Expert consensus on the molecular diagnosis of early-stage pancreatic cancer （2023 edition） / 临床肝胆病杂志

Pancreatic cancer is a relatively common tumor of the digestive system， with difficulties in early-stage diagnosis and an extremely high degree of malignancy. Molecular diagnostic technology based on tumor biomarkers， combined with the existing gold standard in clinical practice， is of great clinical significance to achieve early accurate identification， timely treatment and intervention， and reduction in mortality. Previous studies have shown that miRNAs show high specificity in terms of types and expression levels in different pathological stages of pancreatic cancer and can thus be used in monitoring the development and progression of pancreatic cancer. Since a single miRNA has a limited diagnostic potential， the combination of different miRNAs may effectively improve the diagnostic efficiency of early-stage pancreas carcinogenesis. Based on related research advances in recent years， this consensus document aims to fill the gap in molecular diagnostic technology in the guidelines for the clinical diagnosis and treatment of pancreatic cancer and provide expert guidance and recommendations.

Research progress on non-coding small RNA in oral submucosal fibrosis / 口腔疾病防治

@#Oral submucous fibrosis (OSF) is one of the most common precancerous lesions of the oral mucosa, and its pathogenesis has not been fully elucidated. Small noncoding RNAs (SncRNAs), a class of RNA molecules that do not code for proteins, have been widely reported to be involved in the regulation of a variety of human diseases. An increasing number of studies have shown that a variety of SncRNAs play important roles in the pathogenesis of OSF. Current studies have shown that microRNAs (miRNAs) are involved in OSF disease progression by regulating the expression of related transcription factors and genes or epithelial mesenchymal transformation to regulate the activation of fibroblasts (FBs). Long noncoding RNAs (lncRNAs) that transform growth factor-β/suppressor of mothers against decapentaplegic (TGF-β/Smad) signaling pathways or interact with miRNAs are involved in the development of OSF. Circular RNAs (circRNAs) play a role in OSF by interacting with miRNAs. tRNA-derived small RNAs (tsRNAs) are involved in the progression of various fibrotic diseases, but their specific mechanism of action in OSF still needs to be further explored. In the future, it is still necessary to focus on the targets of SncRNAs mediating OSF progression and explore their function and molecular mechanism in OSF to provide new ideas for the diagnosis and treatment of OSF.

Progressive Changes of Colitis Into Colorectal Cancer Associated With Changes in Neutrophil Lymphocyte Ratio, Serum, Micrornas and Infiltrate Neutrophils

@#Introduction: Understand the progression of colorectal cancer from the beginning until the advance stages is difficult and challenging. However, this could be overcome with a good animal model. Methods: In this study, a modified approach had been used to develop colorectal cancer model. The model was developed and monitored from colitis formation until the late stage of colorectal cancer. The changes of neutrophil lymphocyte ratio (NLR), serum microRNAs and infiltrate neutrophil in different stages of colorectal cancer were assessed in this study. Results: Results showed that the progression of the disease is correlated with NLR as early as the formation of colitis (r=0.121, p<0.026). Meanwhile, the size of the tumor at each stage is also associated with the NLR value (r=0.185, p<0.0012). In the serum microRNAs study, it was found microRNAs expression in blood serum change in different stages of colorectal cancer. In the early stage of colitis formation, miR223 (> 3 fold expression, p < 0.0025) were abundantly found in the blood serum. Meanwhile in others stage mild (miRNA345 > 2.5 fold, p<0.0011), moderate (miRNA347 & miR512 > 3 fold, p<0.002) and severe (miR31 & miR145 > 2 fold, p<0.0001) microRNAs were also found expressed differently. The quantities of infiltrate neutrophil were varied in different stages of the disease. Conclusion: This study provides an insight into the immunity and molecular level of colorectal cancer and it allows a progressive monitoring on the changes in the molecular, cellular and histological level.

Genetic variants in miR-146a and miR-196a2 in endometriosis: a Brazilian study

SUMMARY OBJECTIVE: The aim of this study was to determine the allelic and genotypic frequencies of the polymorphisms, rs2910164 miR-146a and rs11614913 miR-196a2, by investigating their association with endometriosis. METHODS: This is a case-control study performed with approximately 120 women. The polymorphisms were determined by real-time polymerase chain reaction. For the statistical analysis, the chi-square and logistic regression tests were used. RESULTS: There were no significant differences in the genotype and allele frequencies of rs2910164 and rs11614913 between cases and controls. The frequencies in both polymorphisms are in accordance with Hardy-Weinberg equilibrium regarding miR-146a (patients: χ2=1.64, p=0.20; controls: χ2=0.25, p=0.62) and miR-196a2 (patients: χ2=0.58, p=0.44; controls: χ2=2.78, p=0.10). No relationship was observed between rs2910164 and rs11614913 and endometriosis in the inheritance models analyzed. CONCLUSION: In this study, our results show that the studied polymorphisms are not implicated in the development of endometriosis.

