Journal: Cellular and Molecular Life Sciences

Article Title: LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

doi: 10.1007/s00018-024-05172-9

Figure Lengend Snippet: LUESCC regulates the expression of NRSN2, which is highly expressed in ESCC tumor samples and predicts poor prognosis in ESCC patients. A The subcellular distribution of LUESCC in KYSE510 (left panel) and KYSE140 (right panel) cells was determined by nuclear and cytoplasmic fractionation experiment followed by RT-qPCR analysis. B The nuclear and cytoplasmic fractionations as described in A were subjected to immunoblotting analysis. C KYSE510 and KYSE140 cells transfected with negative control siRNA (si NC) and siRNA specifically targeting LUESCC (si LUESCC) for three days were subjected to RNA-seq analysis, and differentially expressed genes are presented by volcano plot. Red and blue dots represent upregulated and downregulated genes, respectively, in both cell lines ( P < 0.05). D The expression of differentially expressed genes ( P < 0.05) as described in C is represented by heat map. E CeRNA network consists of LUESCC-miRNAs-mRNAs (genes positively regulated by LUESCC, n = 401) is shown. Nodes in green, yellow, and light blue represent LUESCC, miRNAs, and target mRNAs, respectively. F The flowchart to identify clinically relevant target genes of LUESCC in ESCC is shown. G The expression of NRSN2 in esophageal carcinoma tissues ( n = 182) and normal tissues ( n = 286) obtained from GEPIA database is shown. H The correlation between the expression of NRSN2 and prognosis of ESCA patients predicted by Oncolnc database is shown. I KYSE510 and KYSE140 cells transfected with negative control siRNA (si NC) or siRNA targeting LUESCC (si LUESCC#1 and si LUESCC#2) were subjected to RT-qPCR analysis to measure the expression of NRSN2. J The expression of NRSN2 in a cohort of ESCC tumor samples ( n = 140) and normal samples ( n = 140) in house is shown. K The correlation between the expression of NRSN2 and prognosis of ESCC patients as described in J is shown. Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were applied: NRSN2 (A14425, ABclonal), Lamin B1 (A11495, ABclonal), GAPDH (#5174, Cell Signaling Technology).

Techniques: Expressing, Fractionation, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, RNA Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

doi: 10.1007/s00018-024-05172-9

Figure Lengend Snippet: NRSN2 promotes the malignant behaviors of ESCC cells. A KYSE510 and KYSE140 cells transfected with negative control siRNA (si NC) or siRNA targeting NRSN2 (si NRSN2#1 and si NRSN2#2) were subjected to RT-qPCR analysis to examine the expression of NRSN2. B KYSE150 cells transfected with control vector or vector expressing NRSN2 were subjected to RT-qPCR analysis to examine the expression of NRSN2. C–V MTS ( C , D ), colony formation ( E , G , I ), wound healing ( K , M , O ), and transwell assays ( Q , S , U ) were performed in si NRSN2-transfected KYSE510 ( E , K , Q ) and KYSE140 ( G , M , S ) cells or NRSN2-transfected KYSE150 cells ( I , O , U ). F , H , J , L , N , P , R , T , V Quantification analysis results for colony formation ( F , H , J ), would healing ( L , N , P ), and transwell ( Q , S , U ) assays as shown in ( E , G , I ), ( K , M , O ), and ( Q , S , U ), respectively. Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were applied: NRSN2 (A14425, ABclonal), Lamin B1 (A11495, ABclonal), GAPDH (#5174, Cell Signaling Technology).

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

doi: 10.1007/s00018-024-05172-9

Figure Lengend Snippet: LUESCC regulates the malignant phenotypes of ESCC cells is partially dependent on the expression of NRSN2. A–N KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or siRNA targeting LUESCC (si LUESCC) in the presence or absence of vector expressing NRSN2 followed by western blot ( A ), MTS ( B ), colony formation ( C , E ), wound healing ( G , I ), and transwell assays ( K , M ). D , F , H , J , L , N Quantification analysis results for colony formation ( D , F ), would healing ( H , J ), and transwell assays ( L , N ) as shown in C , E , G , I , and K , M , respectively. Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were applied: NRSN2 (A14425, ABclonal), Lamin B1 (A11495, ABclonal), GAPDH (#5174, Cell Signaling Technology).

Techniques: Expressing, Transfection, Negative Control, Plasmid Preparation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

doi: 10.1007/s00018-024-05172-9

Figure Lengend Snippet: LUESCC acts as a miRNA sponge for miR-6785-5p to regulate the expression of NRSN2 and the malignant behaviors in ESCC cells. A , B Schematic representation of miR-6785-5p binding sites on wild-type (WT) LUESCC ( A ) and NRSN2-3′ UTR ( B ) as well as the corresponding mutant form (MT) with the predicted miR-6785-5p binding sites mutated. C , D KYSE510 and KYSE140 cells were transfected with reporters containing wild-type (WT- luc ) or mutated (MT- luc ) LUESCC ( C ) or NRSN2 3′ UTR ( D ) in the presence or absence of negative control miRNA mimic (miR-NC) or miR-6785-5p mimic (miR-6785-5p) followed by luciferase activity measurement. E–S KYSE510 and KYSE140 cells were transfected with negative control siRNA (si NC) or siRNA targeting LUESCC in the presence or absence of miR-6785-5p inhibitor followed by RT-qPCR ( E ), western blot ( F ), MTS ( G ), colony formation ( H , J ), wound healing ( L , N ), and transwell assays ( P , R ). I , K , M , O , Q , S Quantification analysis results for colony formation ( I , K ), would healing ( M , O ), and transwell assays ( Q , S ) as shown in H , J , L , N , and P , R , respectively. Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were applied: NRSN2 (A14425, ABclonal), Lamin B1 (A11495, ABclonal), GAPDH (#5174, Cell Signaling Technology).

Techniques: Expressing, Binding Assay, Mutagenesis, Transfection, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis

doi: 10.1007/s00018-024-05172-9

Figure Lengend Snippet: ASO specifically targeting LUESCC is effective in suppressing the malignant phenotypes of ESCC cells. A – D , F , H , J , L , N KYSE510 and KYSE140 cells were transfected with negative control ASO (ASO NC) or ASO specifically targeting LUESCC (ASO LUESCC#1 and ASO LUESCC#2) followed by RT-qPCR ( A ), immunoblotting analysis ( B ), MTS ( C ), colony formation ( D , F ), wound healing ( H , J ), and transwell assays ( L , N ). E , G , I , K , M , O Quantification analysis results for colony formation ( E , G ), would healing ( H , J ), and transwell assays ( L , N ) as shown in D , F , H , J , and L , N , respectively. P BALB/c nude mice were subcutaneously inoculated with KYSE510 cells and treated with or without ASO LUESCC in the presence or absence of antagomiR-6785-5p. Mice were then euthanized and tumors were collected. Q , R The weight ( Q ) and growth curve ( R ) of tumors as described in P are shown. S , T The expression of NRSN2 in tumors as described in P were examined by RT-qPCR ( S ) and immunoblotting ( T ) analysis. U A proposed model of LUESCC function in ESCC progression. The highly expressed LUESCC in ESCC cells functions as a miRNA sponge to sponge miR-6785-5p to release its repression on NRSN2 expression, leading to the aberrant expression of NRSN2 and tumorigenesis. Data were shown as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were applied: NRSN2 (A14425, ABclonal), Lamin B1 (A11495, ABclonal), GAPDH (#5174, Cell Signaling Technology).

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Expressing