The Mechanism Of MiR-155 And MiR-21 Synergistically Promote Lung Cancer Progression By Targeting SOCS6

Background:Lung cancer remains the leading cause of cancer-associated death worldwide.MiR-155 and miR-21 are critical promoters of lung cancer progression. However, it remains unclear how MiR-155 and miR-21 induce lung cancer progression.Purpose:To confirm the miR-155 and miR-21 promote lung cancer progression by directly targeting SOCS6; In addition, Comparing the role and regulation of miR-155, miR-21, and 155/21 in lung cancer progression, we found that treatment with 155/21 inhibitors in combination was always more effective against lung cancer than treatment with a single inhibitor.Methods:(1) The levels of miR-155, miR-21 as well as SOCS6 mRNA were measured in lung cancer and adjacent normal tissues from 80 paired patients. We analyzed the relationship among miR-155, miR-21 and SOCS6.(2) Cell lines (A549 and H1299 cells) were transfected withmiR-155, miR-21, or miR-155/21 mimics or inhibitors. Immunoblot and real-time PCR analyses were performed to measure the SOCS6 mRNA and protein levels, respectively. The activities of miR-155,miR-21and 155/21 in these cell lines were analyzed via cell proliferation, cell viability,flow cytometry analyses;(3) Tumor growth was measured in mice injected with A549 cells transfected with miR-21, miR-155, or miR-21/155 antagomirs. The protein levels of SOCS6 were detected in xenografted tumors by immunohistochemical staining.Results:(1) The levels of miR-155, miR-21 as well as SOCS6 mRNAs were measured in lung cancer and adjacent non-tumor tissues from 80 paired patients. MiR-155 and miR-21 are highly expressed and SOCS6 is lowly expressed in lung cancer. Furthermore, the expression of miR-155 and miR-21 gradually increased as lung cancer stages progressed,whereas the expression of SOCS6 gradually decreased.(2) In vitro experiment, treatment with miR-155, miR-21, or miR155/21 mimics or inhibitors potently inhibits or promotes SOCS6 expression. Notably, the combination treatment of MiR-155 and miR-21 mimics or inhibitors is always better than treatment of a single mimics or inhibitor against SOCS6. Furthermore, treatment with miR-155 or miR-21 mimics reduced the luciferase activities of SOCS6-3’-UTR reporter containing the predicted binding sites of miR-21 or miR-155. On the other hand, mimics containing mutations in the seed sequences of miR-155 or miR-21 did not change the luciferase activities of the SOCS6-3’-UTR reporters. These results demonstrate that miR-155 and miR-21 down-regulate the expression of SOCS6 by directly binding to its 3’-UTRs.(3) In vitro experiment, treatment with miR-155 and miR-21 inhibitors induced the apoptosis of A549 and H1299 cells. In addition, the cell proliferation rate of A549 and H1299 cells was significantly inhibited, as shown by reducing cell numbers, viability, and colony formation. Notably, combination treatment with miR-155 and miR-21 inhibitors was more effective than treatment with a single miRNA inhibitor.(4) In vivo experiment, tumor growth was suppressed in mice back injected with A549 cells transfected with miR-155, miR-21, or miR-155/21 antagomirs. Elevated protein levels of SOCS6 were detected in xenografted tumors from cells treated with miR-155,miR-21, or miR-155/21 antagomirs by immunohistochemical staining. Notably,combination treatment with miR-155 and miR-21 inhibitors was more effective than treatment with a single miRNA inhibitor.Conclusion:(1) In clinic, in vitro and vivo, our study indicate that miR-155 and miR-21 are major promoters of lung cancer progression.(2) MiR-155 and miR-21 promote lung cancer progression by directly targeting SOCS6-3,UTR.(3) The inhibiting effect of combination treatment with miR-155 and miR-21 inhibitors was more effective than treatment with a single miRNA inhibitor against lung cancer progession.