A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway

ABSTRACT Introduction Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. Objective This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. Results Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. Comments Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

Association between microRNA expression and risk of male idiopathic infertility in Iraq

SUMMARY OBJECTIVE: The World Health Organization defines infertility as the inability to get pregnant after 12 months of unprotected sexual activity. This study was conducted to estimate the levels of gene expression for two mature miRNAs (i.e., miR-122 and miR-34c-5p) to evaluate susceptibility to male infertility. METHODS: This study included 50 male patients with idiopathic infertility who were admitted to hospital from the period November 2021 to May 2022 and another group consisting of 50 apparently healthy individuals used as controls. RESULTS: miR-122 level was significantly highest in azoospermia and followed by oligospermia, 39.22 (31.88) versus 37.34 (20.45), respectively. In addition, there was a very significant difference in miR-34c-5p levels between the study groups (p<0.05). CONCLUSION: Two miRNAs, namely, miR-34c-5p and miR-122, can be used as predictive and diagnostic biomarkers for infertility.

3D-Structural Characterization of MicroRNA Expressed in Leprosy / Caracterização Estrutural em 3D de MicroRNAs Expressos na Hanseníase

Introduction: Hansen's disease, or leprosy is caused by Mycobacterium leprae (M. leprae), is a major public health problem in developing countries, and affecting the skin and peripheral nerves. However, M. leprae can also affect bone tissue, mucous membranes, liver, eyes, and testicles, producing a variety of clinical phenotypes. MicroRNAs (miRNAs) have been expressed in the various clinical forms of leprosy and could potentially be used for its diagnosis. Objective: in silico design of the molecular structure of miRNAs expressed in leprosy. Methodology: we performed a nucleotide sequence search of 17 miRNAs expressed in leprosy, designing in silico the molecular structure of the following miRNAs: miRNA-26a, miRNA-27a, miRNA-27b, miRNA-29c, miRNA-34c, miRNA-92a-1, miRNA- 99a-2, miRNA-101-1, miRNA-101-2, miRNA-125b-1, miRNA-196b, miRNA-425-5p, miRNA-452, miRNA-455, miRNA-502, miRNA-539, and miRNA-660. We extracted the nucleotides were from the GenBank of National Center for Biotechnology Information genetic sequence database. We aligned the extracted sequences with the RNA Folding Form, and the three-dimensional molecular structure design was performed with the RNAComposer. Results: we demonstrate the nucleotide sequences, and molecular structure projection of miRNAs expressed in leprosy, and produces a tutorial on the molecular model of the 17 miRNAs expressed in leprosy through in silico projection processing of their molecular structures. Conclusion: we demonstrate in silico design of selected molecular structures of 17 miRNAs expressed in leprosy through computational biology.

Introdução: a doença de Hansen, ou hanseníase é causada pelo Mycobacterium leprae (M. leprae), é um grande problema de saúde pública nos países em desenvolvimento e afeta, a pele e os nervos periféricos. Entretanto, o M. leprae também pode comprometer o tecido ósseo, membranas mucosas, fígado, olhos e testículos, produzindo uma variedade de fenótipos clínicos. MicroRNAs (miRNAs) têm sido expressos nas várias formas clínicas da hanseníase e podem ser potencialmente utilizados para seu diagnóstico. Objetivo: objetivou-se com esse experimento modelar computacionalmente a estrutura molecular dos miRNAs expressos na hanseníase. Metodologia: realizou-se como metodologia uma pesquisa das sequências nucleotídicas de 17 miRNAs expressos na hanseníase, desenhando em modelo computacional a estrutura molecular dos seguintes miRNAs: miRNA-26a, miRNA-27a, miRNA-27b, miRNA- 29c, miRNA-34c, miRNA-92a-1, miRNA-99a-2, miRNA-101-1, miRNA-101-2, miRNA-125b-1, miRNA-196b, miRNA-425-5p, miRNA-452, miRNA-455, miRNA-502, miRNA-539, e miRNA-660. Extraiu-se os nucleotídeos do banco de dados do GenBank of National Center for Biotechnology Information . Alinhou-se as sequências extraídas com o RNA Folding Form, e o projeto da estrutura molecular tridimensional foi realizado com o RNAComposer. Resultados: demonstrou-se como resultados as sequências dos nucleotídeos e a projeção da estrutura molecular dos miRNAs expressos na hanseníase, e produzimos um tutorial sobre o modelo molecular dos 17 miRNAs expressos em hanseníase através do processamento de suas estruturas moleculares em projeção computacional. Conclusão: foi demonstrado computacionalmente o projeto de estruturas moleculares selecionadas de 17 miRNAs expressos em hanseníase através da biologia computacional.

miR-27a/b Level is Associated with ABCA1 Expression and is a Potential PBMC-Based Biomarker for Coronary Artery Disease

