scholarly journals Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Real Time ◽  
Three Dimensional ◽  
Housekeeping Gene ◽  
Carcinoma Cells ◽  
Gene Profiling ◽  
Hepatocellular Carcinoma Cells ◽  
Cells Cultured

scholarly journals Identification and evaluation of an appropriate housekeeping gene for real-time gene profiling of hepatocellular carcinoma cells cultured in three-dimensional scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Culture ◽  
Real Time ◽  
Internal Control ◽  
18S Rrna ◽  
Three Dimensional ◽  
Melting Curve ◽  
3D Culture ◽  
Gene Profiling ◽  
3D Microenvironment

Abstract Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analyzed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

scholarly journals CD147 promotes collective invasion through cathepsin B in hepatocellular carcinoma

2020 ◽  
Author(s):  
Shi-Jie Wang ◽  
Dong Chao ◽  
Wei Wei ◽  
Gang Nan ◽  
Jia-Yue Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Carcinoma Cells ◽  
Human Hepatocellular Carcinoma ◽  
Migration And Invasion ◽  
Hepatocellular Carcinoma Cells ◽  
Collective Invasion ◽  
Invasion Model

Abstract Background: Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma.Methods: Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors.Results: Human hepatocellular carcinoma undergone collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activation. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma.Conclusions: CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

scholarly journals CD147 promotes collective invasion through cathepsin B in hepatocellular carcinoma

2020 ◽  
Author(s):  
Shi-Jie Wang ◽  
Dong Chao ◽  
Wei Wei ◽  
Gang Nan ◽  
Jia-Yue Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Carcinoma Cells ◽  
Human Hepatocellular Carcinoma ◽  
Migration And Invasion ◽  
Hepatocellular Carcinoma Cells ◽  
Collective Invasion ◽  
Invasion Model

Abstract Background Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma. Methods Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors. Results Human hepatocellular carcinoma undergone collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activation. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma. Conclusions CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

scholarly journals Identification and Evaluation of an Appropriate Housekeeping Gene for Real-Time Gene Profiling of Hepatocellular Carcinoma Cells Cultured in Three-Dimensional Scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Culture ◽  
Real Time ◽  
Internal Control ◽  
18S Rrna ◽  
Three Dimensional ◽  
Melting Curve ◽  
3D Culture ◽  
Gene Profiling ◽  
3D Microenvironment

Abstract Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

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