Metformin Suppresses Hypopharyngeal Cancer Growth by Epigenetically Silencing Long Non-coding RNA SNHG7 in FaDu Cells

Abstract

Local recurrence after therapy remains a challenging problem for hypopharyngeal cancer (HPC) due to the chemotherapy resistance. Metformin is associated with reduced cancer risk through promoting global DNA methylation in cancer cells by controlling S-adenosylhomocysteine (SAHH) activity. However, the mechanisms by which metformin inhibits HPC remain elusive. In this study, we aim to investigate the role of metformin in HPC and illustrate the mechanism by which metformin regulates long non-coding RNAs (lncRNAs) expression. CCK-8 and annexin-V/PI double staining were performed to analyze the cell viability and apoptosis. LncRNA microarray analysis, QPCR, methylation specific PCR, Western blot and RNA Immunoprecipitation were performed to analyze the molecular mechanism, Here, we report that metformin inhibits FaDu cell proliferation in time- and dose-dependent manner by suppressing lncRNA SNHG7. Further investigations revealed that SNHG7 interacted with SAHH and metformin decreased SNHG7 expression by activating SAHH activity. Increased SAHH activity resulted in upregulating DNMT1 expression, leading to hypermethylation of SNHG7 promotor. In addition, upregulation of SNHG7 was associated with advanced stage. The patients with high SNHG7 have lower overall survival than that of with low SNHG7. Interestingly, SNHG7 levels were higher in taxol resistant patients than in taxol sensitive patients. Metformin sensitizes FaDu cells to taxol and irradiation through decreasing SNHG7. In conclusion, our recent study demonstrates that metformin inhibits FaDu cell proliferation by decreasing SNHG7 expression via SAHH-mediated DNA methylation. These findings indicate that combined metformin with paclitaxel or irradiation would be a novel therapeutic strategy to overcome resistance and prevent recurrence in HPC.

Keywords: DNA methylation; SNHG7; hypopharyngeal cancer; long non-coding RNAs; metformin.

FIGURE 1

Metformin inhibits cell viability and induces apoptosis in FaDu cells. (A) CCK-8 assay was used to determine the cell viability after 48 h of a serial concentrations of metformin (left) treatment and after 8 mM metformin treatment for a serial of time points (right) in FaDu cells. (B) BrdU assay was used to determine the cell proliferation rate in FaDu cells. (C) Flow cytometry was used to measured cell apoptosis after 8 mM metformin treatment for 48 h in FaDu cells. ^∗P < 0.05.

FIGURE 2

Rescue of SNHG7 reverse metformin-mediated inhibitory effects in vitro and in vivo. (A) The heatmap showed the differentially expression lncRNAs in metformin (8 mM for 48 h) treated FaDu cells. SNHG7 was significantly decreased by metformin compared with vehicle treated cells. (B) QPCR was performed to measure the SNHG7, LINC01504, and LINC00189 expression after 48 h of a serial concentrations of metformin treatment in FaDu cells. (C) QPCR was performed to measure the SNHG7, LINC01504, and LINC00189 expression after 8 mM metformin treatment for a serial of time points in FaDu cells. (D) QPCR was performed to measure the SNHG7 expression after SNHG7 lentivirus infection. (E) CCK-8 assay was used to determine the cell viability after metformin treatment (8 mM for 48 h) or combined with SNHG7 lentivirus. (F) Flow cytometry was used to measured cell apoptosis after metformin treatment (8 mM for 48 h) or combined with SNHG7 lentivirus. (G) FaDu cells treated with metformin or together with SNHG7 lentivirus were injected into nude mice. The cells untreated were used as negative control. Tumor volumes were calculated and shown. (H) QPCR was performed to measure the SNHG7 expression in tumor tissues from nude mice. (I) Western blot was performed to measure the p-AMPK and AMPK expression in tumor tissues from nude mice. ^∗P < 0.05.

FIGURE 3

Metformin epigenetically regulates SNHG7. (A) qMSP test results indicated that methylation of SNHG7 promoter was higher in metformin treated FaDu cells than that of in vehicle treated cells. (B) FaDu cells were treated with or without metformin (metf) at a final concentration of 8 mM for 48 h. SAHH activity were assessed by SAHH activity assay. (C) SAHH protein levels were assessed by Western blot. (D) RNA immunoprecipitation (RIP) was performed. The levels of SNHG7 and GAPDH mRNA are presented as fold enrichment in anti-SAHH relative to IgG immunoprecipitants. (E) qPCR was performed to measure the expression of DNMT1, DNMT2, DNMT3a and DNMT3b after metformin treatment. (F,G) DNMT1, DNMT2, DNMT3a, and DNMT3b protein levels were assessed by Western blot in FaDu cells treated with or without metformin (metf) (F) and the quantification of bands (G). (H) Confirmation of Dnmt1 knockdown by RT–qPCR. (I) Confirmation of Dnmt1 knockdown by Western blot analysis. (J) qMSP analysis of SNHG7 promoter methylation. (K,L) Cell viability (K) and cell apoptosis rate (L) were assessed by CCK8 and flow cytometry in FaDu cells treated metformin with or without shDNMT1. (M) RT-qPCR analysis for GALNT1, Cyclin D1, FAIM2, p15, p16 in FaDu cells after treated with or without metformin. ^∗P < 0.05.

FIGURE 4

The expression of SNHG7 in hypopharyngeal cancer tissues. (A) RT-qPCR was used to determine the expression of SNHG7 in hypopharyngeal cancer tissues (N = 73) and matched adjacent control (N = 73). (B) The expression of SNHG7 in patients who sensitive (N = 38) or primary resistant (N = 33) to taxol. (C) Overall survival analysis in hypopharyngeal cancer patients with low or high SNHG7 expression. ^∗P < 0.05.

FIGURE 5

Metformin sensitizes FaDu cells to gefitinib through inhibiting SNHG7. (A) CCK-8 assay was used to determine the IC50 in FaDu/taxol cells (control vs. metformin: 79 vs. 31 mM). (B) CCK-8 assay was used to determine the change of viability after taxol, metformin and SNHG7 treatment. (C) Flow cytometry was used to measure cell apoptosis after taxol, metformin and SNHG7 treatment. (D) RT-qPCR analysis for SNHG7 in FaDu cells after treated with taxol. (E) RT-qPCR analysis for MDR1, MRP7, LRP, BCRP, and TRAG3 in FaDu cells after treated with or without metformin. (F) CCK-8 assay was used to determine the change of viability after irradiation, metformin, and SNHG7 treatment. (G) Flow cytometry was used to measure cell apoptosis after irradiation, metformin, and SNHG7 treatment. ^∗P < 0.05 vs. control, ^#p < 0.05 vs. irradiation, ^$p < 0.05 vs. irradiation plus metformin; ns, no significance.