Hsa-miR-425-5p Inhibits Hypertrophic Scar Formation Through Suppressing the Growth of Human Hypertrophic Scar Fibroblasts and Extracellular Matrix Deposition by Targeting Smad2

2020 ◽  
Vol 10 (3) ◽  
pp. 352-359
Author(s):  
Pengju Fan ◽  
Zhen Li ◽  
Wuyuan Tan ◽  
Man Fang
Keyword(s):  
Extracellular Matrix ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Hypertrophic Scar ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Matrix Deposition ◽  
Qrt Pcr ◽  
Apoptosis Protein ◽  
Extracellular Matrix Deposition

The current study aimed to explore the role and mechanism of microRNA-425-5p (miR-425-5p) in hypertrophic scar (HS) development. Firstly, we used reverse transcription-quantitative polymerase chain reaction (qRT-PCR)to detect the expression of miR-425-5p in human hypertrophic scar fibroblasts (hHSFs) and HS tissues. qRT-PCR assay showed that miR-425-5p level significantly down-regulated in HS tissues and hHSFs. Next, we performed TargetScan and dual-luciferase reporter assay to predict and verify Smad2 was the target gene of miR-425-5p. In order to determine the role of miR-425-5p in HS formation, miR-425-5p was over-expressed or knockdown in hHSFs through transfection with miR-425-5p mimic or miR-425-5p inhibitor. CCK-8 assay and cell apoptosis analysis were carried out to measure cell viability and apoptosis. Protein expression was assessed by Western blotting. The findings indicated that miR-425-5p mimic transfection inhibited cell viability, promoted cell apoptosis and repressed Smad2, Col I, and Col III expression in hHSFs. Notably, the transfection of Smad2-plasmid eliminated the effects of miR-425-5p mimic on hHSFs. However, miR-425-5p inhibitor transfection had opposite effects on hHSFs, and were eliminated by the transfection of Smad2-siRNA. In conclusion, these findings suggested that miR-425-5p inhibited the hHSFs viability, induced hHSFs apoptosis and repressed extracellular matrix deposition of hHSFs through regulating Smad2. Therefore, miR-425-5p might be a novel therapeutic target for HS treatment.

scholarly journals The Inhibition of Long Non-Coding RNA CAR10 and The Regulation of miR-203 Expression Suppresses Cell Viability and Induces Cell Apoptosis in Prostate Cancer

2020 ◽  
Author(s):  
Liansheng Zhang ◽  
Yougan Chen ◽  
Zhenjie Wang ◽  
Qiang Xia
Keyword(s):  
Prostate Cancer ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Gene Assay ◽  
Long Non Coding Rna

Abstract Background: Prostate cancer (PC) is one of the most common malignant tumors. Recently, it has been reported that long noncoding RNAs (lncRNAs) play key roles in tumor progression. Studies have revealed that long non-coding RNA CAR10 (CAR10) can regulate tumor cell behaviors through sponging miR-203. In this study, we examined the effects of CAR10 in PC cells. Methods: Firstly, real time-quantitative polymerase chain reaction (qRT-PCR) was used to explore CAR10 expression in tumor tissues, peripheral blood of PC patients, and PC cells. We used the dual-luciferase reporter gene assay to analyze the relationship between CAR10 and miR-203. Moreover, flow cytometry, MTT assay, and western blot assay were used to determine cell apoptosis, cell viability, and apoptosis-related protein expression. Results: The results showed that CAR10 expression was remarkably higher in PC samples compared with that of control, and CAR10 regulated miR-203 negatively in PC cells. The qRT-PCR results also showed that miR-203 expression was significantly decreased in PC samples. Moreover, knockdown of CAR10 inhibited PC cell viability and promoted cleaved caspase-3 expression but induced PC cell apoptosis and, reduced pro-caspase-3 expression; miR-203 inhibitor reversed these effects. Conclusion: Our study found that CAR10 is a potential oncogene in PC and suggests that CAR10 inhibition could inhibit PC cell viability but promote PC cell apoptosis through regulating miR-203 expression. Our results show that CAR10 is a potential target for the treatment of PC.