Abstract Background: Coronary artery disease (CAD) is the most common form of cardiac disease with high morbidity and mortality rates. Objectives: In this study, we evaluated the expression of miR-27a and miR-27b as biomarkers in peripheral blood mononuclear cells (PBMCs) of patients with CAD and investigated its correlation with cholesterol-efflux transporter, ATP-binding cassette transporter A1 (ABCA1). Method: This study was performed on 54 men with CAD and 51 healthy, sex- and age-matched control participants. The expression of miR-27a/b and ABCA1 genes in PBMCs were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of ABCA1 was assessed by Western blotting. Concurrently, the specificity and sensitivity of miR-27a/b was evaluated through receiver operating characteristic (ROC) curve. The significance level adopted in the statistical analysis was 5%. Results: We found that miR-27a and miR-27b expression were significantly increased, while both mRNA and protein expression of ABCA1 were markedly reduced in the PBMCs of CAD patients in comparison to non-CAD controls. miR-27a/27b expression was also shown to be inversely correlated with ABCA1. ROC analysis showed that the miR-27a had an area under the ROC curve (AUC) of about 92.6 (sensitivity 83.3٪ and specificity 86.6٪) and miR-27b had an AUC of about 93.0 (sensitivity 86.6٪ and specificity 80.0 (%, suggesting the diagnostic potential of miR-27a/b in CAD patients. Conclusions: Our data suggested a possible role of miR-27a/b in CAD pathogenesis. Additionally, we proposed that miR-27a/b expression in PBMCs may have potential clinical implications in the diagnosis of CAD patients, but further validations in large cohorts are required.

The Role of MicroRNAs in the Pathogenesis of Chagas Disease

Abstract Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi and affects about six to seven million individuals worldwide. The distribution of cases is concentrated mainly throughout Latin America, especially in rural areas. This study aims to evaluate microRNAs (miRNAs) as indicators in CD diagnosis for possible contributions to its management. This is a literature review study, carried out in the PubMed, SciELO, Bireme Library, NCBI, Science Direct, and Embase databases, through which a total of 12 articles were included for qualitative analysis. The discussion of this review was based on the thematic axes regarding the modulation of T. cruzi in the immune system and the expression of miRNAs, their production and action, the modulation mechanism of host gene expression, how they act as biomarkers, the importance of miRNAs in the diagnosis of CD, and how their regulation occurs in Chronic Chagas Cardiomyopathy (CCC). Moreover, T. cruzi infection is associated with the downregulation of several miRNAs, which directly related to the findings of hypertrophy and fibrosis. When quantified, these could be used as consistent indicators for CD to support the diagnosis of patients with CD complications, as well as a possible therapeutic target. However, the need for clinical studies that evaluate the usefulness of this biomarker in humans is emphasized, considering that in the present study, only experimental in vitro studies were evaluated, reflecting a lack of studies with practical applicability.

MicroARN circulantes y su potencialidad como futuros biomarcadores óseos / Circulating microRNAs and their potential use as future bone biomarkers / MicroRNAs circulantes e a sua potencialidade como futuros biomarcadores ósseos

Resumen El remodelamiento óseo es ejercido por la actividad de osteoblastos y osteoclastos y puede evaluarse por marcadores bioquímicos de formación y resorción ósea. Sin embargo, el nivel de los marcadores óseos está sometido a una enorme cantidad de variables y, además, carece o presenta escaso valor pronóstico. Los microARN (miARN) fueron recientemente estudiados como una alternativa potencial para ser utilizados como nuevos marcadores óseos. Los miARN son pequeñas moléculas de ARN no codificantes (15-25 nucleótidos) que, a través de la inhibición o degradación de ARN mensajeros, modifican una serie de funciones biológicas. Los miARN específicos de hueso ejercen funciones reguladoras sobre factores transcripcionales involucrados en la osteoblastogénesis y osteoclastogénesis, modificando el remodelamiento óseo. La mayoría de los miARN permanecen dentro de la célula, pero algunos son liberados a la circulación donde pueden ser dosados. Los miARN circulantes presentan gran estabilidad en fluidos biológicos, lo que los hace potenciales candidatos a ser utilizados como nuevos biomarcadores óseos. Cambios en el patrón normal de miARN circulantes específicos de hueso reflejarán modificaciones en el metabolismo óseo y señalan el posible inicio o progresión de enfermedades óseas, como la osteoporosis. Si bien es promisorio, el uso en la práctica clínica de los miARN específicos circulantes como nuevos biomarcadores óseos, ello implica primeramente cumplir con una serie de requisitos que permitan estandarizar las condiciones preanalíticas, analíticas y posanalíticas de estas moléculas. La presente revisión brinda información reciente sobre los estudios clínicos tendientes a determinar el posible uso de los miARN circulantes como nuevos biomarcadores óseos, ya que cuentan con elevada sensibilidad y especificidad diagnósticas, valor predictivo positivo y valor predictivo negativo.

