BACKGROUND: Gastric cancer (GC) is a common cancer worldwide; however, its molecular and pathogenic mechanisms remain unclear. MicroRNAs (miRNAs), which target key genes in GC, are associated with tumor promotion or suppression. Therefore, identifying new miRNA mechanisms could improve the novel diagnostic and therapeutic strategies for patients with GC.
METHODS: To explore the biological functions of miR-135b-5p in GC, bioinformatic analysis and in vitro functional assays, including colony formation, wound healing, Transwell, and EdU assays, were used to assess the proliferative, invasive, and migratory capacities of GC cells. Target genes were predicted using RNA-seq and online databases. Dual-luciferase reporter assay, fluorescence in situ hybridization and western blotting were used to confirm the regulatory relationship between miR-135b-5p and CLIP4. The role of CLIP4 in tumor progression was assessed using clinical samples and both in vitro and in vivo assays. The tumor-suppressive mechanism of CLIP4 in GC was elucidated using rescue assays.
RESULTS: Our study identified that miR-135b-5p as one of the top three over-expressed miRNAs in GC tissues, with RT-qPCR confirming its upregulation. Functional analysis showed that upregulated miR-135b-5p promoted malignant phenotypes in GC cells. Mechanistic research indicated that miR-135b-5p acts as a cancer promoter by targeting CLIP4. Moreover, our study suggested that CLIP4 exerts its tumor-suppressive function by inhibiting the JAK2/STAT3 signaling pathway.
CONCLUSIONS: This study reveals a novel mechanism by which miR-135b-5p exerts its tumor-promoting functions by targeting CLIP4. The tumor-suppressive function of CLIP4 by inactivating the JAK2/STAT3 pathway is also elucidated. Regulatory mechanism of CLIP4 by miR-135b-5p provides a promising novel therapeutic strategy for GC patients.

Despite advances in targeted therapies, primary and acquired resistance make the treatment of colorectal cancer (CRC) a pressing issue to be resolved. According to reports, the development of CRC is linked to miRNA dysregulation. Multiple studies have demonstrated that miR-135b-5p has an aberrant expression level between CRC tissues and adjacent tissues. However, it is unclear whether there is a correlation between miR-135b-5p and cetuximab (CTx) resistance in CRC. Use the GEO database to measure miR-135b-5p expression in CRC. Additionally, RT-qPCR was applied to ascertain the production level of miR-135b-5p in three human CRC cells and NCM460 cells. The capacity of cells to migrate and invade was examined utilizing the wound-healing and transwell assays, while the CCK-8 assay served for evaluating cell viability, as well as colony formation assays for proliferation. The expected target protein of miR-135b-5p in CRC cell cetuximab resistance has been investigated using western blot. Suppression of miR-135b-5p could increase the CTx sensitivity of CTx-resistant CRC cells, as manifested by the attenuation of proliferation, migration, and invasion ability. Mechanistic studies revealed miR-135b-5p regulates the epithelial-to-mesenchymal transition (EMT) process and Wnt/β-catenin signaling pathway through downgulating FOXN3. In short, knockdowning miR-135b-5p could increase FOXN3 expression in CRC cells, promote the EMT process, and simultaneously activate the Wnt/β-catenin signaling pathway to elevate CTx resistance in CRC cells.

UNASSIGNED: The main purpose of this paper is to explore the interaction between GAS5 and miR-135b-5p to understand their function in the metastasis, invasion, and proliferation of glioma. This may provide new ideas for the pathogenesis and treatment of glioma.
UNASSIGNED: Western blotting assays and RT‑qPCR were employed to investigate the expression of related genes in glioma tissues or cell lines. CCK-8 was used to examine the impact of GAS5 on cell viability. Motile activities were adopted by the transwell and wound healing experiments. A double luciferase experiment was performed to elucidate transcriptional regulation.
UNASSIGNED: GAS5 showed low expression in glioma cells and tissues, and up-regulation of GAS5 could depress the invasion, proliferation, and metastasis of glioma. GAS5 negatively regulates miR-135b-5p, which can counteract the cellular effects caused by GAS5. APC was the target of miR-135b-5p, and GAS5 can regulate the expression of APC by sponging miR-135b-5p. APC overexpression reversed the effects of miR-135b-5p promotion on glioma cells, while miR-135b-5p has the opposite function. As a downstream target gene of GAS5, miR-135b-5p was negatively regulated by GAS5. The restoration of miR-135b-5p can remarkably reverse the impact of GAS5 on glioma cells. In addition, GAS5 increased the expression of APC in glioma cells by inhibiting miR-135b-5p.
UNASSIGNED: GAS5 increased APC expression by restraining miR-135b-5p and partially blocked the progression of glioma, suggesting that it could be an advantageous therapeutic target for glioma intervention.

