<i>LINC00609</i> inhibits A549 cells progression through the regulation of <i>miR-128-3p/RND3</i> axis

Background: Long-chain non-coding RNA (lncRNA) LINC00609 is a potential tumor suppressor, but the mechanism of action in non-small cell lung cancer (NSCLC) is yet to be understood.Objectives: The effects of LINC00609 on A549 cell proliferation, apoptosis, and cell cycle arrest were investigated. Methods: The LINC00609 levels in NSCLC and normal tissues were analyzed by bioinformatics. Expressions of LINC00609, miR-128-3p, and Rho family GTPase 3 (RND3) in NSCLC cells (A549) were determined by qRT-PCR. Bioinformatics analysis predicted target genes and dual-luciferase reporter assays to ensure that LINC00609 targeted miR-128-3p and miR-128-3p targeted RND3. The proliferation of cells was determined using EDU and CCK-8. Flow cytometry was used to evaluate cell apoptosis rate and cell cycle. The western blotting assay identified proteins related to proliferation and apoptosis. Results: In NSCLC tissues, LINC00609 was expressed in low levels, while its high expression was associated with a higher survival rate. LINC00609 affected cell proliferation, apoptosis, cell cycle arrest, and expression of related proteins. Dual-luciferase reporter assay showed that LINC00609 binds specifically to miR-128-3p, and miR-128-3p binds to RND3. MiR-128-3p overexpression could neutralize the effects of LINC00609. A siRNA targeting RND3 could reverse the effect of the miR-128-3p inhibitor. Silencing RND3 resulted in a decrease in apoptosis rate and the number of cells in the S-phase and an increase in the number of cells in the G1-phase. Furthermore, phosphorylation levels of the AKT protein and mTOR protein, and Bcl2 expression, increased; however, the expression of RND3, Bax, and caspase3 decreased. Conclusions: LINC00609 regulated miR-128-3p/RND3 axis to modulate A549 cell proliferation, apoptosis, and cell cycle arrest. In the case of NSCLC, LINC00609 could be a potential target for therapy.     

MiR-194-5p suppresses the warburg effect in ovarian cancer cells through the IGF1R/PI3K/AKT axis

Background: The Warburg effect is considered as a hallmark of various types of cancers, while the regulatory mechanism is poorly understood. Our previous study demonstrated that miR-194-5p directly targets and regulates insulin-like growth factor1 receptor (IGF1R). In this study, we aimed to investigate the role of miR-194-5p in the regulation of the Warburg effect in ovarian cancer cells. Methods: The stable ovarian cell lines with miR-194-5p overexpression or silencing IGF1R expression were established by lentivirus infection. ATP generation, glucose uptake, lactate production and extracellular acidification rate (ECAR) assay were used to analyze the effects of aerobic glycolysis in ovarian cancer cells. Gene expression was analyzed by quantitative polymerase chain reaction (qPCR) and western blot. Immunohistochemistry assays were performed to assess the expression of the IGF1R protein in ovarian cancer tissues. Results: Overexpression of miR-194-5p or silencing IGF1R expression in ovarian cancer cells decreases ATP generation, glucose uptake, lactate production, and ECAR and inhibits both the mRNA and protein expression of PKM2, LDHA, GLUT1, and GLUT3. While the knockdown of miR-194-5p expression led to opposite results. Overexpression of miR-194-5p or silencing IGF1R expression suppressed the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, whose activation can sustain aerobic glycolysis in cancer cells, and the knockdown of miR-194-5p expression promoted the activation of the PI3K/AKT pathway. Conclusion: Our results suggest that miR- 194-5p can inhibit the Warburg effect by negative regulation of IGF1R and further repression of the PI3K/AKT pathway, which provides a theoretical basis for further test of miR-194-5p as a target in the treatment of ovarian cancer.     

