scholarly journals miR-145-5p Overexpression Inhibits the Proliferation, Migration and Invasion of Esophageal Carcinoma Cells by Targeting ABRACL

2020 ◽  
Author(s):  
Shengming Fan ◽  
Pei Chen ◽  
Shugang Li
Keyword(s):  
Carcinoma Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Regulatory Relationship ◽  
Ec Cells ◽  
Differentially Expressed Mirna ◽  
Invasion Assays ◽  
Dual Luciferase Reporter Assay

Abstract Background: The study aimed to investigate the regulatory relationship between miR-145-5p and ABRACL, and tried to clarify the mechanisms underlying the proliferation, migration and invasion of esophageal cancer (EC) cells.Methods: Gene expression data related to EC were accessed from TCGA database and the “edgeR” package was used to screen the differentially expressed miRNA (DEmiRNAs) and genes (DEGs). TargetScan, miRDB and miRTarBase databases were used to predict potential targets for the target miRNA miR-145-5p. qRT-PCR and Western blot were performed to assess the expression of miR-145-5p and ABRACL in EC cells. Dual-luciferase reporter assay was performed for verification of the targeting relationship between miR-145-5p and ABRACL. Functional experiments including colony formation assay, Transwell migration and invasion assays were used to detect the proliferation, migration and invasion abilities of EC cells. Results: The expression of miR-145-5p was significantly decreased in EC, while ABRACL was remarkably increased. In addition, there was a negative correlation identified between miR-145-5p and ABRACL expression levels. Overexpressing miR-145-5p was able to suppress cell proliferation, migration and invasion, whereas silencing miR-145-5p posed an opposite effect. In the meantime, ABRACL was identified as a direct target of miR-145-5p by dual-luciferase reporter assay. Furthermore, miR-145-5p could inhibit the expression of ABRACL, in turn inhibiting the proliferation, migration and invasion of EC cells. Conclusion: miR-145-5p functions on the proliferation, migration and invasion of EC cells via targeting ABRACL, and it may be a novel therapeutic target for EC treatment.

scholarly journals miR-145-5p Inhibits the Proliferation, Migration, and Invasion of Esophageal Carcinoma Cells by Targeting ABRACL

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Shengming Fan ◽  
Pei Chen ◽  
Shugang Li
Keyword(s):  
Esophageal Carcinoma ◽  
Carcinoma Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Regulatory Relationship ◽  
Ec Cells ◽  
Invasion Assays ◽  
Dual Luciferase Reporter Assay

Objective. The study is aimed at investigating the regulatory relationship between miR-145-5p and ABRACL, and has tried at clarifying the mechanisms underlying the proliferation, migration, and invasion of esophageal carcinoma (EC) cells. Methods. Gene expression data related to EC were accessed from TCGA database, and the “edgeR” package was used to screen differentially expressed genes. TargetScan, miRDB, and miRTarBase databases were used to predict potential targets for the target miRNA miR-145-5p. qRT-PCR and Western blot were performed to assess the expression of miR-145-5p and ABRACL in EC cells. Dual-luciferase reporter assay was performed to validate the targeting relationship between miR-145-5p and ABRACL. Functional experiments including CCK-8 assay, Transwell migration, and invasion assays were used to detect the proliferation, migration, and invasion of EC cells. Results. The expression of miR-145-5p was significantly decreased in EC, while ABRACL was remarkably increased. In addition, there was a negative correlation identified between miR-145-5p and ABRACL mRNA. Overexpressing miR-145-5p was able to suppress cell proliferation, migration, and invasion, whereas silencing miR-145-5p posed an opposite effect. In the meantime, ABRACL was identified as a direct target of miR-145-5p by dual-luciferase reporter assay. Furthermore, miR-145-5p could inhibit the expression of ABRACL, in turn inhibiting the proliferation, migration, and invasion of EC cells. Conclusion. miR-145-5p functions on the proliferation, migration, and invasion of EC cells via targeting ABRACL, and it may be a novel therapeutic target in EC treatment.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals FENDRR Sponges miR-424-5p to Inhibit Cell Proliferation, Migration and Invasion in Colorectal Cancer

2020 ◽  
Vol 19 ◽  
pp. 153303382098010
Author(s):  
Chuan Cheng ◽  
Huixia Li ◽  
Jiujian Zheng ◽  
Jie Xu ◽  
Peng Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Differential Analysis ◽  
Inhibit Cell Proliferation ◽  
Dual Luciferase Reporter Assay