BACKGROUND: Thyroid carcinoma is the most frequent malignancy among endocrine-related tumours. Papillary thyroid carcinoma (PTC) is the main type of thyroid carcinoma, and almost 80% cases of thyroid carcinoma are diagnosed as PTC. The molecular mechanism underlying PTC progression is unclear. This study aims to investigate the potential mechanisms of zinc finger antisense 1 (ZFAS1) function in PTC.
METHODS: The expression of ZFAS1 and p53 was determined by quantitative polymerase chain analysis (qPCR) in PTC tissues derived from 20 PTC patients. Quantitative chromatin immunoprecipitation assay (qChIP) analysis was performed to validate the target of ZFAS1/p53 and miRNAs/p53. The Gene Expression Omnibus (GEO) dataset GSE94908 was analysed to obtain the differentially expressed p53-associated microRNAs (miRNAs). Luciferase assay validated the target of ZFAS1/miRNAs, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cell proliferation.
RESULTS: The expression of ZFAS1 was up-regulated in the tissues derived from PTC patients, and the expression of ZFAS1 was negatively associated with p53 expression in PTC. The expression of ZFAS1 was significantly higher in the MDA-T120 cells harbouring mutant p53. We validated that ZFAS1 is a direct target of p53. In PTC cells, p53 directly repressed the ZFAS1 expression. In addition, we determined that miR-135b-5p and miR-193a-3p are directly induced by p53 in PTC cells. Interestingly, p53-targeted miR-135b-5p, miR-193a-3p, and miR-34b repressed the expression of ZFAS1 via the seed-matching sequences in the 3\'-untranslated region (3\'-UTR) of ZFAS1, and thereby suppressed PTC cell proliferation induced by ZFAS1.
CONCLUSIONS: The oncogenic lncRNA ZFAS1 is directly repressed by p53 in PTC. p53-mediated miRNAs including miR-135b 5p, miR-193a-3p, and miR-34b repress ZFAS1 expression, and thereby inhibit the proliferation of PTC.

BACKGROUND: Preeclampsia was a serious complication often leaded to adverse pregnancy outcomes. Abnormal placental miR-135b-5p expression in preeclampsia was observed in our preliminary investigation. However, the role of miR-135b-5p in preeclampsia was unclear.
METHODS: We determined the miR-135b-5p expression pattern at the fetomaternal interface and levels in placental tissue and exosomes. MiR-135b-5p expression in the trophoblast cell line HTR8/SVneo was manipulated by transient agomir or antagomir transfection or establishment of HTR8/SVneo cell line stably overexpressing miR-135b or miR-135b-5p-sponger. Then the function of miR-135b-5p on the motility of HTR8/SVneo cells, and its effects on cell viability was determined. Finally, we confirmed the relationship between miR-135b-5p and ADAM12.
RESULTS: MiR-135b-5p exclusively expressed in the villous cytotrophoblast, and extravillous trophoblast. Significant miR-135b-5p upregulation was observed in the placenta and peripheral plasma exosomes in preeclampsia, and could be a highly sensitive molecular marker for preeclampsia. Elevated miR-135b-5p expression significantly promoted apoptosis and inhibited HTR8/SVneo cell invasion and migration. Binding of miR-135b-5p to the ADAM12 mRNA 3\'-untranslated region was predicted by bioinformatics analysis and confirmed using a dual-luciferase reporter assay. High miR-135-5p levels inhibit the invasion and migration of trophoblastic cells, possibly by directly binding to the 3\'-UTR of DADM12 and suppressing its translation efficiency, thereby nullifying the promotion of trophoblast invasion and migration via ADAM12.
CONCLUSIONS: Abnormal upregulation of miR-135b-5p may be involved in preeclampsia through triggering trophoblast apoptosis and impeding trophoblast invasion and migration by targeting ADAM12.