Effect of microRNA-143-3p-mediated CTNND1 on the biological function of lung cancer cells

Lung cancer poses a serious threat to human life with high incidence and miRNA is an important biomarker in tumors. This study aimed to explore the effect of miR-143-3p on the biological function of lung cancer cells and the underlying mechanism. Eighty-seven samples of lung cancer tissues and 81 samples of tumor-adjacent tissues from patients undergoing radical lung cancer surgery in our hospital were collected. The lung cancer cells and lung fibroblast cells (HFL-1) were purchased, and then miR-143-3p-mimics, miR-NC, si-CTNND1, and NC were transfected into A549 and PC-9 cells to establish cell models. MiR-143-3p and CTNND1 expression levels were measured by the qRTPCR, Bax, Bcl-2, and CTNND1 expression levels by the Western Blot (WB), and cell proliferation, invasion, and apoptosis by the MTT assay, Transwell assay, and flow cytometry. Dual luciferase report assay was used to determine the relationship between miR-143-3p and CTNND1. In this study, miR-143-3p was lowly expressed in lung cancer and CTNND1 was highly expressed in lung cancer. The overexpression of miR-143-3p inhibited cell proliferation and invasion, promoted cell apoptosis, significantly increased Bax protein expression, and decreased Bcl-2 protein expression. The inhibition of CTNND1 led to opposite biological characteristic in cells. The dual luciferase reporter assay demonstrated that miR-143-3p was a target region of CTNND1. Such results suggest that miR-143-3p can inhibit the proliferation and invasion of lung cancer cells by regulating the expression of CTNND1 and promote the apoptosis of lung cancer cells, sott is expected to be a potential target for lung cancer.     

Oncolytic adenovirus targeting LASP-1 inhibited renal cell cancer progression

Recent studies suggested that LIM and SH3 protein 1 (LASP-1) is a promising therapeutic target for renal cell cancer (RCC). This study aimed to explore the role of LASP-1 in RCC. For this purpose, LASP-1 expression in RCC tissues was analyzed by immunohistochemistry and Western blot analysis. Cell proliferation, migration, invasion, and gene expression were detected by CCK-8 assay, Transwell assay, and Western blot analysis. The results showed that LASP-1 was highly expressed in RCC, and its expression level,t was positively correlated with lymph node metastasis and tumor, nodes, and metastases (TNM) stage. The knockdown of LASP-1 expression significantly inhibited the proliferation of RCC cells, increased the apoptosis rate, and inhibited RCC cell invasion and migration by inhibiting epithelial–mesenchymal transition. We conclude that LASP-1 promotes RCC progression and metastasis and is a promising therapeutic target for RCC.     

MicroRNA-708 inhibits the proliferation and chemoresistance of pancreatic cancer cells

Pancreatic cancer is one of the most aggressive malignancies with poor prognosis and high mortality. Recent studies showed that microRNAs are dysregulated and involved in the initiation and progression of pancreatic cancer. In this study, we found that miR-708 was significantly downregulated in pancreatic cancer tissues and cell lines. Lentivirus-mediated overexpression of miR-708 could significantly inhibit the proliferation and invasion, while enhanced chemosensitivity to gemcitabine in both Panc-1 and SW1990 cells. Luciferase reporter assay showed that miR-708 bound the 3’-untranslated region of survivin and suppressed the expression of survivin in pancreatic cancer cells. In pancreatic cancer tissues, survivin protein was highly expressed and negatively correlated with miR-708 expression. Furthermore, the restoration of survivin expression could partially antagonize proliferation inhibition and apoptosis induction by miR-708 in pancreatic cancer cells. The Panc-1 cells with overexpression of miR-708 also showed decreased proliferation capability in nude mouse model compared with parental cells. In conclusion, our results suggest that miR-708 inhibits pancreatic cancer and could be a novel potential candidate to treat pancreatic cancer.     