Objective: LncRNAs are non-coding RNAs exerting vital roles in the occurrence and development of various cancer types. This study tended to describe the expression pattern of FENDRR in colorectal cancer (CRC), and further investigate the role of FENDRR in CRC cell biological behaviors. Methods: Gene expression profile of colon cancer was accessed from the TCGA database, and then processed for differential analysis for identification of differentially expressed lncRNAs and miRNAs. Some in vitro experiments like qRT-PCR, MTT, colony formation assay, wound healing assay and Transwell assay were performed to assess the effect of FENDRR on cell biological behaviors. Dual-luciferase reporter assay was conducted to further validate the targeting relationship between FENDRR and miR-424-5p, and rescue experiments were carried out for determining the mechanism of FENDRR/miR-424-5p underlying the proliferation, migration and invasion of CRC cells. Results: Bioinformatics analysis suggested that FENDRR was significantly down-regulated in CRC tissue, and low FENDRR was intimately correlated to poor prognosis. FENDRR overexpression could greatly inhibit cell proliferation, migration and invasion. Besides, there was a negative correlation between FENDRR and miR-424-5p. Dual-luciferase reporter assay indicated that miR-424-5p was a direct target of FENDRR. Rescue experiments discovered that FENDRR exerted its role in cell proliferation, migration and invasion in CRC via targeting miR-424-5p. Conclusion: FENDRR is poorly expressed in CRC tissue and cells, and low FENDRR is responsible for the inhibition of cell proliferation, migration and invasion of CRC by means of targeting miR-424-5p.

scholarly journals Long Non­coding RNA SNHG16 Functions as Tumor Activator by Sponging hsa-miR-373-3p to Regulate TGFBR2/SMAD Pathway in Prostate Cancer

2020 ◽  
Author(s):  
WuBin Weng ◽  
ChangMing Liu ◽  
GuoMin Li ◽  
QiongFang Ruan ◽  
HuiZhang Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Gleason Score ◽  
Cell Apoptosis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Smad3 Expression ◽  
Dual Luciferase Reporter Assay

Abstract Background: Long noncoding RNAs (lncRNAs) are one of the major causes of tumorigenesis. However, the roles and mechan­­isms of lncRNA SNHG16 in prostate cancer (PCa) remain unknown. The purpose of this study was to elucidate the mech­­anisms of lncRNA SNHG16 in the proliferation and metastasis of human PCa cells.Material and Methods: First, the quantitative polymerase chain reaction (qPCR) was used to measure SNHG16 expression in PCa tissues and adjacent normal tissues (n=80). Down-regulate and over-express SNHG16 in human PCa DU-145 cell. Then cell proliferation was detected by CCK8 assay, cell apoptosis was analyzed by flow cytometry, cell migration were determined by wound healing, and cell invasion was examined by transwell. Western blot assays were used to examine the expression of the TGFBR2, c-MYC, E2F4, SMAD2, p-SMAD2, SMAD3, and p-SMAD3. Second, the targeting relationship between SNHG16 and hsa-miR-373-3p was verified by dual-luciferase reporter assay and rescue experiments. Third, the targeting relationship between hsa-miR-373-3p and TGFBR2 was verified by dual-luciferase reporter assay and rescue experiments. Results: The expression of SNHG16 was significant increase in PCa tissues (Z=-8.405, P<0.001), and with significant correlation with patient's age (<60 and ≥60 years old, P=0.007). Silencing SNHG16 inhibited DU-145 cell proliferation, migration, and invasion, while induced cell apoptosis significantly (P<0.01, respectively). Overexpressing SNHG16 promoted cell proliferation, migration and invasion, and reduced cell apoptosis rate (P<0.05, respectively). SNHG16 overexpression observably increased TGFBR2, c-MYC, E2F4, p-SMAD2, and p-SMAD3 expression (P<0.001, respectively), but SNHG16 inhibition was opposite. However, SNHG16 did not regulate SMAD2 and SMAD3 expression. Next, hsa-miR-373-3p was found down-regulated in PCa tissues (Z=-8.344, P<0.001), and the down-regulation of hsa-miR-373-3p were closely linked to Gleason score (Gleason score: <7 and >7, P = 0.024). Hsa-miR-373-3p expression of hsa-miR-373-3p was negatively correlated with SNHG16 (r=-0.544, P<0.001). The result of dual-luciferase reporter assay and qPCR test revealed that hsa-miR-373-3p was a target of SNHG16. Hsa-mir-373-3p inhibitor could rescue sh-SNHG16-inhibited cell proliferation, migration and invasion by promoting TGFBR2, C-MYC, E2F4, P-Smad2, and P-smad3 expression. Finally, we found that TGFBR2 may be the target gene of hsa-mir-373-3p through TargetScan and starbase. Further research found that TGFBR2 was markedly up-regulated in PCa tissues (Z=-5.945, P<0.001), and the expression of TGFBR2 was negatively correlated with hsa-miR-373-3p (r=-0.627, P<0.001). Dual-luciferase reporter assay and qPCR test showed that TGFBR2 was a target of hsa-miR-373-3p. TGFBR2 knockdown could inhibit hsa-mir-373-3p inhibitor-induced cell proliferation, migration and invasion, and reversed the effect of hsa-mir-373-3p inhibitor on cell apoptosis. Based on the data, sh-TGFBR2 partially disabled hsa-mir-373-3p inhibitor effect. Conclusion: LncRNA SNHG16 might act as a ceRNA to regulate the proliferation and migration of DU-145 cells by modulating the hsa-miR-373-3p/TGFBR2/SMAD axis.