Objective: To investigate the expression status of miR-135b-5p in the sepsis induced acute lung injury (ALI) mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues. Methods: The cecal ligation puncture (CLP) method was employed to construct sepsis-induced ALI mice models. The C57BL/6 mice were randomly divided into 6 groups with 8 mice in each group. The sepsis mouse models were constructed by performing CLP surgery: mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital, the abdomen was cut longitudinally to expose the cecum, the cecum was ligated and perforated with syringe needle, the wound was sutured after extruding part of the intestinal contents. The sham operation group (Sham group) did not undergo any treatment and suture wounds after laparotomy, and no CLP operation was performed. The treatment groups were divided into CLP+NC mimic group, CLP+ miR-135b-5p mimic group, CLP+NC mimic+ Empty vector group, CLP+GSDMD group, and CLP+ miR-135b-5p mimic+GSDMD group. One week before CLP surgery, mice in the treatment group were injected subcutaneously with 200 μ L NC mimic (200 nmol/L), miR-135b-5p mimic (200 nmol/L), Empty vector (100 nmol/L), GSDMD Vector (100 nmol/L), and miR-135b-5p mimic (200 nmol/L), once a day for a week. The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation. The qRT-PCR was utilized to determine miR-135b-5p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues. The mice right lung tissues were flushed with 5 ml saline for 3 times, and each flush lasted for 3~5 min to collect the BALF, and the levels of GSDMD, IL-1β and IL-18 in BALF were determined by ELISA. The protein levels of NLRP3, caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis; Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135b-5p and GSDMD. Results: Compared with the control group mice, there were a large number of inflammatory cell infiltration, alveolar damage, interstitial edema and alveolar collapse in the lung tissues of the CLP group mice (P<0.01), and the expression levels of the pyroptosis-associated proteins (NLRP3, caspase-1 and GSDMD) were all increased, while miR-135b-5p was down-regulated in the CLP group (P<0.01). Further experiments confirmed that overexpression of miR-135b-5p significantly reduced CLP-induced pyroptotic cell death in mice lung tissues (P<0.01), and dual-luciferase reporter gene system assay confirmed that miR-135b-5p targeted GSDMD for its degradation. Moreover, the rescuing experiments validated that up-regulation of GSDMD abrogated the inhibition effects of miR-135b-5p overexpression on cell pyroptosis in CLP mice lung tissues (P<0.01). Consistently, we verified that miR-135b-5p also suppressed the expression levels of IL-1β and IL-18 in mice BALF via degrading GSDMD (P<0.05). Conclusion: Overexpression of miR-135b-5p attenuated sepsis-induced ALI by inhibiting GSDMD-mediated pyroptotic cell death, and this study provided potential therapeutic target and theoretical foundation for sepsis-induced ALI treatment.
目的: 探究miR-135b-5p在小鼠脓毒症(sepsis)引起的急性肺损伤(ALI)模型中的表达水平及其对小鼠肺部炎症反应和细胞焦亡的影响。方法: 将C57BL/6小鼠随机分为6组,每组8只,通过盲肠结扎穿刺法(CLP)手术构建CLP诱导的脓毒症小鼠模型:腹腔注射0.1 mg/kg的巴比妥麻醉,腹部纵向切开暴露盲肠,结扎盲肠并用注射器针头进行穿孔,挤出部分肠道内容物后缝合伤口。假手术组(Sham组)开腹后不做任何处理缝合伤口,无CLP手术处理。治疗组分为CLP+NC mimic组,CLP+miR-135b-5p mimic组,CLP+NC mimic+empty vector组,CLP+消皮素D (GSDMD)组,CLP+miR-135b-5p mimic+GSDMD组。治疗组小鼠在CLP手术前一周皮下注射200 μl溶解于生理盐水的NC mimic(200 nmol/L),miR-135b-5p mimic(200 nmol/L),empty vector(100 nmol/L),GSDMD vector(100 nmol/L),每天注射1次,连续一周。术后24 h采用二氧化碳窒息法实施安乐死。采用qRT-PCR检测小鼠肺组织样本中miR-135b-5p和GSDMD mRNA的表达水平;苏木精-伊红(HE)染色检测小鼠肺组织形态和损伤状态;采用5 ml生理盐水冲洗小鼠右肺3次,每次持续约3~5 min,收集肺泡灌洗液(BALF),酶联免疫吸附实验(ELISA)检测小鼠肺泡灌洗液(BALF)中GSDMD、白介素1β(IL-1β)和白介素18(IL-18)的表达水平;蛋白免疫印迹法检测小鼠肺组织内含NLR家族PYRIN域蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶1(caspase 1)以及切割后的N-端GSDMD端蛋白结构域(cleaved-GSDMD-N)的表达水平。双荧光素酶报告基因检测系统验证miR-135b-5p与GSDMD的靶向结合关系。结果: 与对照组相比,CLP组小鼠肺组织中有大量的炎症细胞浸润,肺泡损伤,细胞间质水肿及肺泡塌陷等病理特征,小鼠肺组织内细胞焦亡相关蛋白(NLRP3,caspase-1和GSDMD)的表达水平明显增加(P<0.01),但miR-135b-5p的表达水平明显下调(P<0.01);与CLP组相比,超表达miR-135b-5p能够明显抑制CLP诱导的小鼠肺组织内细胞焦亡(P<0.01),靶向抑制GSDMD的表达水平(P<0.01);超表达GSDMD能够逆转超表达miR-135b-5p对肺组织细胞焦亡的抑制作用(P<0.01),超表达miR-135b-5p能够通过靶向GSDMD抑制小鼠BALF中IL-1β及IL-18的表达水平(P<0.01)。结论: miR-135b-5p靶向下调GSDMD抑制细胞焦亡,改善脓毒症引起的ALI,为脓毒症诱导的ALI治疗提供了潜在的治疗靶点和理论依据。.