scholarly journals MicroRNA-9-5p inhibits proliferation and induces apoptosis of human hypertrophic scar fibroblasts through targeting peroxisome proliferator-activated receptor β

Biology Open ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. bio051904
Author(s):  
Chi-Yung Chai ◽  
I.-Chun Tai ◽  
Rui Zhou ◽  
Junlong Song ◽  
Chaoying Zhang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hypertrophic Scar ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Western Blot Assay ◽  
Peroxisome Proliferator Activated Receptor ◽  
Matrix Deposition ◽  
Induced Apoptosis

ABSTRACTHypertrophic scar (HS) is a dermal fibro-proliferative disorder result from abnormal wound healing after skin injury. MicroRNA-9-5p (miR-9-5p) has been reported to be upregulated and closely related to collagen proteins in human dermal fibroblasts. However, the correlation and possible mechanism between miR-9-5p and HS require further investigation. The expressions of miR-9-5p in HS tissues and HS fibroblasts were detected by quantitative real-time PCR (RT-qPCR). The expression level of peroxisome proliferator-activated receptor β (PPARβ) was measured by RT-qPCR assay. The protein levels of PPARβ, α-SMA, Vimentin, COL1A, cyclin D1, bcl-2, and bax were detected by western blot assay. The effect of miR-9-5p and PPARβ on HS fibroblasts proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry assays. The interaction between miR-9-5p and PPARβ was predicted by TargetScan, and then confirmed by dual-luciferase reporter assay. MiR-9-5p expression was downregulated in HS tissues and HS fibroblasts. MiR-9-5p inhibited the levels of extracellular matrix-associated genes (α-SMA, Vimentin, COL1A) in HS fibroblasts. MiR-9-5p repressed proliferation and induced apoptosis of HS fibroblasts. PPARβ is a target gene of miR-9-5p. The silencing of PPARβ expression hindered proliferation and expedited apoptosis of HS fibroblasts. MiR-9-5p suppressed proliferation and promoted apoptosis of HS fibroblasts by targeting PPARβ. In this paper, we firstly disclosed that miR-9-5p hampered extracellular matrix deposition and proliferation, and induced apoptosis by targeting PPARβ in HS fibroblasts. Our findings provided a new role of miR-9-5p/PPARβ in the occurrence and development of HS fibroblasts, promising a new target for HS.

p75NTR silencing inhibits proliferation, migration and extracellular matrix deposition of hypertrophic scar fibroblasts by activating autophagy through inhibiting PI3K/Akt/mTOR pathway

2020 ◽  
Author(s):  
Wen Shi ◽  
Yan Wu ◽  
Donghui Bian
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Hypertrophic Scar ◽  
Mtor Pathway ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Inhibitory Effects ◽  
Matrix Deposition ◽  
Extracellular Matrix Deposition

Hypertrophic scar (HS) results from abnormal wound healing, accompanied by excessive hypercellularity, migration and extracellular matrix (ECM) deposition. Autophagy dysregulation plays crucial roles during HS formation. The overexpressed p75 neurotrophin receptor (p75NTR) in injured skin tissue after wound healing becomes a factor aggravating scar. The study was designed to investigate the role of p75NTR and p75NTR-mediated autophagy in the process of HS. The results revealed that p75NTR expression was significantly upregulated while that of autophagy proteins was downregulated in cicatrix at 3 and 6 months after burn, which was recovered at 12 months. p75NTR silencing inhibited proliferation, migration and ECM deposition of hypertrophic scar fibroblasts (HSF), whereas p75NTR overexpression presented the opposite results. Silencing of p75NTR reduced the expression of PI3K/Akt/mTOR signaling molecules while enhanced that of autophagy proteins. Importantly, PI3K agonist (IGF-1) intervention notably decreased the levels of LC3B II/I and Beclin-1, and restored the inhibitory effects of p75NTR silencing on proliferation, migration and ECM deposition of HSF. Concurrently, autophagy inhibitor 3-methyladenine (3-MA) treatment exhibited the same variation trends with IGF-1. Taken together, these findings demonstrated that p75NTR silencing inhibits proliferation, migration and ECM deposition of HSF by activating autophagy through inhibiting PI3K/Akt/mTOR pathway.