Abstract Osteoblasts and osteoclasts activity determines the level of the bone remodelling process which can be assessed by biochemical markers of bone formation and resorption. However, bone marker levels are subject to a series of variables resand, furthermore, they lack or have little prognostic values. MicroRNAs (miRNAs) were recently studied as a potential alternative to be used as new bone biomarkers. The miRNAs are endogenous small noncoding RNA molecules (15-25 nucleotides) that regulate many biological functions by inhibiting or degrading specific messenger RNAs. Bone-specific miRNAs exert regulatory functions on key transcriptional factors involved in osteoblastogenesis and osteoclastogenesis, modifying the bone remodelling process. Most miRNAs remain within the cell, but some of them are released into circulation. Circulating miRNAs show great stability in biological fluids, which makes them excellent candidates to be used as new bone biomarkers. Modifications in the normal pattern of bone-specific circulating miRNA might reflect changes in bone metabolism, signalling the possible onset or progression of bone diseases, such as osteoporosis. Although promising, the use of specific circulating miRNAs as new bone biomarkers in clinical practice implies fulfilling a series of requirements that lead to standardising the pre-analytical, analytical and post-analytical conditions of these molecules. The present review gives an overview on the clinical studies related to the possible use of specific circulating miRNAs as new bone biomarkers.

Resumo A remodelação óssea é exercida pela atividade dos osteoblastos e osteoclastos e pode ser avaliada para a medição dos marcadores bioquímicos de formação e reabsorção óssea. No entanto, o nível dos marcadores ósseos está sujeito a grande quantidade de variáveis e, além disso, carece ou tem pouco valor prognóstico. Os microARN (miARN) foram estudados recentemente como uma alternativa potencial para serem utilizados como novos marcadores ósseos. Os MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificantes (15-25 nucleotídeos) que, através da inibição ou degradação de RNA mensageiros modificam uma série de funções biológicas. Os miRNAs específicos de osso exercem funções reguladoras sobre fatores transcricionais envolvidos na osteoblastogênese e na osteoclastogênese, modificando o processo de remodelação óssea. A maioria dos miRNAs permanece dentro da célula, mas de RNA mensageiros alguns são liberados na circulação, onde podem ser determinados bioquimicamente. Os miRNAs circulantes apresentam grande estabilidade em fluidos biológicos, o que os torna excelentes candidatos para serem usados como novos biomarcadores ósseos. Mudanças no padrão normal de miRNA circulantes específicos do osso mostrarão mudanças no metabolismo ósseo, sinalizando o possível início ou progressão de doenças ósseas, como osteoporose. Embora promissor, o uso de miRNAs específicas circulantes na prática clínica como novos biomarcadores ósseos, implica primeiramente, atender uma série de requisitos que permitem padronizar as condições pré-analíticas, analíticas e pós-analíticas dessas moléculas. A presente revisão fornece informações recentes sobre estudos clínicos destinados a determinar o possível uso dos miRNAs circulantes como novos biomarcadores ósseos, visto que contam com elevada sensibilidade e especificidade diagnósticas, valor preditivo positivo e valor preditivo negativo.

Evaluation of microRNA let-7b-3p expression levels in methamphetamine abuse

SUMMARY OBJECTIVE: In this study, we aimed to identify a microRNA expression signature that could be used to distinguish methamphetamine from control samples. We also utilized the existing bioinformatics tools in order to predict the candidate microRNAs that could play potential key roles in regulating drug addiction-related genes. METHODS: Methamphetamine samples from 21 ventral tegmental area and 21 nucleus accumbens samples and their control regions were obtained from the Council of Forensic Medicine (Istanbul). Quantitative analysis of let-7b-3p was studied using quantitative reverse transcription PCR. Statistical analysis was carried out using Student's t-test. The receiver operating characteristic curves were plotted with Statistical Package for the Social Sciences (SPSS 20.0). RESULTS: Our quantitative reverse transcription PCR results revealed that let-7b-3p was significantly overexpressed in brain tissues of the methamphetamine-user group. Let-7b-3p had significant power to discriminate methamphetamine from control samples in the ventral tegmental area (AUC; 0.922) and nucleus accumbens (AUC; 0.899) regions. CONCLUSION: We have shown for the first time in the literature the differential expression of let-7b-3p in samples from methamphetamine-addicted individuals. We suggest that let-7b-3p could be a powerful marker for the diagnosis of methamphetamine addiction. Our results showed that differentially expressed let-7b-3p in methamphetamine users could be used as a diagnostic and therapeutic marker.

Mesenchymal stem cells-derived exosomal miR-653-5p suppresses laryngeal papilloma progression by inhibiting BZW2

Abstract Objectives: Although miR-653-5p has been validated to participate in the progression of multiple types of cancer, the functional role of exosomal miR-653-5p derived from Mesenchymal Stem Cells (MSCs) in Laryngeal Papilloma (LP) has still remained elusive. Hence, this study aimed to investigate the role of MSCs-derived exosomal miR-653-5p in LP. Methods: LP tissues (n = 15) and adjacent normal tissues (n = 10) were collected to examine the expression level of miR-653-5p. The expression level of miR-653-5p in LP cells and normal cells was also detected. Then, miR-653-5p was overexpressed or silenced to explore its effects on the proliferation, migration, invasion, and apoptosis of LP cells. Thereafter, the effects of exosomal miR-653-5p derived from MSCs on LP cell progression and the potential regulatory mechanism of miR-653-5p were assessed. Results: It was revealed that the expression level of miR-653-5p was downregulated in LP tissues and cells. In addition, miR-653-5p suppressed the proliferation, migration, invasion, and apoptosis of LP cells. Exosomes derived from MSCs played a suppressive role in LP development and mediated the transmission of miR-653-5p to LP cells. Further exploration identified Basic leucine Zipper and W2 domains 2 (BZW2) as the target of miR-653-5p. More importantly, the rescue experiments revealed that MSCs-secreted exosomal miR-653-5p efficiently inhibited the aggressive phenotypes of LP cells, which could be significantly reversed by BZW2 overexpression in LP cells. Conclusion: MSCs-derived exosomal miR-653-5p exerted inhibitory effects on LP progression through targeting BZW2, which provided a novel idea for the therapy of LP. Clinical Trial registration number: chictr-ior-17011021.