Ovarian cancer (OC) is a common gynecologic cancer with high incidence and mortality. We attempted to investigate the role of circular RNA_0000471 (circ_0000471) in OC progression and its associated mechanism.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assay were conducted to measure RNA and protein expression, respectively. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK8) assay, colony formation assay, and 5-Ethynyl-2\'-deoxyuridine (EdU) assay. Cell apoptosis was assessed by flow cytometry. Cell migration and invasion were analyzed by wound healing assay and transwell assay, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the target relationships. Xenograft tumor model was established to assess the role of circ_0000471 on tumor growth in vivo.
Circ_0000471 expression was down-regulated in OC tissues and cell lines. Circ_0000471 overexpression blocked the proliferation, migration, and invasion and triggered the apoptosis of OC cells. Circ_0000471 served as a molecular sponge for microRNA-135b-5p (miR-135b-5p), and circ_0000471 overexpression-mediated anti-tumor influences in OC cells were largely reversed by the overexpression of miR-135b-5p. Dual specificity phosphatase 5 (DUSP5) was a target of miR-135b-5p, and miR-135b-5p silencing-induced anti-tumor effects were largely counteracted by the interference of DUSP5. Circ_0000471 increased DUSP5 expression by sponging miR-135b-5p in OC cells. Circ_0000471 overexpression restrained the growth of xenograft tumors in vivo.
Overexpression of circ_0000471 inhibited OC development by targeting miR-135b-5p/DUSP5 axis, indicating that circ_0000471 may be a new potential target for OC treatment.

Parkinson\'s disease (PD) is a neurodegenerative disease resulted from the loss of dopaminergic neurons. Here, we analyzed the role of long noncoding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in PD using 1-methyl-4-phenyl pyridine (MPP+)-induced PD cell model.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were performed to determine RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry (FCM) analysis were conducted to analyze cell viability and apoptosis. Enzyme-Linked Immunosorbent Assay (ELISA) was conducted to analyze the release of inflammatory cytokines. Cytotoxicity was assessed using reactive oxygen species (ROS) assay kit, superoxide dismutase (SOD) activity assay kit and lactate dehydrogenase (LDH) activity assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between microRNA-135b-5p (miR-135b-5p) and SNHG14 or karyopherin subunit alpha 4 (KPNA4).
MPP+ treatment elevated the expression of SNHG14 in SK-N-SH cells in a dose and time-dependent manner. SNHG14 knockdown alleviated MPP+-induced apoptosis, inflammation, and cytotoxicity in SK-N-SH cells. SNHG14 interacted with miR-135b-5p, and SNHG14 silencing-mediated effects were partly overturned by miR-135b-5p knockdown in PD cell model. Besides, miR-135b-5p interacted with the 3\' untranslated region (3\'UTR) of KPNA4, and KPNA4 overexpression partly reversed miR-135b-5p overexpression-induced effects in PD cell model. SNHG14 knockdown reduced the protein level of KPNA4 partly by up-regulating miR-135b-5p in SK-N-SH cells.
SNHG14 promoted MPP+-induced neuro injury in PD cell model through mediating miR-135b-5p/KPNA4 axis.