MicroRNA-449b-5p suppresses cell proliferation, migration and invasion by targeting TPD52 in nasopharyngeal carcinoma

2019 ◽  
Vol 166 (5) ◽  
pp. 433-440 ◽  
Author(s):  
Wei Yin ◽  
Lei Shi ◽  
Yanjiao Mao
Keyword(s):  
Cell Proliferation ◽  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Nasopharyngeal carcinoma (NPC) is an important type of head and neck malignant cancer with geographical distribution. MicroRNA-449b-5p (miR-449b-5p) is related to the development of various cancers, while its function in NPC remains unknown. The present study aimed to investigate the role and target gene of miR-449b-5p in NPC. Expressions of miR-449b-5p in NPC cell lines and clinical tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was determined by MTT and colony formation assays. Migration and invasion abilities after different treatment were evaluated by wound healing and Transwell assays, respectively. Dual-luciferase reporter assay was performed to explore the relationship between miR-449b-5p and tumour protein D52 (TPD52). TPD52 expression was determined by qRT-PCR and western blot assay. miR-449b-5p was significantly downregulated in NPC cell lines and clinical tissues than the matched control. Overexpression of miR-449b-5p inhibited proliferation, migration and invasion of NPC cells. Dual-luciferase reporter assay indicated that miR-449b-5p directly targeted TPD52. Furthermore, shRNA-mediated downregulation of TPD52 rectified the promotion of cell migration and invasion by miR-449b-5p inhibition. In conclusion, the present study suggests that miR-449b-5p, as a novel tumour-suppressive miRNA against NPC, inhibits proliferation, migration and invasion of NPC cells via inhibiting TPD52 expression.

scholarly journals Long non-coding RNA MCM3AP-AS1 promotes growth and migration through modulating FOXK1 by sponging miR-138-5p in pancreatic cancer

2019 ◽  
Vol 25 (1) ◽  
Author(s):  
Ming Yang ◽  
Shijuan Sun ◽  
Yao Guo ◽  
Junjie Qin ◽  
Guangming Liu
Keyword(s):  
Pancreatic Cancer ◽  
Suppressive Effect ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Non Coding Rna ◽  
And Migration ◽  
Long Non Coding Rna ◽  
Dual Luciferase Reporter Assay

Abstract Background Pancreatic cancer (PC) is a type of malignant gastrointestinal tumor. Long non-coding RNA MCM3AP antisense RNA 1 (MCM3AP-AS1) has been reported to stimulate proliferation, migration and invasion in several types of tumors. However, the role of MCM3AP-AS1 in PC remains unclear. Methods MCM3AP-AS1, microRNA miR-138-5p (miR-138-5p) and FOXK1 levels were detected using quantitative real time PCR. Cell proliferation, migration and invasion were analyzed. Dual luciferase reporter assay was used to confirm the relationship between MCM3AP-AS1 and miR-138-5p, between miR-138-5p and FOXK1. Protein levels were identified using western blot analysis. Results MCM3AP-AS1 overexpression promoted proliferation, migration and invasion in PC cells. MCM3AP-AS1 silencing showed a suppressive effect on cell growth in PC cells. Moreover, MCM3AP-AS1 knockdown suppressed tumor growth in mice. Dual luciferase reporter assay demonstrated MCM3AP-AS1 could sponge microRNA-138-5p (miR-138-5p), and FOXK1 could bind with miR-138-5p. Positive correlation between MCM3AP-AS1 and FOXK1 was testified, as well as negative correlation between miR-138-5p and FOXK1. MCM3AP-AS1 promoted FOXK1 expression by targeting miR-138-5p, and MCM3AP-AS1 facilitated growth and invasion in PC cells by FOXK1. Conclusion MCM3AP-AS1 promoted growth and migration through modulating miR-138-5p/FOXK1 axis in PC, providing insights into MCM3AP-AS1/miR-138-5p/FOXK1 axis as novel candidates for PC therapy from bench to clinic.

