Upregulation of miR-345-5p suppresses cell growth of lung adenocarcinoma by regulating ras homolog family member A (RhoA) and Rho/Rho associated protein kinase (Rho/ROCK) pathway / 中华医学杂志(英文版)

BACKGROUND@#Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells.@*METHODS@#In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance.@*RESULTS@#MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells.@*CONCLUSIONS@#MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.

MicroRNA-23a reduces lipopolysaccharide-induced cellular apoptosis and inflammatory cytokine production through Rho-associated kinase 1/sirtuin-1/nuclear factor-kappa B crosstalk / 中华医学杂志(英文版)

BACKGROUND@#MicroRNAs are closely associated with the progression and outcomes of multiple human diseases, including sepsis. In this study, we examined the role of miR-23a in septic injury.@*METHODS@#Lipopolysaccharide (LPS) was used to induce sepsis in a rat model and H9C2 and HK-2 cells. miR-23a expression was evaluated in rat myocardial and kidney tissues, as well as H9C2 and HK-2 cells. A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability, apoptosis, and the secretion of inflammatory cytokines. Furthermore, the effect of Rho-associated kinase 1 (ROCK1), a miR-23a target, on cell damage was evaluated, and molecules involved in the underlying mechanism were identified.@*RESULTS@#In the rat model, miR-23a was poorly expressed in myocardial (sham vs. sepsis 1.00 ± 0.06 vs. 0.27 ± 0.03, P < 0.01) and kidney tissues (sham vs. sepsis 0.27 ± 0.03 vs. 1.00 ± 0.06, P < 0.01). Artificial overexpression of miR-23a resulted in increased proliferative activity (DNA replication rate: Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 34.13 ± 3.12 vs. 12.94 ± 1.21 vs. 13.31 ± 1.43 vs. 22.94 ± 2.26, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), decreased cell apoptosis (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 11.39 ± 1.04 vs. 32.57 ± 2.29 vs. 33.08 ± 3.12 vs. 21.63 ± 2.35, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), and decreased production of inflammatory cytokines, including interleukin-6 (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 59.61 ± 5.14 vs. 113.54 ± 12.30 vs. 116.51 ± 10.69 vs. 87.69 ± 2.97 ng/mL; P < 0.05, F = 12.67, HK-2 cells: 68.12 ± 6.44 vs. 139.65 ± 16.62 vs. 143.51 ± 13.64 vs. 100.82 ± 9.74 ng/mL, P < 0.05, F = 9.83) and tumor necrosis factor-α (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 103.20 ± 10.31 vs. 169.67 ± 18.84 vs. 173.61 ± 15.91 vs. 133.36 ± 12.32 ng/mL, P < 0.05, F = 12.67, HK-2 cells: 132.51 ± 13.37 vs. 187.47 ± 16.74 vs. 143.51 ± 13.64 vs. 155.79 ± 15.31 ng/mL, P < 0.05, F = 9.83) in cells. However, ROCK1 was identified as a miR-23a target, and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells. Moreover, ROCK1 suppressed sirtuin-1 (SIRT1) expression to promote the phosphorylation of nuclear factor-kappa B (NF-κB) p65, indicating the possible involvement of this signaling pathway in miR-23a-mediated events.@*CONCLUSION@#Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1, mediated through the potential participation of the SIRT1/NF-κB signaling pathway.

lncRNA OIP5-AS1 targets ROCK1 to promote cell proliferation and inhibit cell apoptosis through a mechanism involving miR-143-3p in cervical cancer

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury / 亚洲男科学杂志(英文版)

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.