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

Abstract

miRNAs are 20-22 nt long post-transcriptional regulators in metazoan cells that repress protein expression from their target mRNAs. These tiny regulatory RNAs follow tissue and cell-type specific expression pattern, aberrations of which are associated with various diseases. miR-122 is a liver-specific anti-proliferative miRNA that, we found, can be transferred via exosomes between human hepatoma cells, Huh7 and HepG2, grown in co-culture. 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. Our observations suggest existence of a reciprocal interaction between two different hepatic cells with distinct miR-122 expression profiles. This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal. According to our data, human hepatoma cells use IGF1 to prevent intercellular exosomal transfer of miR-122 to ensure its own proliferation by preventing expression of growth retarding miR-122 in neighbouring cells.

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 ( = C_{t sample}- C_{t 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.

Figure 2.

HepG2 cells receiving miR-122 from the donor Huh7 cells exhibit a decreased growth rate, increased senescence and increased sensitivity to apoptosis inducing agent. (A) HepG2 cells expressing GFP were co-cultured with equal number of Huh7 cells expressing DsRed. As a control, equal numbers of GFP-HepG2 and DsRed-Huh7 cells were cultured separately for same durations of time. Cells were collected by trypsinization and fixed and analysed by FACS. The percentage of GFP positive and DsRed positive cells in each sample were scored and relative growth rates were plotted. Relative growth rates were calculated by dividing the number of green (GFP) or Red (DsRed) cells in co-cultured sets by their number in the corresponding control sets. P values were determined by paired t test between 0 and 72 h replicates. n = 4. (B) Number of PCNA positive DsRed expressing HepG2 cells in co-culture either with HepG2 cells (control, not expressing DsRed) or with Huh7 cells (untreated or expressing anti-miR-122 or anti-let-7a oligos). PCNA (mitosin) positive cells were then detected by indirect immunofluorescence. Data shown represents three independent slides with five fields from each slide. P values were calculated using unpaired t test. (C) HepG2 cells treated with miR-122 containing Huh7 exosomes showed reduced growth and size of colonies. HepG2 cells were incubated with exosomes from HepG2, Huh7, anti-miR-122 or anti-let-7a transfected Huh7, and HepG2 cells expressing miR-122, for 7 days with changes after every 48 h. The cells were then reseeded at 1 × 10³ cells/cm². After 96 h the numbers of colonies formed in each case were counted. For each set n = 3. P values were calculated using unpaired t test. (D) Experiments were redone with HepG2 cells transfected with anti-miR-122 and anti-let7 oligonucleotides. For each set n = 3. P values were calculated using unpaired t test. (E) Tritiated thymidine incorporation in HepG2 cells, incubated with exosomes from control HepG2, Huh7 and HepG2 expressing miR-122 and grown in presence of ³H labelled thymidine. Experiment was done in three sets. P value was calculated by using unpaired t test. (F) Similar experiments were done with Huh7 exosomes isolated from anti-miRNA transfected Huh7 cells. For each condition n = 3. P value was calculated by using unpaired t test. (G) Estimation of number of senescent HepG2 cells per field. Cells were incubated with exosomes from control HepG2, Huh7, Huh7 transfected with anti-miR-122 or anti-let-7a oligos for 24 h. Cells were then fixed and senescence assays were performed. Data represents three independent slides with five fields from each slide. P value is calculated using unpaired t test. (H) Western analysis of cleaved PARP and Caspase 9 in HepG2 cells treated with 50 μg/ml doxorubicin along with exosomes isolated from HepG2 (Control), Huh7, or anti-miR-122 and anti-let7 transfected Huh7. Exosome treatment was done for 48 h with one change after 24 h when Doxorubicin was added. Data are presented as mean ±SEM in all results obtained from multiple experiments (minimum three) when ns: non-significant, *P < 0.05, **P < 0.01 and ***P < 0.001.

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 ΔC_t was calculated by subtracting the C_t 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.

Figure 4.