Research progress on the role of non-coding RNA in chemotherapy resistance of osteosarcoma / 中华骨科杂志

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. With the emergence of chemotherapy resistance in recent years, the survival rate of osteosarcoma patients has reached a bottleneck. Therefore, exploration of its chemoresistance mechanism is one of the popular research directions currently. Non-coding RNA (ncRNA) is a class of RNA without the ability to encode proteins, which is classified into microRNA, long non-coding RNA, and circular RNA based on length and shape. With the development of high-throughput sequencing technology, there is increasing evidence that some non-coding RNAs are abnormally upregulated or downregulated in osteosarcoma cells and affect the response of osteosarcoma to four commonly used chemotherapeutic drugs (methotrexate, doxorubicin, cisplatin and ifosfamide) through mechanisms such as regulation of apoptosis, cell cycle, signaling pathways, intracellular drug concentration, and cellular autophagy. Therefore, thesenon-coding RNAs are expected to be novel targets for osteosarcoma treatment. In this paper, the current studies were searched and reviewed on the above three non-coding RNAs mediating chemoresistance in osteosarcoma, aiming to provide a reference for breaking the bottleneck of survival rate of osteosarcoma patients.

Effects of adriamycin resistance cell-derived exosomes on the proliferation and migration of osteosarcoma cells through drug resistance transmission / 中华骨科杂志

Objective:To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs. Methods:MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains, and the 50% inhibitory concentration (half maximal inhibitory concentration, IC 50) of drug-resistant and sensitive strains was detected by MTT, and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity. RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells. Exosomes were identified by particle size analysis and Western Bolt detection. The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed, and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay. The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay. Tumor growth, serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group, drug-resistant+normal exosome group, drug-resistant+drug-resistant+drug-resistant exosome group were observed. MDR1 expression level in tumor tissue was detected by RT-PCR, Western Blot and immunohistochemistry. Results:The IC 50 of two adriamycin resistant strains were 2.21 vs. 11.81 μg/ml and 0.93 vs. 11.81 μg/ml, respectively, and the fluorescence intensity decreased faster than that of normal strains. The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16, 1.02±0.11 and drug-resistant 2.15±0.10, 2.127±0.12, respectively. The relative protein expression of P-gp was normal 0.92±0.11, 0.73±0.10 and drug-resistant 0.46±0.03, 0.30±0.04, the differences were statistically significant ( P<0.05). Drug-resistant exosomes can enter osteosarcoma cells through endocytosis and concentrate in the cytoplasm when co-cultured with normal strains. Osteosarcoma cells were co-cultured with drug-resistant exosomes at 2, 4, 6, and 8 μg/ml adriamycin, respectively. Compared with normal group, the proliferation level in drug-resistant group was significantly increased. Compared with the normal cell group 35.95±3.92, 6.72±3.55 and the normal exosome group 51.22±5.55, 19.31±1.93, the drug-resistant cell group 54.20±9.32, 19.24±2.88 and drug-resistant exosome group 76.40±5.41, 30.26±4.87, all had significantly higher cell mobility, the difference was statistically significant ( P<0.05). Exosome sequencing and biogenic analysis of 10 highly upregated miRNAs to validate mRNA expression differences between normal and drug-resistant strains by RT-PCR, showing a significant increase in miR-21-5p expression level of drug-resistant strains (5.89±0.26 vs. 0.99±0.06; 1.05±0.07 vs. 8.80±0.93, P<0.05), the difference was statistically significant ( P<0.05). In MG63 and U2OS, the normal cell group and drug-resistant cell group, and the normal exosome group and drug-resistant exosome group were compared, the tumor volume and the terminal tumor weight of nude mice were increased to varying degrees. MRNA relative expression levels of miR-21-5p in serum exosomes of nude mice after drug intervention were 0.86±0.07 and 0.86±0.05 in normal cell group, respectively. The values were 1.13±0.12, 1.14±0.12 in drug-resistant cell group, 0.71±0.05, 0.75±0.03 in normal exosome group, and 0.90±0.07, 0.93±0.04 in drug-resistant exosome group. Compared with normal and drug-resistant strains, the expression levels of normal and drug-resistant exosome groups were increased, with statistical significance ( P<0.05). Conclusion:The exosomes of drug-resistant cells in osteosarcoma could enhance the proliferation level and migration ability of cells through intercellular transfer of MDR1 and miRNAs. The expression of MDR1 and miR-21-5p in drug-resistant cells and tumor-forming nude mouse serum and tumor tissues were up-regulated which suggested that it might be involved in regulating the drug resistance process of osteosarcoma.

