LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p / 中华肿瘤杂志

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.

Let -7i : A key player and a promising biomarker in diseases / 中南大学学报(医学版)

MicroRNAs (miRNAs) are endogenous non-coding single-stranded small RNAs that regulate gene expression by recognizing homologous sequences and interfering with transcriptional, translational or epigenetic processes. MiRNAs are involved in a variety of disease processes, and regulate the physiological and pathological status of diseases by modulating target cell activity, migration, invasion, apoptosis, autophagy and other processes. Among them, let-7i is highly expressed in various systems, which participates in the process of tumors, cardiovascular and cerebrovascular diseases, fibrotic diseases, inflammatory diseases, neurodegenerative diseases and other diseases, and plays a positive or negative regulatory role in these diseases through different signal pathways and key molecules. Moreover, it can be used as an early diagnosis and prognostic marker for a variety of diseases and become a potential therapeutic target. As a biomarker, let-7i is frequently tested in combination with other miRNAs to diagnose multiple diseases and evaluate the clinical treatment or prognosis.

Effects of irinotecan combined with XELOX regimen on prognosis and serum CD cells, miR-34a and let-7i in patients with advanced gastric cancer / 中国医师杂志

Objective:To investigate the effect of irinotecan combined with XELOX regimen on serum CD cells, miR-34a and let-7i in patients with advanced gastric cancer.Methods:A total of 72 patients with advanced gastric cancer treated in the Affiliated Hospital of Hubei University of Arts And Science from January 2015 to January 2020 were prospectively selected, and they were divided into control group and observation group with 36 cases in each group by random number table method. The control group was treated with XELOX regimen, while the observation group was treated with irinotecan on the basis of the control group. The efficacy, serum immune level, serum miR-34a and let-7i contents, incidence of toxic and side effects and life cycle of the two groups were compared.Results:The objective response rate (ORR) of the observation group was 33.33% , which were significantly higher than that of the control group (13.89%, χ 2=3.770, P>0.05). There was no significant difference in serum immune level, miR-34a and let-7i between the two groups before treatment (all P>0.05). After treatment, the levels of CD3 + , CD4 + and CD8 + in the two groups were significantly increased (all P<0.05), and the improvement of CD3 + , CD4 + and CD8 + in observation group was significantly better than that in control group (all P<0.05). After treatment, the serum miR-34a level increased significantly and serum let-7i level decreased significantly in the two groups (all P<0.05), and the serum miR-34a level in the observation group was higher than that in the control group, and the serum let-7i level was significantly lower than that in the control group (all P<0.05). Among the grade Ⅰ-Ⅱ adverse reactions, nausea and vomiting and leukocyte decline were the most common; The incidence of grade Ⅲ-Ⅳ leukopenia in the control group and the observation group were 5.56%(2/36) and 5.56%(2/36), respectively; The incidence of grade Ⅲ-Ⅳ thrombocytopenia was 5.56%(2/36) and 2.78%(1/36), respectively; One patient in the control group had grade Ⅲ-Ⅳ abnormal liver function, and one patient in the observation group had grade Ⅲ-Ⅳ nausea and vomiting. All patients with adverse reactions were effectively relieved after symptomatic treatment, and there were no treatment-related deaths. The progression free survival (PFS) of the observation group was 24.90 months (95% CI: 0.469-1.955), and that of the control group was 21.85 months (95% CI: 0.512-2.131). The PFS of the observation group was significantly longer than that of the control group (log rank=1.357, P=0.007). Conclusions:Irinotecan combined with XELOX regimen in the treatment of advanced gastric cancer can effectively improve the level of serum miR-34a and immune function, reduce the content of let-7i, and has good safety.