Puerarin inactivates NLRP3-mediated pyroptotic cell death to alleviate cerebral ischemia/reperfusion (I/R) injury through modulating the LncRNA DUXAP8/miR-223-3p axis

NLRP3 inflammasome-mediated cell pyroptosis aggravates the development of cerebral ischemia/reperfusion (I/R) injury, and the aim of this study is to investigate the potential utilization of the Chinese medicine, Puerarin, in treating this disease. Through conducting in vitro and in vivo experiments, the present study illustrated that Puerarin regulated LncRNA double homeobox A pseudogene 8 (DUXAP8)/miR-223-3p axis to inactivate NLRP3-mediated pyroptotic cell death, resulting in the attenuation of I/R injury. Specifically, the cerebral I/R injury in rat models and hypoxia/reoxygenation (H/R) in primary hippocampus neuron (PHN) cells were inducted, which were subsequently exposed to Puerarin treatment. As expected, we validated that Puerarin suppressed cell pyroptosis and rescued cell viability in I/R rat hippocampus tissues and H/R PHN cells. Next, through bioinformatics analysis, we noticed that miR-223-3p targeted both LncRNA DUXAP8 and NLRP3 mRNA, and both LncRNA DUXAP8 ablation and miR-223-3p overexpression inactivate NLRP3-mediated cell pyroptosis to rescue cell viability in H/R PHN cells. Interestingly, we evidenced that Puerarin restrained LncRNA DUXAP8 expressions, but upregulated miR-223-3p in I/R rat tissues and H/R PHN cells, and the protective effects of Puerarin on H/R PHN cells were abrogated by overexpressing LncRNA DUXAP8 and silencing miR-223-3p. Collectively, we concluded that Puerarin regulated LncRNA DUXAP8/miR-223-3p/NLRP3 signaling cascade to attenuate I/R injury.     

Comparison of inflammatory microRNA expression in healthy and periodontitis tissues

MicroRNAs (miRNAs) are short RNA molecules that negatively regulate gene expression primarily by degrading target mRNA or inhibit the translation of protein product. Recently, many reports have shown the altered miRNA expression in various diseases. However, there are no reports on miRNA expression related to periodontitis. Thus, this study aimed to compare the miRNAs differentially expressed in healthy and chronic periodontitis tissues and to determine the miRNAs closely associated with chronic periodontitis. To find out the miRNAs differentially induced in healthy and chronic periodontitis tissues, miRNA microarray was carried out and the expression of miRNAs was confirmed by real-time PCR. According to miRNA microarray analyses, six miRNA genes, let-7a, let-7c, miR-130a, miR301a, miR-520d, and miR-548a, were up-regulated more than 8 fold compared to the healthy gingiva. The expression of twenty-two miRNAs was increased more than 4 fold. Among these miRNAs, eight miRNAs which are known to be closely related to inflammation were selected. Six of these miRNA genes, miR-181b, miR-19b, miR-23a, miR-30a, miR-let7a, and miR-301a, were amplified successfully and increased much more in periodontitis gingivae than in healthy ones. In summary, this study indicate that six miRNAs up-regulated in periodontitis gingiva may play a key role in chronic periodontitis.     

MicroRNA-181a is elevated by 10-hydroxycamptothecin and represses lung carcinoma progression by downregulating FOXP1

Tumor progression is usually characterized by proliferation, migration, and angiogenesis, which is essential for supplying both nutrients and oxygen to the tumor cells. Therefore, targeting angiogenesis has been considered a promising therapeutic strategy for cancer prevention and treatment. In the present study, we demonstrated that in addition to suppressing lung cancer cell proliferation and migration in vitro, 10-hydroxycamptothecin (10-HCPT) is also capable of inhibiting angiogenesis in vivo with a miR-181a-dependent manner. Mechanistically, by upregulating miR-181a, which in turn downregulating FOXP1, 10-HCPT can inhibit the PI3K/Akt/ERK signaling pathwaymediated angiogenesis. Furthermore, reduced levels of miR-181a have been found in both lung cancer cell lines and xenograft with concurrently elevated levels of FOXP1, VEGF, bFGF, and HDGF. Consistent with the findings from the in vitro experiments, miR-181a impairs neovascularization in our xenograft model. In summary, our findings have not only established the anti-oncogenic role of miR-181a in lung cancer angiogenesis but also suggest that 10-HCPT could be a potential therapeutic reagent for lung cancer treatment.     

