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Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells.

Basu S, Bhattacharyya SN - Nucleic Acids Res. (2014)

Bottom Line: Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells.Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells.This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal.

View Article: PubMed Central - PubMed

Affiliation: RNA Biology Research Laboratory, Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700032, India.

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HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. (A) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. (B) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. (C) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (D) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (E) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (F) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. (G) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (H) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (I) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (J) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2−ΔCt where ΔCt was calculated by subtracting the Ct for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.
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Figure 3: HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. (A) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. (B) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. (C) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (D) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (E) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (F) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. (G) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (H) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (I) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (J) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2−ΔCt where ΔCt was calculated by subtracting the Ct for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

Mentions: Huh7 is a hepatoma cell that exhibits constitutive expression of miR-122 which is associated with low Cyclin G1 expression level in hepatic cells (29,38,39). Previous experiments indicated that in the presence of HepG2, growth rate of Huh7 increases (Figure 2A). Does HepG2 reduce miR-122 in neighbouring Huh7 cells to increase their proliferation? To investigate this further, Huh7 cells expressing miR-122 reporter were co-cultured either with non-transfected control Huh7 cells or HepG2 cells at a ratio of 1:1. In the presence of HepG2, there was a decrease in the relative fold repression of miR-122 reporter in Huh7 cells as compared to control (Figure 3A and B). Quantitative PCR indicated that there was a decrease in the miR-122 level in HepG2 co-cultured, as opposed to the control Huh7 cells (Figure 3C). This tells us that the observed decrease in miR-122 activity is because of a reduced miR-122 level in Huh7 cells co-cultured with HepG2. Real-time quantification further revealed a similar change in the precursor form of miR-122 (pre-miR-122) in HepG2 exposed Huh7 cells, suggesting a reduced production of miR-122 (Figure 3D). This decrease in transcription appears to be specific for the miR-122 promoter and does not depend on miR-122 identity. When Huh7 cells were transfected with plasmids expressing pre-miR-122 under a U6 promoter (31), there was no change in the miR-122 level in control and HepG2 co-cultured Huh7 cells (Figure 3E). Therefore, it may be concluded that HepG2 cells exert a reciprocal effect on the miR-122 level in co-cultured Huh7 cells primarily by reducing the production of pre-miR-122.


Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells.

Basu S, Bhattacharyya SN - Nucleic Acids Res. (2014)

HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. (A) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. (B) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. (C) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (D) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (E) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (F) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. (G) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (H) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (I) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (J) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2−ΔCt where ΔCt was calculated by subtracting the Ct for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.
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Figure 3: HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. (A) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. (B) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. (C) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (D) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (E) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (F) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. (G) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (H) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (I) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (J) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2−ΔCt where ΔCt was calculated by subtracting the Ct for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.
Mentions: Huh7 is a hepatoma cell that exhibits constitutive expression of miR-122 which is associated with low Cyclin G1 expression level in hepatic cells (29,38,39). Previous experiments indicated that in the presence of HepG2, growth rate of Huh7 increases (Figure 2A). Does HepG2 reduce miR-122 in neighbouring Huh7 cells to increase their proliferation? To investigate this further, Huh7 cells expressing miR-122 reporter were co-cultured either with non-transfected control Huh7 cells or HepG2 cells at a ratio of 1:1. In the presence of HepG2, there was a decrease in the relative fold repression of miR-122 reporter in Huh7 cells as compared to control (Figure 3A and B). Quantitative PCR indicated that there was a decrease in the miR-122 level in HepG2 co-cultured, as opposed to the control Huh7 cells (Figure 3C). This tells us that the observed decrease in miR-122 activity is because of a reduced miR-122 level in Huh7 cells co-cultured with HepG2. Real-time quantification further revealed a similar change in the precursor form of miR-122 (pre-miR-122) in HepG2 exposed Huh7 cells, suggesting a reduced production of miR-122 (Figure 3D). This decrease in transcription appears to be specific for the miR-122 promoter and does not depend on miR-122 identity. When Huh7 cells were transfected with plasmids expressing pre-miR-122 under a U6 promoter (31), there was no change in the miR-122 level in control and HepG2 co-cultured Huh7 cells (Figure 3E). Therefore, it may be concluded that HepG2 cells exert a reciprocal effect on the miR-122 level in co-cultured Huh7 cells primarily by reducing the production of pre-miR-122.

Bottom Line: Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells.Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells.This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal.

View Article: PubMed Central - PubMed

Affiliation: RNA Biology Research Laboratory, Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700032, India.

Show MeSH
Related in: MedlinePlus