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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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Related in: MedlinePlus

Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. (A) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. (B, C) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. (D) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. (E) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (F) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = Ct sample- Ct 18S) values were plotted. (G) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). (H) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. (I) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (J) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or >100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. (K) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. (L) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. (M) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments (n = 3) when ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001. All luciferase experiments have been conducted in triplicate. For data presented in panels E, F, G, I and K the ΔΔCt method for RQ of gene expression was used and generated using the equation 2−ΔΔCt. P values were determined by paired t test.
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Figure 1: Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. (A) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. (B, C) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. (D) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. (E) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (F) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = Ct sample- Ct 18S) values were plotted. (G) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). (H) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. (I) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (J) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or >100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. (K) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. (L) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. (M) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments (n = 3) when ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001. All luciferase experiments have been conducted in triplicate. For data presented in panels E, F, G, I and K the ΔΔCt method for RQ of gene expression was used and generated using the equation 2−ΔΔCt. P values were determined by paired t test.

Mentions: For the experiment in Figure 1J, the supernatant from Huh7 cells was centrifuged to clear cellular debris and this constituted the Huh7 CM. This was then further centrifuged in a centricon with a molecular weight cutoff of 100 kDa such that constituents having molecular weight of <100 kDa were filtered out and those which were >100 kDa were retained. The latter was then filter sterilized and added to HepG2 cells.


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)

Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. (A) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. (B, C) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. (D) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. (E) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (F) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = Ct sample- Ct 18S) values were plotted. (G) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). (H) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. (I) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (J) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or >100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. (K) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. (L) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. (M) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments (n = 3) when ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001. All luciferase experiments have been conducted in triplicate. For data presented in panels E, F, G, I and K the ΔΔCt method for RQ of gene expression was used and generated using the equation 2−ΔΔCt. P values were determined by paired t test.
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Figure 1: Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. (A) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. (B, C) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. (D) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. (E) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (F) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = Ct sample- Ct 18S) values were plotted. (G) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). (H) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. (I) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. (J) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or >100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. (K) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. (L) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. (M) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments (n = 3) when ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001. All luciferase experiments have been conducted in triplicate. For data presented in panels E, F, G, I and K the ΔΔCt method for RQ of gene expression was used and generated using the equation 2−ΔΔCt. P values were determined by paired t test.
Mentions: For the experiment in Figure 1J, the supernatant from Huh7 cells was centrifuged to clear cellular debris and this constituted the Huh7 CM. This was then further centrifuged in a centricon with a molecular weight cutoff of 100 kDa such that constituents having molecular weight of <100 kDa were filtered out and those which were >100 kDa were retained. The latter was then filter sterilized and added to HepG2 cells.

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