Downregulation of hsa_circ_0002198 inhibits keloid fibroblast activities <i>in vitro</i> by reducing NLRP3 inflammasome activity

The levels of hsa circular RNA_0002198 (hsa_circ_0002198) have been found to be significantly upregulated in keloid dermal fibroblasts. However, the functional role of hsa_circ_0002198 in keloid fibroblasts and the underlying molecular mechanism for its effects have not been reported. In this study, the levels of hsa_circ_0002198 and nucleotide-binding and oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) expression in keloid scar tissues and adjacent normal skin tissues were determined by quantitative real-time PCR and western blotting, respectively. In vitro models of keloid tissue were created by culturing primary keloid fibroblasts obtained from patients. A series of functional experiments, including CCK-8 assays, Transwell assays, and ELISA assays were performed to analyze the functional role of hsa_circ_0002198/NLRP3. Our data showed that hsa_circ_0002198 and NLRP3 were upregulated in keloid scar tissues when compared with adjacent normal tissues. Knockdown of hsa_circ_0002198 expression significantly suppressed cell proliferation, migration, and invasion, and those effects could be partially reversed by forced NLRP3 overexpression in keloid fibroblasts. At the molecular level, knockdown of hsa_circ_0002198 downregulated the levels of Col I, α-SMA, and NLRP3 proteins, as well as the levels of TGF-β, IL-1ß, and IL-33, but upregulated caspase 3 expression in keloid fibroblasts. All those effects were partially reversed after NLRP3 overexpression. In conclusion, our results suggest hsa_circ_0002198 as a potential target for treating keloid lesions.     

Effect of RBM10 on regulating the proliferation and metastasis activity of human hepatocellular carcinoma cells by affecting the stability of miR-21

In recent decades, RNA binding motif (RBM) proteins have been widespread concerned by researchers. Among them, RBM5 is considered as a potential tumor suppressor gene in HCC. RBM10, also belonging to the RBM family, have similar structure and high homology with RBM5, indicating its potential as potential tumor suppressor genes. However, the role of RBM10 in tumors is controversial. The purpose of this study was to analyze the expression correlation and functional relationship of miR-21 and RBM10 in human hepatocellular carcinoma (HCC) tissues and corresponding tumor cells. Bioinformatics analysis showed that miR-21 and RBM10 were both highly expressed in HCC; similarly, the expression levels of miR-21 and RBM10 in HCC cells were significantly higher than those in normal hepatocytes. There was a positive correlation between miR-21 and RBM10. Furthermore, knockdown of RBM10 inhibited the proliferation, migration and invasion of SNU-398 cells, and simultaneous overexpression of miR-21 attenuated this inhibitory effect. Meanwhile, overexpressing RBM10 could promote the proliferation, migration and invasion of Hep G2 cells; while the stability of miR-21 could be reduced by knocking down RBM10. On the whole, the findings of this study indicate that RBM10 is involved in regulating the function and activity of human HCC cells by affecting the stability of miR-21. RBM10 cannot be simply summarized as a tumor suppressor or oncogene. Further studies are needed to clarify the role of RBM10 in tumors.     