Highly expressed miRNA let-7i-5p in renal cell carcinoma tissues regulates the malignant biological behaviors of 769-P cells through hyaluronan-binding protein 4 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miRNA let-7i-5p在肾细胞癌（renal cell carcinoma，RCC）组织中的表达水平及其对RCC细胞769-P的增殖、迁移、侵袭和透明质酸结合蛋白4（hyaluronan-binding protein 4，HABP4）表达的影响。方法：利用TCGA RCC数据库及GEO数据库对let-7i-5p在RCC组织中的表达进行meta分析。体外常规培养人RCC细胞769-P并对其进行转染，根据转染物不同分为过表达组（转染let-7i-5p模拟物）、抑制组（转染let-7i-5p抑制物）和对照组（转染NC序列）。采用CCK-8、细胞划痕实验、Transwell实验及WB法检测let-7i-5p对769-P细胞增殖活力、划痕愈合率、穿膜侵袭细胞数及HABP4表达水平的影响。双荧光素酶报告基因实验验证let-7i-5p与HABP4 mRNA的靶向关系。结果：分析TCGA RCC数据库及5个GEO数据集（GSE23085、GSE47582、GSE95385、GSE16441和GSE71302）中数据结果显示，let-7i-5p在RCC组织中的表达水平显著高于正常肾组织（均P<0.05）。体外实验结果显示，与对照组相比，过表达组在24、48及72 h时细胞增殖活力显著升高，而抑制组显著降低（均P<0.01）；过表达组的划痕愈合率[（37.276±2.058）% vs（15.663±2.949）%，P<0.01]和穿膜侵袭细胞数[（377.000±34.044） vs （255.667±25.384）个，P<0.01]均显著升高，而抑制组的划痕愈合率[（8.791±2.568）% vs（15.663±2.949）%，P<0.05]和穿膜侵袭细胞数[（170.333±14.978）vs（255.667±25.384）个，P<0.01]均显著降低。在野生型HABP4-3’ URT质粒组中，过表达let-7i-5p可显著抑制细胞的荧光素酶活性（P<0.01）；而在突变型HABP4-3’ URT质粒组中，过表达let-7i-5p对细胞的荧光素酶活性无影响（NS，P>0.05）。WB检测结果显示，与对照组相比，过表达组的HABP4和E-cadherin的水平均降低（均P<0.01）、CDK2的水平升高（P<0.01），而抑制组则相反（均P<0.01）。结论：Let-7i-5p在RCC组织中呈高表达，其可能通过靶向HABP4基因来促进769-P细胞的增殖、迁移和侵袭。

IcarisideII facilitates the differentiation of ADSCs to SCs via let-7i/STAT3 axis to preserve erectile function

BACKGROUND: IcarisideII (ICAII) could promote the differentiation of adipose tissue-derived stem cells (ADSCs) to Schwann cells (SCs), leading to improvement of erectile function (EF) and providing a realistic therapeutic option for the treatment of erectile dysfunction (ED). However, the underlying molecular mechanisms of ADSCs and ICAII in this process remain largely unclear. METHODS: ADSCs were treated with different concentrations of ICAII. Cell proliferation was determined by MTT assay. qRT-PCR and western blot were performed to detect expressions of SCs markers, signal transducer and activator of transcription-3 (STAT3), and microRNA-let-7i (let-7i). Luciferase reporter assay was conducted to verify the regulatory relationship between let-7i and STAT3. The detection of intracavernosal pressure (ICP) and the ratio of ICP/mean arterial pressure (MAP) were used to evaluate the EF in bilateral cavernous nerve injury (BCNI) rat models. RESULTS: ICAII promoted cell proliferation of ADSCs in a dose-dependent manner. The mRNA and protein levels of SCs markers were increased by ICAII treatment in a dose-dependent manner in ADSCs. Moreover, let-7i was significantly decreased in ICAII-treated ADSCs and upregulation of let-7i attenuated ICAII-induced promotion of SCs markers. In addition, STAT3 was a direct target of let-7i and upregulated in ICAII-treated ADSCs. Interestingly, overexpression of STAT3 abated the let-7i-mediated inhibition effect on differentiation of ADSCs to SCs and rescued the ICAII-mediated promotion effect on it. Besides, combination treatment of ADSCs and ICAII preserved the EF of BCNI rat models, which was undermined by let-7i overexpression. CONCLUSION: ICAII was effective for preserving EF by promoting the differentiation of ADSCs to SCs via modulating let-7i/STAT3 pathway.

Let-7i regulates glioma U251 stem cell differentiation through LIN28 protein / 中华神经医学杂志

Objective To investigate the effect of MicroRNA (miRNA) let-7i on differentiation ofglioma U251 stem cells and the potential mechanisms.Methods Glioma U251 cells were cultured in vitro and CD133 positive cells were sorted by magnetic cell separation.Composite let-7i mimics and let-7i controls were transfected into U251 cells; the let-7i expression was detected by real time-PCR; the expressions ofCD133,nestin and LIN28 were detected by Western blotting; the differentiation status was assessed via glial fibrillary acidic protein (GFAP) immunocytochemistry.Composite LIN28 siRNA and siRNA control were transfected into U251 cells; the expressions of CD133 and nestin were detected by Western blotting.Targetscan software analysis and luciferase reporter system were employed to verified the possibility of LIN28 being the target gene of let-7i.Results let-7i in the U251 cells were over-expressed in group of let-7i mimics,enjoying 17.9 fold of group of let-7i control; the expressions of CD 133,nestin and LIN28 were 13.9％,43.7％ and 53.6％,separately,of group of let-7i control; GFAP positive labeling index in group oflet-7i mimics (83.0±1.93)％ was significantly higher than that of group oflet-7i control (39.7±6.73)％ (P＜0.05).The expressions of CD133 and nestin in group of LIN28 siRNA decreased to 23.7％ and 37.9％ of those in groups of siRNA control.LIN28 3'UTR existed let-7i paired binding sites and LIN28 could be the target gene of let-7i.Conclusion over-expressing let-7i could significantly down-regulate the expressions of CD133 and nestin,and enhance the glioma stem cell differentiation through the potential mechanism of down-regulating the expression of LIN28.