Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: RT-qPCR primers used in this study.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: Transfection efficiency of pcDNA3.1-STC2 and pCD5-circRNA8220 vectors and STC2-siRNA, circRNA8220-siRNA and miR-8516 mimics/inhibitors. ( a , b ) mRNA levels of circRNA8220. ( c ) mRNA levels of miR-8516. ( d ) The mRNA levels of STC2. ( e ) Protein levels of STC2. ** p < 0.01, * p < 0.05.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Transfection

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: Antibody used in this study.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: MiR-8516 down-regulated the expression level of STC2 via the 3′UTR. ( a ) The seed sequence of miR-8516 could match with WT-STC2-3′UTR and could not match with Mut-STC2-3′UTR. ( b ) Luciferase reporter assay of 293T cells co-transfected with WT-STC2-3′UTR or Mut-STC2-3′UTR and miR-8516 mimic, NC (negative control), miR-8516 inhibitor or NCH (NC-inhibitor). ( c ) miR-8516 decreased STC2 protein level in GMECs. ( d ) miR-8516 decreased STC2 mRNA level in GMECs. ** p < 0.01, * p < 0.05.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Expressing, Sequencing, Luciferase, Reporter Assay, Transfection, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: circRNA8220 increased the expression of STC2 in GMEC in vitro. ( a ) si-circRNA8220 reduced the mRNA expression of STC2. ( b ) circRNA8220 increased the mRNA expression of STC2. ( c ) si-circRNA8220 blocked the protein expression of STC2. ( d ) circRNA8220 raised the protein expression of STC2. ** p < 0.01.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Expressing, In Vitro

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: STC2 inhibited apoptosis and promoted proliferation of GMEC in vitro. ( a , b ) Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay after transfection with pc3.1-STC2 or si-STC2. ( c , d ) Cell proliferation indices were assessed after treatment with Edu after transfection with pc3.1-STC2 or si-STC2. ( e , f ) Apoptosis analysis of GMEC was detected with flow cytometry method after transfection with pc3.1-STC2 or si-STC2. ( g , h ) Protein levels of Bcl-2, Bax, caspase 3 and caspase 9 in GMEC after transfection with pc3.1-STC2 or si-STC2. Protein levels were measured by WB (western blot) densitometry was normalised to the β-actin density from the same lane. Data were expressed as the means ± SEM. ** p < 0.01, * p < 0.05.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: In Vitro, Cell Counting, CCK-8 Assay, Transfection, Flow Cytometry, Western Blot

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: STC2 enhanced the synthesis of β-casein and triglycerides in GMEC. ( a , b , c , d ) Secretion of β-casein and triglycerides (TG) after transfection with pc3.1-STC2 or si-STC2 was measured by enzyme-linked immunosorbent assay kit. ** p < 0.01, * p < 0.05.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells

doi: 10.3390/ani10081347

Figure Lengend Snippet: STC2 activated the signaling pathways of Ras/MEK/ERK and PI3K/AKT/mTOR in GMEC. ( a , b ) Western blot analysis was performed to detect the protein expression of Ras, p-MEK, MEK, p-ERK44/42, ERK44/42, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, p-S6K, S6K and β-actin in GMEC after transfection with pc3.1-STC2 or si-STC2. Protein levels were measured by WB densitometry was normalised to the β-actin density from the same lane. Data were expressed as the means ± SEM. ** p < 0.01, * p < 0.05.

Article Snippet: To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively.

Techniques: Protein-Protein interactions, Western Blot, Expressing, Transfection