LncRNA LINC01772 promotes metastasis and EMT process in cervical cancer by sponging miR-3611 to relieve ZEB1

Cervical cancer (CC), has been identified as one of the most frequent malignant tumors all over the world, with high mortality in females. A growing number of investigations have confirmed that long noncoding RNAs (lncRNAs) play a crucial role in the progression of multiple cancers. Nonetheless, the biological function and regulatory mechanism of LINC01772 in CC haven’t been explored so far. In this study, LINC01772 expression was found to be upregulated in tissues and cells of CC. Knocking down LINC01772 suppressed CC cell proliferation, migration and epithelial-mesenchymal transition (EMT) process. Through molecular mechanism assays, LINC01772 was verified to be bound with miR-3611 and LINC01772 acted as a sponge for miR-3611. Zinc finger E-box binding homeobox 1 (ZEB1) was a downstream target gene of miR-3611. ZEB1 was negatively regulated by miR-3611 but positively regulated by LINC01772. Rescue assays confirmed that miR-3611 inhibitor or ZEB1 overexpression offset the inhibitive effect of LINC01772 depletion on cell proliferation, migration and EMT process in CC. In a word, our study validated that LINC01772 promoted cell metastasis and EMT process in CC by sponging miR-3611 to upregulate ZEB1 expression, indicating that LINC01772 could serve as a new therapeutic target for patients with CC.     

miRNA-148b-3p targeting SOCS3 inhibits macrophage M2 polarization by JAK2/STAT3 pathway in immune thrombocytopenia

Aberrant expression of miRNAs is significantly correlated with the occurrence of immune thrombocytopenic purpura (ITP). The immune imbalance of M1/M2 macrophage contributes to the development of ITP. However, the role of miR-148b-3p in macrophage phenotype imbalance remains unknown in ITP. In this study, we aimed to explore whether miR-148b-3p inhibits M2 macrophage polarization in ITP and to investigate the underlying mechanism. Peripheral blood from 22 ITP patients were collected, and real-time PCR confirmed that miR-148b-3p was up-regulated and Western blot analyses detected the expression of SOCS3 was down-regulated. Subsequent dual-luciferase reporter gene assay indicated that miR-148b-3p could bind to SOCS3. Furthermore, we found significant correlation between miR-148b-3p expression and platelet count. Applying gain and lose the function experiments of miR-148b-3p and SOCS3, we demonstrated that suppression of miR-148b-3p or up-regulation of SOCS3 promoted macrophage M2 polarization by inhibiting JAK2/STAT3 pathway. Together, our findings demonstrate that that miR-148b-3p targeting SOCS3 inhibits M2 macrophage polarization via JAK2/STAT3 signaling in ITP.     

Exosomes derived from osteoclasts under compression stress inhibit osteoblast differentiation

Orthodontic tooth movement is triggered by orthodontic force loading on the periodontal ligament and is achieved by alveolar bone remodeling, which is regulated by intimate crosstalk between osteoclastogenesis and osteoblast differentiation. Whether the communication between osteoclasts and osteoblasts is influenced by orthodontic compression stress requires further clarification. In this study, osteoclasts were differentiated for 10 days. On day 4 of differentiation, the number of pre-osteoclasts peaked, as determined by the increased expression of RANK and the number of multinucleated cells. After 24 h of compression stress loading, on day 4, the number of osteoclasts increased, and the optimal magnitude of stress to promote osteoclastogenesis was determined as 1 g/cm2. Moreover, the results of RNA-sequencing analysis showed that the miRNA expression profile changed markedly after compression loading and that many of the altered miRNAs were associated with cell communication functions. A series of indirect co-culture experiments showed an inhibitory effect of osteoclasts on osteoblast differentiation, especially after compression. Next, we added osteoclast-derived exosomes to hPDLSCs during osteoblast differentiation. Exosomes derived from osteoclasts under compression (cEXO) showed a greater inhibitory effect on osteoblast differentiation, compared to exosomes from osteoclasts without compression (EXO). Therefore, we analyzed differentially expressed miRNAs associated with bone development functions in exosomes: miR-223-5p and miR-181a-5p were downregulated, whereas miR-133a-3p, miR-203a-3p, miR-106a-5p, and miR-331-3p were upregulated; these altered expressions may explain the enhanced inhibitory effect of compression stress.     