The level of miR‑135b-5p is lower in patients with preeclampsia (PE) superimposed on chronic hypertension than in healthy controls. However, the function of miR‑135b-5p in PE progression remains unknown. In the present study, we investigated the role of miR‑135b-5p in PE development and its possible mechanism for the first time. HTR8/SVneo cells (trophoblast cell line) were exposed to hypoxia/reoxygenation (H/R) to mimic PE in vitro. Hypoxia-inducible factor-1α (HIF-1α), forkhead box O3A (FOXO3a), and miR-135b-5p levels were measured using Real-time PCR. Cell proliferation, apoptosis and migration/invasion were evaluated using the Cell Counting Kit-8 (CCK-8), flow cytometry and transwell assays, respectively. Real-time PCR and Western blotting were performed to determine the levels of several pro- and anti-angiogenic factors. The binding of miR-135b-5p to the PIK3R2-3\' untranslated region (3\'UTR) was confirmed by bioinformatics analysis and a dual-luciferase reporter assay. H/R exposure greatly upregulated HIF-1α, FOXO3a, and PIK3R2 levels, while downregulating miR-135b-5p levels in HTR8/SVneo cells. H/R exposure resulted in the inhibition of proliferation, migration, invasion, angiogenesis, and the induction of apoptosis. MiR-135b-5p overexpression reversed the effects of H/R on trophoblast cell function, while miR-135b-5p knockdown enhanced the effects. PIK3R2 knockdown had similar effects as miR-135b-5p overexpression on proliferation, apoptosis and angiogenesis. The effect of miR-135b-5p overexpression on H/R-exposed cells was enhanced by PIK3R2 knockdown. MiR-135b-5p downregulated PIK3R2 expression by pairing with its 3\'UTR. Therefore, miR-135b-5p may regulate trophoblast function by targeting PIK3R2 in PE and could serve as a novel therapeutic target for PE.

Colorectal adenocarcinoma (COAD) is a prevalent malignant tumor. Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) (CAFs-EVs) are implicated in COAD treatment. This study explored the mechanism of CAFs-EVs in COAD. CAFs and normal fibroblast (NFs) were isolated from COAD tissues and adjacent normal tissues. Vimentin, α-SMA, and FAP expressions were detected. EVs were isolated from CAFs and identified. SW480 and HCT116 cells were co-incubated with EVs. The EV uptake and COAD cell malignant behaviors were assessed. EV-treated SW480 and HCT116 cells were co-cultured with human umbilical vein endothelial cells (HUVECs). Extensive analyses were conducted to examine HUVEC proliferation, migration, and angiogenesis, and miR-135b-5p expression in COAD cells, and SW480 and HCT116 cells. CAFs were transfected with the miR-135b-5p inhibitor. miR-135b-5p downstream targets were predicted. FOXO1 expression in the co-culture system was determined and then overexpressed to evaluate its role in HUVECs mediated by COAD cells. COAD mouse model was established by transplanting SW480 cells into nude mice and injecting with EVs. Tumor growth rate, volume, and weight were examined. Ki67, VEGF, CD34, FOXO1 expressions, and VEGF content were detected. CAFs-EVs promoted COAD cell malignant behaviors and COAD cells-mediated HUVEC proliferation, migration, and angiogenesis. CAFs-EVs delivered miR-135b-5p into COAD cells. miR-135b-5p targeted FOXO1. Inhibition of miR-135b-5p in EVs or overexpression of FOXO1 partially reversed the effect of EVs on promoting COAD-induced angiogenesis. CAFs-EVs promoted tumor proliferation and angiogenesis of COAD in vivo. CAFs-EVs delivered miR-135b-5p into COAD cells to downregulate FOXO1 and promote HUVECs proliferation, migration, and angiogenesis.