Effect of microRNA-21 on Proliferation and Apoptosis of Osteosarcoma Cells via Phosphatase and Tensin Homologue (PTEN)-Phosphoinositide 3-Kinase (PI3K)/AKT Pathway

2020 ◽  
Vol 10 (4) ◽  
pp. 512-517
Author(s):  
Lei Huang ◽  
Yongheng Xie ◽  
Zilong Yao ◽  
Bin Yu
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Osteosarcoma Cells ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Phosphoinositide 3 Kinase ◽  
Phosphatase And Tensin Homologue

Objective: PTEN can inhibit the activity of PI3K/AKT signaling and regulate cell proliferation and apoptosis. Increased expression of microRNA-21 is associated with osteosarcoma. Bioinformatics analysis showed a targeted binding site between microRNA-21 and PTEN 3 -UTR. Our study assessed whether microRNA-21 regulates PTEN-PI3K/AKT signaling and affects the proliferation, cloning and apoptosis of osteosarcoma cells. Methods: Dual luciferase reporter gene assay was used to assess the targeted interaction between microRNA-21 and PTEN. Expression of microRNA21 and PTEN was measured in human normal osteoblasts hFOB1.19, osteosarcoma Saos-2 and MG-63. Saos-2 cells were cultured and divided into microRNA-NC group and microRNA-21 inhibitor group followed by measuring the expression of microRNA-21, PTEN and p-AKT, cell apoptosis by flow cytometry, cell proliferation by EdU staining and cloning ability by plate cloning. Results: There was a targeted relationship between microRNA-21 and PTEN. Compared with hFOB1.19 cells, microRNA-21 level in Saos-2 and MG-63 cells was increased and PTEN was decreased. Transfection of microRNA-21 inhibitor significantly reduced microRNA-21 level in Saos-2 cells, increased PTEN, decreased p-AKT, cell proliferation and cloning ability, as well as promoted cell apoptosis. Conclusion: The increased microRNA-21 expression may play a role in reducing PTEN level and promoting osteosarcoma pathogenesis. Inhibiting microRNA-21 can inhibit the activity of PTENPI3K/AKT signaling, reduce the proliferation and cloning ability of osteosarcoma cells, and promote cell apoptosis.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals PITPNA-AS1 Activated by H3K27ac Sponged miR-98-5p to Regulate Cisplatin Resistance in Gastric Cancer

2021 ◽  
Author(s):  
Yaping Liu ◽  
Xu Zhao ◽  
Yinnan Chen ◽  
Gang Guo ◽  
Jiansheng Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Platinum Resistance ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Gastric Cancer Cell Lines ◽  
Cancer Tissues

Abstract To evaluate the expression of PITPNA-AS1 and miR-98-5p in gastric cancer tissues as well as their association with progression of gastric cancer, and investigate the role of PITPNA-AS1 and miR-98-5p in developing platinum resistance. RNA sequencing was used to identify candidate lncRNAs and microRNAs related to local recurrence of gastric cancer. qRT-PCR was used to investigate the expression of PITPNA-AS1 and miR-98-5p. CCK-8 and caspase3/7 activity were used to evaluate the cell proliferation and apoptosis rate. Dual luciferase reporter gene assay and RNA pull down were used to evaluate the cross talk between PITPNA-AS1 and miR-98-5p. PITPNA-AS1 and miR-98-5p could regulate cell proliferation and inhibit apoptosis in gastric cancer cell lines. Cisplatin and lobaplatin could significantly suppress the expression of PITPNA-AS1, which interacted with negatively regulated miR-98-5p expression. PITPNA-AS1 overexpression impaired the effect of platinum, which was partially reversed by downregulation of miR-98-5p knock down. In gastric cancer, PITPNA-AS1 and miR-98-5p could regulat cell growth, apoptosis and platinum resistance. They have the potential to be biomarkers and curative therapeutic targets. However, further research on molecular mechanisms are needed.

