MiR-145-5p targeting TLR4 participates in inflammation and oxidative stress of atherosclerosis foam cells / 中华老年心脑血管病杂志

Objective To explore the role of microRNA-145-5p(miR-145-5p)in the regulation of inflammatory response and oxidative stress process in cellular model of atherosclerosis.Methods Human monocytic leukemia THP-1 cells-derived foam cells were constructed in vitro.Then,the relative expression of miR-145-5p in the model and control groups of cells were detected by qRT-PCR.TargetScan database was used to predict the targeting relationship between miR-145-5p and Toll-like receptor 4(TLR4).The 293T cells were divided into wild-type+mimic group,wild-type+mimic negative control(NC)group,and mutant+mimic group,mutant+mimic NC group,and dual luciferase assay was employed to verify the targeting relationship of miR-145-5p and TLR4.Foam cells were cultured in vitro and divided into miR-145-5p mimic group,mimic NC group,miR-145-5p inhibitor group,and inhibitor NC group according to the corresponding treat-ments.The expression of TLR4 at mRNA and protein levels was detected by qRT-PCR and Wes-tern blotting.The contents of TNF-α,IL-1β and IL-6 were detected by ELISA.Biochemical reagent kits were applied for generation of reactive oxygen species(ROS),content of MDA and activity of SOD.Results The expression of miR-145-5p was significantly reduced in the model group than the control group(0.29±0.01 vs 1.00±0.08,t=11.180,P＜0.01).Dual luciferase assay showed that luciferase activity was significantly lower in the miR-145-5p mimic group than the mimic NC group(t=8.612,P＜0.01).Compared with the mimic NC group,the mimic group had obviously lower mRNA and protein levels of TLR4 and contents of TNF-α,IL-1β,lL-6,ROS and MDA,and higher miR-145-5p expression level and SOD activity(P＜0.05,P＜0.01).The treatment of inhibi-tor resulted in increased TLR4 mRNA and protein levels and TNF-α,IL-1β,IL-6,ROS and MDA contents,and decreased miR-145-5p expression and SOD activity when compared with the above levels in the inhibitor NC group(P＜0.05,P＜0.01).Conclusion MiR-145-5p inhibits inflamma-tion and oxidative stress in cellular model of atherosclerosis by targeting TLR4.

miR-145-5p affects the proliferation of colorectal cancer cells and cytotoxicity of NK cells by targeting regulation of RAD18 expression / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探索RAD18影响结直肠癌细胞增殖及调节NK细胞对结直肠细胞的杀伤作用及其可能的机制。方法：采用生物信息学技术分析结直肠癌组织中RAD18和miR-145-5p的表达及两者之间的调控关系、分析RAD18富集通路。采用qPCR法验证RAD18和miR-145-5p在结直肠癌细胞中的表达，双荧光素酶报告基因实验验证miR-145-5p与RAD18的调控关系。按转染物的不同将SW480、HCT-15细胞分为将si-RAD18组、si-NC组，另向SW480细胞分别转染inhibitor-NC+si-NC、miR-145-5p inhibitor+si-NC或miR-145-5p inhibitor+si-RAD18，采用CCK-8法、克隆形成实验分别检测敲降miR-145-5p和/或RAD18对细胞增殖、克隆形成的影响；将各组细胞分别与经IL-2激活的NK92细胞共培养，采用乳酸脱氢酶释放法、ELISA和免疫荧光染色法分别检测NK细胞的细胞毒性、细胞因子分泌及细胞表面穿孔素和颗粒酶B表达的影响。结果：RAD18在结直肠癌组织和细胞中呈高表达（均P<0.01）。敲降RAD18可以抑制结直肠癌细胞增殖能力（P<0.05）和促进NK细胞活力、细胞毒性、IFN-γ、TNF-α、GM-CSF分泌及穿孔素和颗粒酶B的表达（均P<0.05）。双荧光素酶报告实验验证了RAD18-3’UTR与miR-145-5p的结合关系，miR-145-5p在结直肠癌组织和细胞中低表达（P<0.05或P<0.01）。miR-145-5p可以靶向下调RAD18的表达（P<0.05），过表达RAD18可以逆转miR-145-5p过表达对NK细胞杀伤效应的促进作用（均P<0.05）。结论：miR-145-5p可靶向下调RAD18的表达，miR-145-5p/RAD18轴能够影响结直肠癌细胞的增殖和NK细胞对其的细胞毒作用。

Hsa_circ_0000392 affects the radiation sensitivity of cervical cancer by targeting the miR-145-5p/CRKL/MAPK pathway / 中华肿瘤杂志

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.

