necessitates investigation of how individual pathogenic missense variants alter RNA exosome function. Such studies will require access to patient samples, a challenge for these very rare diseases, coupled with modeling the patient variants. Here, we highlight five recent studies that model pathogenic variants in EXOSC3, EXOSC2, and EXOSC5

Inhibition of AR Restores the Sensitivity to Fulvestrant in ER-Positive Breast Cancer Cells: DOI: 10.14800/rd.1143

Endocrine therapy has been widely used in treating estrogen receptor (ER)-positive breast cancer which accounts for nearly 75% of breast cancer. Though endocrine therapy has shown great potency, acquired resistance occurs. Fulvestrant, the ?rst selective ER down-regulator (SERD), was confirmed to completely suppress ER? and notably e?cient. However, it has been observed that some ER-positive breast cancer would eventually develop unresponsiveness and acquired resistance to it, resulting in poor outcome. Several mechanisms have been proposed to be involved in antiestrogen resistance, such as activated pathways and altered expression of microRNAs. Of note, it is postulated androgen receptor (AR) which is often observed in most primary and metastatic breast cancer, might be a crucial protein associated with the efficacy of Fulvestrant, due to its common co-expression and intricate crosstalk with ER. In this study, we demonstrate that treatment suppressing ER would shift tumors from ER dependence to AR dependence, resulting in resistance to Fulvestrant and tumor growth. A blockade of AR could increase the sensitivity to Fulvestrant, and dual inhibition of AR and ER would be more effective than either drug alone, which might provide an insight into choosing optimal therapy for patients with AR-expressing ER-positive breast cancer. Furthermore, activated AR could also upregulate its downstream factor SOX9 to promote cell migration and proliferation

microRNA analysis of colorectal cancer using fecal and tissue samples: DOI: 10.14800/rd.1592

Recently, several microRNAs (miRNAs) have been reported as promising biomarkers for cancer detection and tumor recurrence risk. Due to its stability, miRNA can be accessed from samples stored in severe conditions, such as feces or formalin-fixed paraffin-embedded (FFPE) tissue. Fecal miRNA extracted from the residuum of fecal occult blood tests (FOBTs) was assessed to determine whether a combination of this fecal miRNA test (FmiRT) with FOBT could improve the false-negative rate of colorectal cancer (CRC) screening compared with FOBT alone. Expression of miR-106a in patients with both positive and negative FOBTs was significantly higher than in healthy volunteers. To identify a high-risk group for recurrence, miRNAs were extracted from FFPE samples of patients with stage II CRC. Tumor recurrence occurred at a significantly higher rate in patients with increased miR-181c expression than in those with lower expression. The recurrence rate in patients with stage II CRC with higher expression of miR-181c was similar to that of patients with stage III CRC who had been treated by surgical resection alone. As miRNAs are stable in several severe storage conditions, such as in fecal and FFPE samples, they could be valuable, accessible biomarkers for CRC, for use both in cancer screening and as predictors of recurrence

Colon cancer cell proliferation is inhibited by miR-142a-5p via regulation of the PLK1 gene: DOI: 10.14800/rd.448

The morbidity and mortality of colon cancer are still very high, although medical technology has greatly improved in recent years. This study explored the role of miR-142a-5p in the proliferation of colon cancer cells. The binding between miR-142a-5p and PLK1 was verified by using luciferase assays. Western blotting was employed to determine whether PLK1 is the direct target gene of miR-142a-5p. In addition, a Cell Counting Kit-8 (CCK-8) assay was used to determine whether miR-142a-5p inhibited the proliferation of colon cancer cells by targeting PLK1. Our results show the clear binding between miR-142a-5p and PLK1. Reduced PLK1 expression in colon cancer cells transfected a miR-142a-5p mimic and increased PLK1 expression in colon cancer cells transfected with a miR-142a-5p inhibitor were observed. Moreover, the overexpression of PLK1 promoted cell proliferation, while the miR-142a-5p mimic inhibited cell proliferation. In conclusion, our findings indicate that miR-142a-5p inhibits the proliferation of colon cancer cells by downregulating PLK1