MiR-125b Inhibits Cell Proliferation and Induces Apoptosis in Human Colon Cancer SW480 Cells via Targeting STAT3

2021 ◽  
Vol 16 ◽  
Author(s):  
Junhe Zhang ◽  
Wenwen Yang ◽  
Yunxi Xiao ◽  
Linlin Shan
Keyword(s):  
Colon Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Colon Cancer Cells ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Sw480 Cells

Background: Colon cancer is one of the most common types of cancer worldwide. Multiple studies have unveiled the key role of microRNAs (miRNAs) in the development of various types of cancer. However, the mechanism of action of miR-125b in the development and progression of colon cancer remains unknown. Objective: In this study, we explored the association of miR-125b and signal transducer and activator of transcription 3 (STAT3) and its role in the proliferation and apoptosis of SW480 colon cancer cells. Methods: The miR-125b expression in NCM460, SW480, HT29, and HCT8 cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). SW480 cells were transfected with lentiviruses of GFP–miR–125b and GFP–NC to establish a stable miR-125b overexpression colon cancer cell model and a control model. The targeting relationship between miR-125b and STAT3 was analyzed using bioinformatics and verified by the dual-luciferase reporter gene assay. Cell proliferation and apoptosis were assessed using the Cell Counting Kit-8 assay and TUNEL staining. The expression levels of STAT3, Bcl-2, and Bax were analyzed using Western blot analysis. Results: It was found that the relative mRNA expression of miR-125b was decreased in SW480, HT29, and HCT8 cells compared with that in NCM460 cells (P<0.05). The luciferase reporter gene assay confirmed that miR-125b downregulated the STAT3 gene expression (P<0.05). Overexpression of miR-125b inhibited proliferation and promoted apoptosis in SW480 colon cancer cells and was accompanied by upregulated Bax expression and downregulated Bcl-2 expression (P<0.05). Re-expression of STAT3 promoted cell proliferation and inhibited cell apoptosis, whereas Bcl-2 expression increased, and Bax expression decreased (P<0.05). Conclusion: The miR-125b regulates the expression of Bax and Bcl-2 by downregulating the expression of STAT3, thereby inhibiting proliferation and inducing apoptosis of SW480 colon cancer cells.

scholarly journals miR-196a-2 Promotes Malignant Progression of Thyroid Carcinoma by Targeting NRXN1

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Yaohua Fan ◽  
MingJian Fei ◽  
Yan Li ◽  
Zhenzhen Gao ◽  
Yuzhang Zhu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoting Effect ◽  
Promote Cell Proliferation ◽  
Study Results ◽  
Gene Assay ◽  
Molecular Therapeutic

Thyroid cancer (TC) is the most common endocrine malignant disease with a rising morbidity year by year. Accumulating studies have shown that microRNAs (miRNAs) play a regulatory role in the progression of various tumors, but the molecular regulatory mechanism of miR-196a-2 in TC is still unknown. qRT-PCR was employed to measure the expression of miR-196a-2 and NRXN1 mRNA in TC cells, while western blot was used to detect the protein expression of NRXN1. CCK-8, colony formation and flow cytometry assays were used to measure cell proliferation and apoptosis of TC cells. Dual-luciferase reporter gene assay was used to predict and verify the targeted binding relationship between miR-196a-2 and NRXN1. Our study results manifested that miR-196a-2 was dramatically overexpressed in cells of TC, while NRXN1 was lowly expressed. miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC. Additionally, miR-196a-2 could also target and inhibit the expression of NRXN1. Silencing NRXN1 could reverse the inhibitory effect of miR-196a-2 downregulation on cell proliferation of TC, as well as the promoting effect on cell apoptosis. In a conclusion, we found that miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC by targeting NRXN1. Therefore, miR-196a-2/NRXN1 is potential to be a molecular therapeutic target for TC.

scholarly journals Knockdown of lncRNA HOTTIP Inhibits Retinoblastoma Progression by Modulating the miR-101-3p/STC1 Axis

2021 ◽  
Vol 20 ◽  
pp. 153303382199783
Author(s):  
XiangWen Yuan ◽  
Zhaoyan Sun ◽  
Congxian Cui
Keyword(s):  
Cell Proliferation ◽  
Western Blotting ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Y79 Cells