scholarly journals Knockdown of NCK1-AS1 inhibits the development of atherosclerosis by targeting miR-1197/COX10 axis

2022 ◽  
Vol 16 (1) ◽  
Author(s):  
Bin Zhang ◽  
Juncheng Wang ◽  
Lei Du ◽  
Lufei Shao ◽  
Yourui Zou ◽  
...  
Keyword(s):  
Cell Viability ◽  
Inflammatory Cytokines ◽  
Cell Apoptosis ◽  
Cell Model ◽  
Human Cancer ◽  
Density Lipoprotein ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Blood Samples ◽  
Qrt Pcr

Abstract Background Although long non-coding RNA (lncRNA) NCK1-AS1 plays important roles in human cancer, its function in atherosclerosis (AS) remains unclear. Method The expression of NCK1-AS1 in AS blood samples was detected by qRT-PCR. Oxidized low-density lipoprotein (ox-LDL) was used to construct the AS cell model, and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to evaluate NCK1-AS1 level. Cell phenotypes including proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The malondialdehyde level was measured to evaluate oxidative stress. The expression of apoptosis-related proteins was evaluated by western blot. The expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) was measured by qRT-PCR and ELISA assays. The relationship among NCK1-AS1, miR-1197 and COX10 was determined by bioinformatic analysis and luciferase reporter assay. Results NCK1-AS1 was significantly upregulated in AS blood samples and ox-LDL stimulated vascular smooth muscle cells (VSMCs). Knockdown of NCK1-AS1 increased cell viability, reduced cell apoptosis and MDA level, and also inhibited the expression of inflammatory cytokines (IL-1β, IL-6 and TNK-α) in ox-LDL stimulated VSMCs. NCK1-AS1 could positively regulate COX10 expression by directly sponging miR-1197. Moreover, co-transfection of sh-NCK1-AS1 and miR-1197 inhibitor, or co-transfection of sh-NCK1-AS1 and pc-COX10 (COX10 overexpressing plasmid) obviously reduced cell viability, promoted cell apoptosis, and increased MDA level in VSMCs followed by ox-LDL treatment for 24 h compared to that in sh-NCK1-AS1 transfected VSMCs. Conclusion Our study revealed that knockdown of NCK1-AS1 attenuated the development of AS by regulating miR-1197/COX10 axis, suggesting that this lncRNA might be a potential therapeutic target for AS.

microRNA-133b Regulates Cell Proliferation and Cell Cycle Progression via Targeting HuR in Colorectal Cancer

2022 ◽  
Vol 12 (2) ◽  
pp. 335-345
Author(s):  
Xiaoyan Zhang ◽  
Wei Zhu ◽  
Junjie Lu
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Ht 29