HepG2 CM activates mTOR pathway in Huh7 cells. (A) Western blot analysis was done to detect the levels of phosphorylated and nonphosphorylated mTOR and its downstream substrate kinases in Huh7 cells incubated with either HepG2 CM or Huh7 CM (control) for 48 h with a change after every 24 h. Also the levels of phosphorylated ERK and p38 MAPK were detected. (B) Effect of Rapamycin on phosphorylation of mTOR and its downstream kinases in Huh7 cells incubated with HepG2 CM in absence or in presence of 50 nM Rapamycin. Immunoblotting was done to detect the effect. (C) Effect of Rapamycin on miR-122 activity change in Huh7 cells transfected with RL reporters and incubated with HepG2 CM in presence or absence of 50 nM Rapamycin. After 48 h cells were lysed and luciferase activity was measured. Experiments were done in triplicate. Data are presented as means ±SEM of the Relative fold repression (control mean fold repression = 1) and significance is P = 0.0359.

Figure 5.

IGF1 secreted by HepG2 reduces activity and expression of miR-122 in Huh7 cells. (A) Effect of GW4869 on transfer of anti-miR-122 signal from HepG2 to Huh7 cells. HepG2 cells and Huh7 cells were either co-cultured together or mixed after being cultured separately for 48 h in presence and in absence of GW4869. Real-time quantification of miR-122 was then done to detect the level of miR-122 in both control and co-cultured samples in presence or absence of GW4869. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (B) Effect of different growth factors on miR-122 activity in Huh7 cells. Huh7 cells transfected with RL reporters were incubated for indicated concentrations (ng/ml) of Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor β (TGF-β), Insulin-like Growth Facto1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) overnight in DMEM and luciferase activities were 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. (C, D) Effect of IGF1 on miR-122 activity (C) and Level (D) in Huh7 cells. Cells were incubated with exosome depleted Huh7 CM alone or supplemented with 100 ng/ml IGFI for 72 h with fresh changes after every 36 h. HepG2 CM was used as a positive control. (E) Dose response curve to determine the effect of various concentrations of recombinant IGF1 (ng/ml) on the miR-122 level of Huh7 cells. Huh7 cells were incubated for 24 h with DMEM containing IGF1 (0–50 ng/ml). Total RNA was extracted from the cells and qPCR was done to determine the miR-122 level. We found that the decrease in miR-122 level starts from 5 ng/ml of IGF1. For panel C experiments were performed in triplicate and P value was calculated by using unpaired t test. For panels D and E data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (F) miR-24 and let-7a level change detected by real-time quantification in Huh7 cells treated with IGF1. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (G) Effect of IGF1 on miR-122 level in primary mouse hepatocytes treated with IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (H) Quantification of pre-miR-122, and other hepatic nuclear factor expression in Huh7 cells incubated for 72 h either with HepG2 CM or exosome depleted Huh7 CM containing 0 and 50 ng/ml of IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (I) Effect of IGF1 depletion in HepG2 or IGF1R depletion in Huh7 on miR-122 activity in Huh7 cells in presence of HepG2 CM. Huh7 cells (control or IGF1R depleted), expressing miR-122 RL reporter, were incubated with CM from normal or IGF1 depleted HepG2 for 72 h to determine the specificity of IGF1 to decrease miR-122 activity in Huh7 cells. For control experiments, non-target siRNA was used. Incubation of HepG2 CM with αIGF1 antibody removed the anti-miR-122 activity. nIgG was used as a control. 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 taking the control as 1 and expressing repression values relative to 1. Experiments were performed in triplicate and P value was calculated by using paired t test. (J) Effect of IGF1 depletion on miR-122 level in Huh7 cells incubated with CM from HepG2 cells transfected with a non-target or IGF1 specific siRNAs. The miR-122 level of the cells was detected by RT-PCR. Data represents five independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (K) miR-122 level in Huh7 cells depleted for IGF1R (siIGF1R transfected) against control siRNA transfected cells in presence of HepG2 CM. Cellular miR-122 levels were quantified by RT-PCR. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. (L) Effect of HepG2 CM on IGFR1R expression in Huh7 cells. Huh7 cells incubated for 72 h with HepG2 CM were analysed for IGF1R mRNA levels by qRT-PCR. Normalization was done by 18S rRNA. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated 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.