Objective: Retinoblastoma (RB) is a frequent eye cancer in children. Long non-coding RNA (LncRNA) HOXA transcript at the distal tip (HOTTIP) is aberrantly expressed in cancer tissues. This study explores the underlying mechanism of lncRNA HOTTIP in RB. Methods: HOTTIP expression in normal retinal cells and RB cell lines was detected using qRT-PCR. The proliferation of RB cells was measured using CCK-8 and EdU assays, and apoptosis was detected using flow cytometry and Western blotting after the transfection of si-HOTTIP into Y79 cells and pc-HOTTIP into HXO-RB-44 cells. The target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1 were predicted by bioinformatics website and verified using dual-luciferase reporter gene assay. The binding of HOTTIP and miR-101-3p was verified using RNA pull-down assay. STC1 mRNA and protein in RB cells were measured using qRT-PCR and Western blotting. Moreover, si-HOTTIP and in-miR-101-3p/in-NC, and si-HOTTIP and pc-STC1/pcDNA were co-transfected into Y79 cells respectively to evaluate cell proliferation and apoptosis. Xenograft study was conducted, and Ki67-positive expression was detected using immunohistochemical staining. Results: HOTTIP expression was promoted in RB tissues and cells. Downregulation of HOTTIP inhibited proliferation and promoted apoptosis of Y79 cells, while upregulation of HOTTIP promoted proliferation and inhibited apoptosis of HXO-RB-44 cells. There were target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1. Inhibition of miR-101-3p or overexpression of STC1 reversed the effect of si-HOTTIP on the proliferation and apoptosis of RB cells. Xenograft study showed that knockdown of HOTTIP suppressed the growth of RB in vitro. Conclusion: It could be concluded that HOTTIP sponged miR-101-3p to upregulate STC1 expression, thereby promoting RB cell proliferation and inhibiting apoptosis.

scholarly journals miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaoyi Zhu ◽  
Zhan Shen ◽  
Da Man ◽  
Hang Ruan ◽  
Sha Huang
Keyword(s):  
Colon Cancer ◽  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cancer Occurrence ◽  
Promising Target ◽  
Gene Assay ◽  
The Relationship

Objective. The purpose of this study was to investigate the relationship between miR-152-3p and the KLF4/IFITM3 axis, thereby revealing the mechanism underlying colon cancer occurrence and development, consequently providing a promising target for colon cancer treatment. Methods. Bioinformatics methods were implemented to analyze the differential expression of miRNAs and mRNAs in colon cancer, confirm the target miRNA, and predict the downstream targeted mRNAs. qRT-PCR and Western blot were performed to detect the expression of miR-152-3p, KLF4, and IFITM3. CCK-8 and colony formation assays were conducted for the assessment of cell proliferation, and flow cytometry was carried out for the detection of cell apoptosis. Finally, dual-luciferase reporter gene assay was employed to verify the targeting relationship between miR-152-3p and KLF4. Results. miR-152-3p was highly expressed in colon cancer cells, whereas KLF4 was poorly expressed. Dual-luciferase assay verified that miR-152-3p targeted to bind to KLF4 and suppressed its expression. Moreover, silencing miR-152-3p or overexpressing KLF4 was found to downregulate IFITM3, thereby inhibiting cell proliferation and potentiating cell apoptosis. In rescue experiments, we found that miR-152-3p deficiency decreased the expression of IFITM3 and weakened cancer cell proliferation, and such effects were restored when miR-152-3p and KLF4 were silenced simultaneously. Conclusion. In sum, we discovered that miR-152-3p can affect the pathogenesis of colon cancer via the KLF4/IFITM3 axis.

miR-29b-3p’s Effects on Prostate Cancer

2022 ◽  
Vol 12 (4) ◽  
pp. 681-689
Author(s):  
Zhou Hongyi ◽  
Yan Zhiqiang ◽  
Zhu Leilei ◽  
Li Maolin ◽  
Shao Jianfeng ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Wound Healing ◽  
Cell Apoptosis ◽  
Cell Number ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Nuclear Levels