Expression and clinical significance of miR-143 in gallbladder cancer / 中华肝胆外科杂志

Objective:This study aims to explore the expression of miR-143 in gallbladder cancer tissues and its correlation with tumor angiogenesis, clinicopathologic features and clinical prognosis.Methods:The clinical and pathological data of 78 patients with gallbladder cancer who were surgically resected and pathologically confirmed from January 2008 to June 2016 in the Department of General Surgery, Xuancheng Hospital Affiliated to Wannan Medical College were collected, including 20 males and 58 females, with an average age of (68.0±1.4) years. 78 cases of gallbladder cancer tissue were used as the tumor group, and 32 cases of normal gallbladder tissue adjacent to cancer were randomly selected as the control group. The expression level of miR-143 in the tumor group and the control group was detected by in situ hybridization. Microvessel density (MVD) were evaluated with immunohistochemistry to detect the expression of CD34 in tumor group. The expression of miR-143 and its correlation with tumor MVD, pathological characteristics and prognosis were analyzed by Cox regression analysis.Results:The results of in situ hybridization showed that the positive rate of miR-143 in gallbladder cancer tissues and average expression score were significantly lower than those in adjacent normal gallbladder tissues [29.49% (23/78) vs 62.5% (20/32), χ 2=10.39, P=0.001; (2.705±0.172) vs (4.688±0.405), t=5.33, P<0.001]. Correlation analysis showed that the expression of miR-143 was associated with lymph node metastasis of gallbladder cancer ( P=0.036). The MVD counts in the miR-143 positive group were significantly lower than those in the miR-143 negative group [(126.2±8.483) vs (157.4±6.412), t=2.75, P=0.007)]. The median survival time of gallbladder cancer patients with miR-143 positive and negative was 16 and 9 months, respectively. Cox regression analysis showed that the low expression of miR-143 was an independent risk factor affecting the clinical prognosis of gallbladder cancer. Conclusion:The expression of miR-143 is down-regulated in gallbladder cancer and participates in angiogenesis and clinical prognosis of gallbladder cancer.

Study of miR-506 in M2 macrophage polarization and immune intervention in pancreatic cancer mice / 中华肝胆外科杂志

Objective:To analyze the effect of microRNA-506 (miR-506) on M2 macrophages polarization and immune intervention in pancreatic cancer mice.Methods:Macrophages from peripheral blood of healthy volunteers were cultured in vitro, polarized into M1 or M2 type macrophages, and transfected with miR-506 or control sequence (miR-ctrl), respectively. Polarized macrophages from M1+ miR-ctrl group, M1+ miR-506 group, M2+ miR-ctrl group and M2+ miR-506 group were collected. The relative expression of marker genes of M1 and M2 type macrophages of four groups were analyzed by qRT-PCR. The characteristic functions of M1 and M2 type macrophages of four groups were also detected, such as phagocytosis and nitric oxide (NO) synthesis (characteristic function of M1 type macrophages), arginase 1 activity and the secretion of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), interleukin-10 (IL-10) (characteristic function of M2 type macrophages). Sixty healthy male C57BL/6 mice without specific pathogen, weighing 20-25 g, were randomly divided into miR-ctrl programmed death-1 (PD-1) group, miR-506 PD-1 group, miR-ctrl iso group, and miR-506 iso group. They were injected with miR-506, miR-ctrl, PD-1 antibodies, and isotype control antibodies, with 15 in each group. The tumor volume, tumor weight, Ki-67 and interferon γ expression were analyzed three weeks later. Results:Compared with M2+ miR-ctrl group, the relative expression of M1 type macrophage marker genes increased, and the relative expression of M2 type macrophage marker genes decreased in M2+ miR-506 group, with significant difference (all P<0.05). Compared with M2+ miR-ctrl group, the phagocytic function and NO synthesis of macrophages in M2+ miR-506 group increased, the activity of arginase 1 and the secretion of VEGF, TGF-β and IL-10 decreased, with significant difference (all P<0.05). There was no significant differences in tumor weight, volume, Ki-67, and interferon γ expression between miR-ctrl iso and miR-ctrl PD-1 group (all P>0.05). The tumor weight, tumor volume and Ki-67 in miR-506 PD-1 group were lower than those in miR-ctrl PD-1 group [(0.32±0.13) g vs (0.85±0.24) g; (0.72±0.23) cm 3 vs (2.03±0.21) cm 3; (25.9±10.3)% vs (55.6±12.5)%], while interferon-γ expression was significantly higher than that in miR-ctrl PD-1 group [(122.4±15.3) ng/g vs (82.4±22.2) ng/g] (all P<0.05). Conclusion:miR-506 inhibits the polarization of M2 macrophages and increases the anti PD-1 immunotherapy sensitivity in pancreatic cancer.