MicroRNAs (miRNAs/miRs) have been identified to serve a key role in the development of tumors. However, the role of miR-133b in colorectal cancer (CRC) remains largely unclear. This study will investigate the role and mechanism of miR-133b in CRC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the level of miR-133b in CRC cell lines. Bioinformatics software TargetScan predicted the potential target genes of miR-133b, and a dual luciferase reporter assay was used to confirm this. To investigate the role of miR-133b in CRC cells, miR-133b was upregulated or downregulated in CRC cell lines (SW620 and HT-29) by transfecting with a miR-133b mimic or inhibitor, respectively. Subsequently, cell viability was analyzed using MTT assay, whereas cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. In addition, the associated protein levels were detected using western blot analysis. The results demonstrated that miR-133b was significantly downregulated in CRC cell lines when compared with the normal colonic epithelial NCM-460 cell line. Human antigen R (HuR; also termed ELAVL1) was demonstrated to be a direct target of miR-133b and was negatively regulated by miR-133b. HuR was also notably upregulated in the CRC cell lines when compared with the normal control. Transfection of SW620 and HT-29 cells with the miR-133b mimic significantly inhibited cell viability, and induced cell apoptosis and G1 phase arrest, while upregulation of HuR demonstrated the opposite effects. Furthermore, the present data demonstrated that the miR-133b mimic significantly enhanced the protein levels of p21 and p27, and downregulated cyclin D1 and cyclin A levels in SW620 and HT-29 cells; the opposite effects were observed following treatment with the miR-133b inhibitor. In conclusion, the data indicate that miR-133b suppressed CRC cell growth by targeting HuR.

miR-4262 Targets Anti-Apoptotic Gene B-Cell Lymphoma-2 to Induce Cell Apoptosis and Inhibit Cell Proliferation in Oral Squamous Cell Carcinoma

2020 ◽  
Vol 10 (6) ◽  
pp. 804-811
Author(s):  
Guangyao Hu ◽  
Dianxiu Wu
Keyword(s):  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Viability ◽  
B Cell ◽  
Cell Apoptosis ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Qrt Pcr

Oral squamous cell carcinoma (OSCC), a frequently happened cancer, is still an important threaten to human with unsatisfactory prognosis. Increasing evidence indicated that abnormal miRNA expressions were related to the development of OSCC. microRNA (miR)-4262 has been considered to be a cancer suppressor in various tumors, however its exact role in OSCC remains to be clarified. During the current research, we proposed to probe the biological activity and fundamental mechanism of miR-4262 in OSCC. The expression levels of miR-4262 in SCC9 and HOK cells were analyzed using quantitative real time polymerase chain reaction (qRT-PCR). TargetScan and luciferase reporter assay were carried out to quest the possible target gene of miR-4262. qRT-PCR and Western blotting analysis were employed to measure the expressions of B-cell lymphoma-2 (Bcl-2) in OSCC tissues, adjacent non-cancerous tissues, SCC9 and HOK cells. Cell proliferation and apoptosis of SCC9 cells were detected by Thiazolyl Blue Tetrazolium Bromide (MTT) and flow cytometry (FCM) analysis. The expressions of apoptosis-related proteins Bcl-2 and Bcl2-associated X protein (Bax) were checked by Western blotting analysis. Firstly, we found that miR-4262 expressed lower level in OSCC cells than that in the control. Results from TargetScan and luciferase reporter analysis showed that miR-4262 directly targeted Bcl-2. Then, up-regulated Bcl-2 was detected in OSCC tissues and cells compared with controls. Subsequently, Bcl-2-siRNA was found to be able to decrease cell viability while promote cell apoptosis in SCC9 cells, accompanied with the reduction of Bcl-2 and promotion of Bax. The Bcl-2/Bax ratio was reduced in Bcl-2-siRNA transfected group. In addition, miR-4262 mimic significantly suppressed the Bcl-2 expression, whereas the suppression was reversed by Bcl-2-plasmid. Furthermore, our results presented that miR-4262 mimic notably reduced cell viability and promoted cell apoptosis in SCC9 cell lines. Up-regulated miR-4262 could also downregulate Bcl-2 expression, upregulate the expression of Bax and decrease the Bcl-2/Bax ratio in SCC9 cells. However, Bcl-2-plasmid attenuated all these effects of miR-4262 mimic. Taken together, our findings indicated that miR-4262 exerted tumor-suppressive effects through targeting Bcl-2, resulting in promotion of cell apoptosis and inhibition of cell viability in OSCC cell lines. Therefore, miR-4262 might be a promising prognostic biomarker and novel therapeutic target during the therapy of OSCC.

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