Objection: Our research wanted to discuss miR-29b-3p in PCa occurrence and development and relative mechanisms. Methods: Collecting adjacent and cancer tissues from prostate cancer patients and measuring miR-29b-3p expressions by RT-qPCR and ISH assay. Using DU145 and PC3 cell lines which the miR-29b-3p were high expression in our study. Using miR inhibitor to knockdown miR-29b-3p in DU145 and PC3. Using CCK-8 and flow cytometry to measure cell proliferation and cell apoptosis, invasion cell number by transwell and wound healing rate by wound healing assay. The relative proteins expressions were measured using WB assay. p-AKT nuclear levels were evaluated using Cell immunofluorescence test. Using dual-luciferase reporter gene assay to analysis correlation miR-29b-3p and PTEN. Results: miR-29b-3p gene significantly increased. miR-29b-3p knockdown had effects to depress cell proliferation, increase cell apoptosis, depress invasion cells number and wound healing rates. PTEN proteins were significantly up-regulation and p-AKT and MMP-9 proteins expressions were significantly down-regulation (P < 0.001, respectively). And p-AKT nuclear volume were significantly depressed. And miR-29b-3p could target PTEN. Conclusion: miR-29b-3p played an oncology gene in prostate cancer via regulation PTEN/AKT pathway in vitro study.

LncRNA LINC00858 enhances cervical cancer cell growth through miR-3064-5p/ VMA21 axis

2021 ◽  
pp. 1-11
Author(s):  
Min Wei ◽  
Youguo Chen ◽  
Wensheng Du
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Cancer Cell Growth ◽  
Protein Coding ◽  
Gynecological Malignancy ◽  
Promising Therapeutic Target ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

BACKGROUND: Cervical cancer (CC) is the most common form of gynecological malignancy. Long intergenic non-protein coding RNA 858 (LINC00858) has been identified to participate in multiple cancers. However, the role and mechanism of LINC00858 in CC cells are still elusive. AIM: The aim of this study is to explore the biological functions and mechanisms of LINC00858 in CC cells. METHODS: RT-qPCR analysis was used to examine the expression of LINC00858 in CC cells. EdU and colony formation assay were utilized to assess cell proliferation. TUNEL assay and flow cytometry assay were conducted to assess cell apoptosis. The mechanism regarding LINC00858 was certified through RNA pull down, RIP and luciferase reporter assays. RESULTS: The up-regulated LINC00858 was detected in CC cells. Reduction of LINC00858 effectively subdued CC cells proliferation and stimulated cell apoptosis. LINC00858 was determined to bind with miR-3064-5p and up-regulate VMA21 in CC cells. In rescue assays, miR-3064-5p down-regulation and VMA21 up-regulation were able to counteract the effect caused by LINC00858 decrease on CC cell proliferation and apoptosis. CONCLUSION: LINC00858 enhances cell proliferation, while restraining cell apoptosis in CC through targeting miR-3064-5p/VMA21 axis, implying that LINC00858 may serve as a promising therapeutic target for CC.

scholarly journals MiR-486 protects against acute myocardial infarction via regulation of PTEN