Analysis of the expression levels of microRNA-205 and microRNA -24-3p in cervical cancer tissues and their correlation with survival prognosis / 中国医师进修杂志

Objective:To analyze the expression of microRNA(miR)-205 and miR-24-3p in cervical cancer tissues and their correlation with survival prognosis.Methods:A total of 82 patients with cervical cancer(cervical cancer group), 60 patients with uterine fibroids(uterine fibroids group) and 40 healthy women (healthy control group) treated in Hubei Hospital of Integrated Traditional Chinese and Western Medicine from March 2014 to March 2015 were enrolled. The expression levels of miR-205 and miR-24-3p in the tissues of the three groups were compared. The receiver operative characteristic (ROC) curve was used to analyze the diagnostic value and zero threshold of the two indicators for cervical cancer. Analyzed the 5-year survival status of cervical cancer patients at the upper and lower thresholds, and then further discuss the risk factors that affected the prognosis of cervical cancer.Results:The relative expression levels of miR-205 and miR-24-3p in the cervical cancer group were significantly higher than those in the uterine fibroids group and the healthy control group ( P<0.01). ROC curve analysis showed that the cut-off value was 1.13 and 1.35, the area under the curve was the biggest, and the sensitivity in diagnosis of cervical cancer were 83.87%, 84.38%, the specificity were 75.00%, 82.42%.The survival rate analysis based on these results showed that the 5-year survival rate of patients with miR-205≥1.13 was 69.05%, which was significantly lower than patients with miR-205<1.13 (87.50%) ( P<0.05); the 5-year survival rate of patients with miR-24-3p≥1.35 was 68.89%, which was significantly lower than that of patients with miR-24-3p<1.35 (89.19%)( P<0.05). The results of the Cox regression model showed that moderately and highly differentiated, FIGO stage Ⅱ, depth of invasion ≥1/2, combined lymph node metastasis, and high levels of miR-205 and miR-24-3p were risk factors affecting the prognosis of cervical cancer patients ( P<0.05). Conclusions:Serum miR-205 and miR-24-3p are at high levels in cervical cancer tissues, and are closely related to the prognosis of patients. They are expected to become auxiliary diagnosis and prognostic indicators for cervical cancer.

Inhibitory effect of miR-497 on corneal neovascularization and its regulation mechanism targeting STAT3 / 中华实验眼科杂志

Objective:To investigate the role of microRNA (miR)-497 in the formation of corneal neovascularization (CNV) induced by alkali burn and its mechanism.Methods:Forty-two wild type (WT) C57BL/6 mice aged 6 to 8 weeks, 42 CRISPR/Cas9 mediated miR-497 knockout (KO) and 42 CRISPR/Cas9 mediated overexpression transgenic (TG) C57BL/6 mice were selected and assigned as WT group, KO group and TG group, respectively.The corneal alkali burn model was established.At 3, 7, 14 and 21 days after modeling, corneal epithelium damage and stromal turbidity were scored according to slit lamp microscopy.The area of neovascularization was measured.Corneal structural changes and expression of inflammatory cells were observed by histopathological staining.The expression of CD31 in corneal tissues was detected by immunohistochemistry staining.The targeted binding relationship between miR-497 and signal transducer and activator of transcription 3 (STAT3) was detected by luciferase reporter assay.The relative expressions of miR-497, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and macrophage inflammatory protein (MCP)-1 mRNA were detected by real-time quantitative PCR.At 14 days following modeling, the expression of STAT3 and p-STAT3 proteins in mice corneal tissues was detected by Western blot.The use and care of animals complied with the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No.2019K-K010).Results:Corneal injury, inflammatory cell infiltration and CNV occurred in mice cornea after alkali burn.Corneal epithelial injury score, corneal stromal turbidity score and CNV area increased first and reached the peak on the 14th day after modeling, and then decreased.There were significant differences in corneal epithelial injury score, corneal stromal turbidity score, CNV area and number of CD31-positive cells among various time points after alkali burn ( Fgroup=49.19, 34.56, 44.56, 77.56; all at P<0.01; Ftime=51.62, 65.62, 71.32, 46.12; all at P<0.01). Corneal epithelial injury score, corneal stromal turbidity score, CNV area and the number of CD31-positive cells were greater in KO group at various time points than in WT and TG groups, and those in WT group were greater than in TG group (all at P<0.05). In WT STAT3 co-transfected cells, the luciferase activity of the miR-497 group was significantly lower than that of the miR-negative control group and normal control group (both at P<0.05). In mutant STAT3-transfected cells, there was no significant difference in luciferase activity among all groups ( F=0.69, P=0.56). On the 14th day after modeling, the relative expression levels of miR-497 in corneal tissue of WT, KO and TG groups were 0.68±0.11, 0.41±0.06 and 1.05±0.14, respectively, which were significantly lower than 1.00±0.04, 0.56±0.07 and 1.34±0.11 before modeling (all at P<0.01). The relative expressions of STAT3 and p-STAT3 were higher in KO group than in WT and TG groups, and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.05). The expressions of VEGFA, TNF-α, IL-6, IL-1β and MCP-1 mRNA at various time points after modeling in various groups were significantly higher than before modeling, which were higher in KO group than in WT and TG groups and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.01). Conclusions:MiR-497 inhibits corneal inflammation and CNV formation induced by alkali burn.It might inhibit the activation of the inflammation signal pathway via targeting STAT3.