scholarly journals Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

2020 ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Dual Luciferase Reporter Assay

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1 was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expression assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments verified that BCYRN1 affects CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour tissues and cell lines had significantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1 inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS is a target gene of miR-204-3p and is negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenesis experiments in a CRC mouse model confirmed that BCYRN1 downregulation effectively inhibited tumour growth.Conclusions: Our findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

scholarly journals miR-10b-5p Suppresses the Proliferation and Invasion of Primary Hepatic Carcinoma Cells by Downregulating EphA2

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Xu Niu ◽  
Haitao Sun ◽  
Feng Qiu ◽  
Jing Liu ◽  
Tianchi Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Caspase 3 ◽  
Carcinoma Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Hepatic Carcinoma ◽  
Primary Hepatic Carcinoma ◽  
Tissue Specimens ◽  
Dual Luciferase Reporter Assay

Objective. To analyze the function of miR-10b-5p in suppressing the invasion and proliferation of primary hepatic carcinoma cells by downregulating erythropoietin-producing hepatocellular receptor A2 (EphA2). Material and Methods. Eighty-six hepatic carcinoma (HCC) tissue specimens and 86 corresponding adjacent tissue specimens were collected, and the mRNA expression of miR-10b-5p and Ephrin type-A receptor 2 (EphA2) in the specimens was determined using a reverse transcription-polymerase chain reaction (RT-PCR) assay. Western blot was employed to quantify EphA2, B-cell chronic lymphocytic leukemia/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 in the cells, and CCK8, Transwell assay, and flow cytometry were applied to evaluate the proliferation, invasion, and apoptosis of cells, respectively. Moreover, the dual luciferase reporter assay was utilized for correlation analysis between miR-10b-5p and EphA2. Results. miR-10b-5p was lowly expressed in HCC, while EphA2 was highly expressed. Cell experiments revealed that miR-10b-5p overexpression or EphA2 knockdown could reduce cell proliferation, accelerate apoptosis, strongly upregulate Bax and Caspase-3, and downregulate Bcl-2. In contrast, miR-10b-5p knockdown or EphA2 overexpression gave rise to reverse biological phenotypes. Furthermore, dual luciferase reporter assay verified that miR-10b-5p was a target of EphA2, and the rescue experiment implied that transfection of pCMV-EphA2 or Si-EphA2 could reverse EphA2 expression and cell biological functions caused by miR-10b-5p overexpression or knockdown. Conclusions. miR-10b-5p reduced HCC cell proliferation but accelerate apoptosis by regulating EphA2, suggesting it has the potential to be a clinical target for HCC.

scholarly journals miR-126 Functions as a Tumor Suppressor by Targeting SRPK1 in Human Gastric Cancer

2018 ◽  
Vol 26 (9) ◽  
pp. 1345-1353 ◽  
Author(s):  
Qiaorong Li ◽  
Geng Wang ◽  
Hong Wang
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
In Vitro Assay ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Vitro Assay ◽  
Dual Luciferase Reporter Assay

The expression of miR-126 and serine‐arginine protein kinase 1 (SRPK1) are linked to tumor development; nevertheless, its role in the tumor growth and invasion of gastric cancer (GC) and the underlying mechanism have not been clarified. Here the expression and role of miR-126 and SRPK1 were investigated in GC tissues and cells by in vitro assay, and then targets of miR-126 were identified by dual-luciferase reporter assay. In this study, miR-126 expression was downregulated and associated with lymph node metastasis and poor prognosis as well as SRPK1 expression. In vitro assay revealed that miR-126 obviously inhibited the proliferative and invasive capabilities of GC cells. The dual-luciferase reporter assay showed that miR-126 targets the 3′-UTR of SRPK1 and downregulates its expression. SRPK1 overexpression promoted cell migration and invasion. In conclusion, the reduced expression of miR-126 is suggestive of the risk of GC recurrence and metastasis, and miR-126 functions as a tumor suppressor by targeting SRPK1 expression in the development of GC.

scholarly journals Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer by regulating the miR-204-3p/KRAS axis

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Dual Luciferase Reporter Assay

Abstract Background It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occurrence and progression of CRC. Methods RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1 was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expression assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments verified that BCYRN1 affects CRC by regulating the effect of miR-204-3p on KRAS. Results We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour tissues and cell lines had significantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1 inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS is a target gene of miR-204-3p and is negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenesis experiments in a CRC mouse model confirmed that BCYRN1 downregulation effectively inhibited tumour growth. Conclusions Our findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

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