2021 ◽  
Vol 20 (9) ◽  
pp. 1845-1853
Author(s):  
Qinfeng Han ◽  
Zhong Xu ◽  
Xiaolei Zhang ◽  
Kun Yang ◽  
Zhifei Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Akt Signaling ◽  
Reporter Gene Assay ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Purpose: To investigate the effect of miR-486 on rats with acute myocardial infarction (AMI), and its mechanism of action.Methods: A rat model of AMI was established. They were randomly divided into 4 groups, namely, sham, model, agomiR-486 and antagomiR-486 groups, respectively. Rats in these different groups were treated with agomiR-21 (5 μL, 40 nmol/mL), antagomiR-21 (5 μL, 40 nmol/mL) or agomiR-NC (5 μL, 40 nmol/mL), respectively. Then, key miRNAs were sorted out using gene-chip assay and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Luciferase reporter gene assay was conducted to determine the interaction between miR-486 and gene of PTEN. After intraperitoneal injection of agomiR-486 and antagomiR-486, hemodynamics was measured to determine the effect of miR-486 on myocardial function of the rats. The effect of miR-486 expression level on the expression of myocardial enzymes in serum, the morphology of myocardial tissues, and the apoptosis of myocardial tissues in rats, were investigated. Additionally, the effect of miR-486 expression level on PTEN/AKT signaling pathway in the rats was determined by Western blotting.Results: The results of gene-chip and qRT-PCR assays revealed that there were 8 differentially expressed genes in rat myocardial tissues in the model group when compared with the sham group. MiR-486 improved the cardiac function of rats and the morphology of myocardial tissues, but reduced AMI-induced apoptosis of myocardial cells and the expression of myocardial enzymes (markers of myocardial injury) in a dose-dependent manner (p < 0.05). The results of luciferase reporter gene assay showed that PTEN was a direct target of miR-486. In rat models of AMI, a raised expression of miR-486 remarkably suppressed the protein expression level of PTEN and up-regulated that of p-AKT/AKT (p < 0.05).Conclusion: MiR-486 protects against AMI in rats probably by targeting PTEN and activating the AKT signaling pathway. The results of the current study may provide new insights for the treatment of AMI.

Effect of miRNA-223-3p Targeting Stromal Interaction Molecule 1 on Proliferation and Apoptosis of Hypoxia/Reoxygenation-Applied H9c2 Cardiomyocytes

2021 ◽  
Vol 11 (6) ◽  
pp. 1066-1072
Author(s):  
Bin Guang ◽  
Xiaoqin Liu ◽  
Tingchen Liang
Keyword(s):  
Cell Activity ◽  
Luciferase Reporter ◽  
Cardiomyocyte Proliferation ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
H9c2 Cardiomyocytes ◽  
Cultured Cardiomyocytes ◽  
Hypoxia Reoxygenation

This study was established to determine the effect of miRNA-223-3p on the proliferation and apoptosis of hypoxia/reoxygenation-applied H9c2 cardiomyocytes and the associated mechanisms. A hypoxia/reoxygenation (H/R) model was established, with normal cells also used as a control. miRNA-NC, miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p plasmids were transfected into normally cultured cardiomyocytes, defined as the miRNA-NC, miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p groups. In addition, miRNA-223-3p was co-transfected into normally cultured cardiomyocytes with pcDNA3.1 and pcDNA3.1-STIM1 plasmids, followed by treatment with H/R for cells in the miR-NC and miR-223-3p groups, defined as the H/R+miRNA-NC, H/R+miRNA-223-3p, H/R+miRNA-223-3p+pcDNA3.1, and H/R+miRNA-223-3p+pcDNA3.1-STIM1 groups. A liposome method was adopted for assessing transfection. qRT-PCR was used to detect miRNA-223-3p expression, while western blotting was used to detect protein expression. MTT assay was used to detect cell viability, flow cytometry to detect apoptosis, and dual luciferase reporter gene assay to detect fluorescence activity. After H/R treatment, miR-223-3p, cyclin D1, and Bcl-2 expression of cardiomyocytes decreased, p21 and Bax expression significantly increased, cell activity decreased, and the apoptosis rate increased. miRNA-223-3p achieved the targeted regulation of STIM1 expression. miRNA-223-3p overexpression promoted the H/R-induced cardiomyocyte proliferation and inhibited cardiomyocyte apoptosis. STIM1 overexpression reversed the proliferation-promoting and apoptosis-inhibiting effects of miRNA-223-3p on cardiomyocytes treated with H/R. The findings show that miRNA-223-3p overexpression promotes H/R-induced cell proliferation, inhibits apoptosis, and protects H/R-induced cardiomyocytes from injury, via a mechanism probably associated with STIM1 expression. miRNA-223-3p thus provides a new target for treating cardiomyocyte injury.

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