Exosomal miR-218 regulates the development of endometritis in dairy cows by targeting TGIF2/TGF-β pathway

Endometritis affects the reproductive capacity of dairy cows and leads to serious economic losses in dairy farming. Clarification of the pathogenesis of endometritis is necessary to improve the reproductive efficiency of dairy cows. Exosomes and their miRNAs have been proven to play an important role in inflammatory regulation. Exosomal miR-218 is a differentially expressed miRNA found in endometrial epithelial cells (EECs) under endometrial inflammation. Therefore, we investigated the expression of miR-218 in the uterine tissue of dairy cows, lipopolysaccharide (LPS) treated EECs, exosomal vesicles, and regulation of exosomal miR-218 by targeting TGIF-2 inducible factor homology frame 2 (TGIF2)/transforming growth factor-beta (TGF-β). The expression of miR-218 was suppressed in inflammatory uterine tissues and LPS treated EECs. The expression of TGIF2 and TGF-β in inflammatory uterine tissues and LPS treated EECs was significantly higher than those in healthy uterine tissues and EECs (p < 0.01). Interestingly, miR-218 derived from donor cells was found to regulate the expression of the target gene TGIF2 in recipient cells through the fusion of exosomes. Concurrently, the expression of its target gene TGIF2 was also suppressed by miR-218 in donor cells resulting in fewer TGIF2 being transported into recipient cells with exosomal fusion. This may be a novel mechanism of miRNAs-mediated regulation and provides a new reference for analyzing the pathogenesis of endometritis in dairy cows.     

Hepatitis B virus X protein-mediated upregulation of miR-221 activates the CXCL12-CXCR4 axis to promote NKT cells in HBV-related hepatocellular carcinoma

Backgrounds: Both hepatitis B virus X protein (HBx) and microRNA-221 (miR-221) have been implicated in the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). The present study demonstrates that HBx promotes HCC cell proliferation via the C-X-C motif chemokine ligand 12-C-X-C chemokine receptor type 4 (CXCL12-CXCR4) axis. We predict that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC. Methods: After miR-221 mimic, miR-221 mimic negative control, miR-221 inhibitor, miR-221 inhibitor negative control were transfected into cells, the expression of CXCL12 and miR-221 was detected by qPCR and western blot. Then we constructed a stable HBV-HCC cell line. HBV-HCC cells were injected into the nude mice, thus a HBV-HCC mouse model was constructed. Q-PCR and western blot were used to detect the expression of HBx, miR-221, CXCL12 and CXCR4 in tumor tissues. The expression of CXCL12 was detected by immunohistochemistry, and the expression of CXCR4, CD3 and CD56 was detected by immunofluorescence. The levels of CXCL12, IL-2 and TNF-α in serum of mice were detected by ELISA. Sixty-one patients with HBV-related HCC, 61 patients with HBV-related cirrhosis, 61 patients with chronic hepatitis B (CHB) and 30 healthy people were enrolled. CXCL12, cytokine levels, and clinicopathological parameters were tested. Results: Hepatitis B virus X protein upregulates the expression of miR-221 and CXCL12 in lentivirus (LV5)-HBx-transfected HepG2 cells. HBx protein promotes HepG2 cell proliferation in vitro. HBx protein promoted tumor growth via the miR-221/CXCL12/CXCR4 pathway in a mouse tumor model. HBx protein upregulated natural killer T cell expression via the CXCR4/CXCL12 pathway to promote tumor growth. The data demonstrated a positive correlation between CXCL12 concentration with Cre levels and Child-Pugh scores. CXCL12 had an inferior diagnostic efficiency compared to IL-2 and IL-6 for HBV-related HCC. Conclusions: We present evidence that HBx/miR-221-mediated CXCL12/CXCR4 signaling induces NKT cells to promote HBV-related HCC.