lncRNA FTX silencing inhibits gastric cancer cell proliferation through the miR-22-3p/NLRP3 inflammasome pathway / 国际肿瘤学杂志

Objective:To investigate the regulatory effect of long non-coding RNA (lncRNA) FTX on gastric cancer cell proliferation through miR-22-3p/NOD-like receptor protein 3 (NLRP3) inflammasome pathway.Methods:The gastric cancer cell line NCI-N87 were divided into blank control group, si-FTX-NC group, si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group. Quantitative real-time fluorescent PCR was performed to analyze the expression levels of lncRNA FTX and miR-22-3p, clone formation assay was performed to analyze the proliferation ability of NCI-N87 cells, western blotting was performed to analyze the expressions of NLRP3 inflammasome pathway proteins, and dual-luciferase reporter assay was performed to analyze the targeting relationship between lncRNA FTX and miR-22-3p.Results:The relative expressions of lncRNA FTX in the blank control group, si-FTX-NC group, si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group were 1.03±0.09, 1.01±0.15, 0.42±0.08, 0.45±0.06 and 0.46±0.13 respectively, with a statistically significant difference ( F=52.19, P<0.001). The relative expressions of miR-22-3p were 1.04±0.12, 0.97±0.08, 2.26±0.15, 2.23±0.13 and 1.15±0.11 respectively, with a statistically significant difference ( F=178.53, P<0.001). Compared with the blank control group and si-FTX-NC group, the relative expressions of lncRNA FTX in the si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group decreased (all P<0.001). Compared with the blank control group, si-FTX-NC group and si-FTX+miR-22-3p inhibitor group, the relative expressions of miR-22-3p in the si-FTX group and si-FTX+miR-22-3p inhibitor-NC group increased (all P<0.001). The clones of the five groups were 115.50±7.25, 112.33±8.46, 54.83±5.17, 56.17±6.32 and 85.67±9.43, with a statistically significant difference ( F=91.67, P<0.001). The levels of NLRP3 protein in the five groups were 1.84±0.17, 1.86±0.12, 0.95±0.09, 0.97±0.11 and 1.28±0.19, with a statistically significant difference ( F=60.62, P<0.001). Compared with the blank control group and si-FTX-NC group, the number of clones and the level of NLRP3 protein of the si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group decreased (all P<0.05). Compared with the si-FTX+miR-22-3p inhibitor group, the number of clones and the level of NLRP3 protein in the si-FTX group and si-FTX+miR-22-3p inhibitor-NC group decreased (all P<0.05). The dual-luciferase reporter assay found that miR-22-3p was the target gene of lncRNA FTX. Conclusion:Silencing the expression of lncRNA FTX can inhibit the proliferation of gastric cancer cells, and the mechanism may be related to the regulation of lncRNA FTX on the miR-22-3p/NLRP3 inflammasome pathway.

PCAT4 drives breast cancer progression by up-regulating URGCP expression through sponge miR-508-5p / 国际外科学杂志

Objective:To investigate the driving effect of prostate cancer associated transcript 4 (PCAT4) on the up-regulation of upregulator of cell proliferation (URGCP) expression in breast cancer progression through sponging miR-508-5p.Methods:The microarray data of lncRNA and miRNA with differential expression in breast cancer tissue were analyzed by Cancer Genome Atlas. The expression of PCAT4 in breast cancer was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was measured by MTT and colony formation, cell apoptosis was analyzed by TUNEL, and cell migration and invasion were analyzed by Transwell. The correlation between PCAT4 and miR-508-5p, and miR-508-5p and URGCP was analyzed by RNA pull-down and double luciferase assay. Tumor xenograft studies were performed to analyze the correlation between PCAT4/miR-508-5p/URGCP axis and breast cancer cell growth in vivo. Measurement data were expressed as mean ± standard deviation ( ± s). T-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. The correlation between PCAT4 and URGCP and miR-508-5p expression was evaluated by Pearson correlation analysis. Results:The expression level of PCAT4 was up-regulated in breast cancer tissues and cells. Knockout of PCAT4 inhibited cell proliferation and metastasis and promoted cell apoptosis. miR-508-5p was the target of PCAT4 and was negatively correlated with PCAT4. Overexpression of miR-508-5p in breast cancer can inhibit cell growth, migration and invasion, and promote cell apoptosis. URGCP is the target of miR-508-5p and induces progression of breast cancer. Tumor xenograft studies showed that PCAT4 drives breast cancer progression by affecting miR-508-5p/URGCP.Conclusion:The expression of PCAT4 is up-regulated in breast cancer tissues and cells, and PCAT4 can act as a molecular sponge of miR-508-5p, and significantly promote breast cancer progression by activating URGCP protein expression.