Hsa_circ_0000392 affects the radiation sensitivity of cervical cancer by targeting the miR-145-5p/CRKL/MAPK pathway / 中华肿瘤杂志

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.

Role of miR-145-5p Targeting ARK5 in Regulating the Proliferation and Apoptosis of Human Epithelial Ovarian Cancer Cells / 中国医学科学院学报

Objective To explore the effect of miR-145-5p on the proliferation and apoptosis of human ovarian cancer cells and the possible molecular mechanisms involved.Methods Real-time quantitative PCR was performed to detect the expression of miR-145-5p in ovarian epithelial cells and ovarian cancer cells.CCK-8 and flow cytometry were used to detect the effects of miR-145-5p overexpression on the proliferation and apoptosis of ovarian cancer cells.TargetScan was employed to predict the target genes of miR-145-5p.Western blotting,dual luciferase reporter assay and rescue experiment were employed to predict and verify the underlying molecular mechanism of miR-145-5p function.Results The expression of miR-145-5p in ovarian cancer cells was significantly lower than that in normal ovarian epithelial cells(

Over-expression of miR-145-5p inhibits malignant biological behaviors of esophageal squamous cells carcinoma via down-regulating IGF1R / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the mechanism of miR-145-5p on malignant biological behaviors, such as pro-liferation, invasion, migration and epithelial-mesenchymal transition (EMT), of esophageal squamous cell carcinoma (ESCC) TE-10 cells. Methods: The expression of miR-145-5p in ESCC cell lines and normal cells was detected by PCR. Dual luciferase reporter gene assay was used to detect the targeted regulation between miR-145-5p and insulin-like growth factor 1 receptor (IGF1R). The expres-sions of IGF1R protein and EMT related proteins were detected by Western blotting. Transwell assay and CCK-8 assay were carried out to detect the effects of miR-145-5p/IGF1R axis on the proliferation, migration andinvasionofTE-10 cells. Results: miR-145-5p was down-regulated in ESCC cell lines with the lowest expression in TE-10 cells (P<0.01orP<0.05).Over-expression of miR-145-5p significantly inhibited proliferation, invasion, migration and EMT of TE-10 cells (P<0.01 or P<0.05). Dual luciferase reporter gene assay con-firmed that miR-145-5p targetedly down-regulated IGF1R expression (P<0.01). The restora-tion experiments further confirmed that simultaneous over-expression of miR-145-5p and IGF1R significantly attenuated the promotion effect of IGF1R on proliferation, invasion, migration and EMT of TE-10 cells (P<0.01 or P<0.05). Conclusions: Over-expression of miR-145-5p inhibits proliferation, invasion, migration and EMT of ESCC TE-10 cells by down-regulating IGF1R.

Expression of miR-145-5p in colorectal cancer and its relationship with multidrug resistance / 解剖学报

Objective To investigate the expression and relationship between miR-145-5p and multidrug resistance gene (MDR1) protein P-glycoprotein(P-gp) in colorectal cancer. Methods SP, Real-time PCR and Western blotting were used to detect the expression of P-gp colorectal carcinoma (CRC) in 50 patients and in 30 paracancerous normal tissue patients, the effect of miR-145-5p expression on MDR1 mRNA and P-gp and the correlation between them and clinicopathological features. Results The expression level of miR-145-5p in CRC was significantly lower than that in paracancerous normal tissue, and the expression level of MDR1 mRNA and P-gp was significantly higher than that in paracancerous normal tissue (r=-0. 403, P<0. 01).MiR-145-5p could inhibit the expression of MDR1 mRNA and P-gp in HCT-15 cells(P<0. 05). Conclusion MiR-145-5p plays a regulatory role in the expression of multidrug resistance genes and proteins in colorectal cancer, and is involved in the occurrence, development and multidrug resistance of colorectal cancer.