Figure 6.

HepG2 cells overcome the tumour suppressive effect of miR-122 containing exosomes by secreting IGF1. (A) Effect of co-culture with miR-122 expressing cells on matrigel invasion properties of HepG2. DsRed expressing HepG2 were co-cultured with HepG2 (control) or Huh7 cells (with or without GW4869 treatment) on matrigel coated polycarbonate membrane having a pore size of 8 μm. After 48 h, cells were fixed, and the number of cells which had invaded through the matrigel layer to the outer side was determined. HepG2 cells expressing miR-122 was used as a positive control. DAPI stained the nucleus and the percentage of invaded DsRed cells was determined by counting the number of DAPI and DsRed double positive cells. Results represent data from ≥ 5 independent fields taken from two independent experiments. (B) Effect of miR-122 containing exosome treatment on matrigel invasion properties of HepG2 cells. HepG2 cells stably expressing DsRed were incubated with exosomes isolated either from HepG2, Huh7, anti-miR-122 or let-7a expressing Huh7, or from Huh7 exogenously expressing miR-122. The number of DsRed cells invaded through matrigel was counted. (C) Experiments similar to described in B were done with HepG2 cells pre-transfected with anti-miR-122 and anti-let-7a oligos. Results represent data from ≥6 independent fields taken from two independent experiments. (D) Effect of IGF1 depletion on invasive property of HepG2 co-cultured with miR-122 expressing Huh7 cells. HepG2 cells expressing GFP were transfected either with siRNAs against IGF1 or with a control siRNA. Cells were seeded onto Huh7 cells layered on matrigel coated polycarbonate membranes having pore sizes of 8 μm. The number of invaded GFP positive cells colocalizing with DAPI was counted after 24 h of co-culture. Results represent data from ≥15 independent fields taken from two independent experiments. (E, F) Effect of HepG2 co-culture or HepG2 CM treatment on proliferation of Huh7 cells. GFP transfected Huh7 cells were co-cultured with nontransfected Huh7 (control) or HepG2 cells for 72 h. After 72 h, cells were fixed and immunofluorescence detection of PCNA (mitosin) was done in panel E. The percentage of PCNA positive nuclei (red) colocalizing with GFP (green) was calculated. Results represent data from eight independent fields taken from two independent experiments. The experiment was also done for GFP transfected Huh7 cells incubated with Huh7 CM (control) and HepG2 CM for 72 h. n = 7 independent fields taken from two independent experiments. (G, H) Effect of IGF1 treatment on proliferation of Huh7 cells. Huh7 cells were grown with HepG2 CM and exosome deprived CM from Huh7 cell cultures (with and without added IGFI) and the number of PCNA positive cells was determined. Results represent data from ≥10 independent fields taken from two independent experiments. (I, J) Senescence status of GFP positive Huh7 cells co-cultured with non-transfected Huh7 cells (control), HepG2 cells and Huh7 transfected with anti-miR-122 for 72 h. Cells were then fixed and senescent cells detected by β-galactosidase staining. The number of senescent GFP positive cells was then counted and the percentage calculated. Results represent data from ≥ 6 independent fields. (K) Change in senescence in Huh7 cells after IGF1 exposure. Huh7 cells were incubated with CM from HepG2, exosome depleted CM from Huh7 with or without 100 ng/ml of IGF1. After 72 h β-galactosidase activity was detected and the percentage of senescent cells was calculated. Results represent data from ≥10 independent fields from two independent experiments. All data are presented as mean ±SEM and *P < 0.05, **P < 0.01 and ***P < 0.001. P value was calculated by unpaired t test.