Non-coding RNA in breast cancer: DOI: 10.14800/rd.453

With the availability of next-generation sequencing technologies since the early 2000s, non-coding RNAs have been assessed as potential therapeutic targets for numerous diseases. It is well documented that non-coding RNAs (ncRNAs), including circular RNA (circRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), PIWI-interacting RNAs (piRNA), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), play critical roles in cellular physiology and disease pathogenesis [1]. Since the discovery of ncRNAs, their roles have gained great attention and their biological function have been extensively studied. snRNAs and snoRNAs have been found to participate in mRNA splicing and rRNA maturation respectively [2,3]. circRNAs can act as competing endogenous RNA (ceRNA) for microRNAs, thus reducing miRNA repression of their target mRNAs [4]. Moreover, circRNAs also modulate the stability of mRNAs, interact with RNA binding proteins, and regulate gene transcription by interacting with RNA pol II [5]. piRNAs were reported to work in PIWI–piRNA complexes to regulate gene expression at the epigenetic and post-transcriptional levels [6]. As of today, miRNAs and lncRNAs were the two most widely studied ncRNAs. They have been found to be involved in the regulation of breast cancer cell proliferation, apoptosis, angiogenesis, epithelial to mesenchymal transition (EMT), cancer stem cells (CSCs) phenotype, interplay of tumor microenvironment, and drug resistance [7]. Breast cancer is the most common malignancy, and is the second cause of cancer death among females worldwide, accounting for an estimated 266,120 new cases and 40,920 deaths in 2018 [8]. Various miRNAs and lncRNAs have been established as potential biomarkers for diagnosis and prognosis in breast cancer [9, 10]. Kawaguchi et al. developed a novel three-miRNAs risk score (i.e., miR-19a, miR-93, and miR-106a) using the TCGA cohort to predict prognosis of survival and bone recurrence in breast cancer patients [11]. Meng et al. found that lncRNAs U79277, AK024118, BC040204, and AK000974 might contribute to the selection of high-risk patients for adjuvant therapy, which is independent of age and breast cancer subtypes [12]. The potential of ncRNAs as therapeutic targets has also been explored extensively in breast cancer. The main advantage of ncRNA based therapeutics is that it can target multiple coding or non-coding genes that regulate specific or redundant pathways involved in cancer development. However, therapeutics based on ncRNAs encounter some obstacles, such as problems associated with the delivery, potential off-target effects, and safety. Various studies have implied that emerging gene editing technology such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats [CRISPR]-associated nuclease-9) system in ncRNA editing could not only correct cancer-causing mutations but also has the potential to edit the ncRNA genes in addition to protein-coding genes [13-15]. For example, Ho et al. successfully generated knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 using CRISPR/Cas system through an HR-based selection and/or dual gRNA approach [16]. Excitingly, several miRNA-based therapeutics are underway by various pharmaceutical companies and part of them are in pre-clinical and clinical stages. More than ten clinical trials about miRNA-based therapeutics, including anti-miR-122, anti-miR-103/107, anti-miR-155, miR-29 mimic, miR-16 mimic, and miR-34 mimic, have been registered in the website ClinicalTrials (http://clinicaltrials. gov/ct2/home) [17]. One of the first miRNA?based molecules to enter clinical development was the locked nucleic acid (LNA) miravirsen, antisense RNA oligo of miR?122, for the treatment of hepatitis C virus (HCV). Miravirsen resulted in a dose-dependent reduction in HCV RNA levels and no dose-limiting adverse events and no escape mutations were observed in the miR-122 binding sites of the HCV genome [18]. The success of the phase 2a trial has led to the advancement of miravirsen into additional phase 2 trial with more patients, long-term follow-ups, and multidrug combinations (clinicaltrials. gov/ct2/show/NCT01872936). Therefore, we believe more such plans for clinical trials for breast cancer might be under conception and breakthrough can be expected in the near future