MiR‐16 is a tumour suppressor that is down‐regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR‐16 on macrophage polarization and subsequent T‐cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon‐γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)‐4. The identity of polarized macrophages was determined by profiling cell‐surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus‐expressing miR‐16 to assess the effects of miR‐16. Effects on macrophage–T cell interactions were analysed by co‐culturing purified CD4+ T cells with miR‐16‐expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR‐16 targets and understand its underlying mechanisms. MiR‐16‐induced M1 differentiation of mouse peritoneal macrophages from either the basal M0‐ or M2‐polarized state is indicated by the significant up‐regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin‐1, and increased secretion of M1 cytokine IL‐12 and nitric oxide. Consistently, miR‐16‐expressing macrophages stimulate the activation of purified CD4+ T cells. Mechanistically, miR‐16 significantly down‐regulates the expression of PD‐L1, a critical immune suppressor that controls macrophage–T cell interaction and T‐cell activation. MiR‐16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4+ T cells. This effect is potentially mediated through the down‐regulation of immune suppressor PD‐L1.

Background: Clinically, when the diagnosis of colorectal cancer is clear, patients are more concerned about their own prognosis survival. Special population with high risk of accidental death, such as elderly patients, is more likely to die due to causes other than tumors. The main purpose of this study is to construct a prediction model of cause-specific death (CSD) in elderly patients using competing-risk approach, so as to help clinicians to predict the probability of CSD in elderly patients with colorectal cancer. Methods: The data were extracted from Surveillance, Epidemiology, and End Results (SEER) database to include ≥ 65-year-old patients with colorectal cancer who had undergone surgical treatment from 2010 to 2016. Using competing-risk methodology, the cumulative incidence function (CIF) of CSD was calculated to select the predictors among 13 variables, and the selected variables were subsequently refined and used for the construction of the proportional subdistribution hazard model. The model was presented in the form of nomogram, and the performance of nomogram was bootstrap validated internally and externally using the concordance index (Cindex). Results: Dataset of 19,789 patients who met the inclusion criteria were eventually selected for analysis. The fiveyear cumulative incidence of CSD was 31.405% (95% confidence interval [CI] 31.402-31.408%). The identified clinically relevant variables in nomogram included marital status, pathological grade, AJCC TNM stage, CEA, perineural invasion, and chemotherapy. The nomogram was shown to have good discrimination after internal validation with a C-index of 0.801 (95% CI 0.795-0.807) as well as external validation with a C-index of 0.759 (95% CI 0.716-0.802). Both the internal and external validation calibration curve indicated good concordance between the predicted and actual outcomes. Conclusion: Using the large sample database and competing-risk analysis, a postoperative prediction model for elderly patients with colorectal cancer was established with satisfactory accuracy. The individualized estimates of CSD outcome for the elderly patients were realized.

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IL‐10‐producing B cells (B10) are associated with autoimmune diseases, infection and tumours. MiR‐15a/16 as a tumour‐suppressive gene is down‐regulated in several tumours, such as chronic lymphocytic leukaemia, pituitary adenomas and prostate carcinoma. Here, increased frequency of IL‐10‐producing CD19+ Tim‐1+ cells was seen in both aged miR‐15a/16−/− mice (15‐18 months) with the onset of B cell leukaemia and young knockout mice (8‐12 weeks) transplanted with hepatic cancer cells. CD19+ Tim‐1+ cells down‐regulated the function of effector CD4+CD25low T cells ex vivo dependent on IL‐10 production, and adoptive transfer of CD19+ Tim‐1+ cells promoted tumour growth in mice. IL‐10 production by CD19+ Tim‐1+ cells was involved with the STAT3 activation. Bioinformatics analysis shows that miR‐16 targets the 3′‐untranslating region (3′‐UTR) of STAT3 mRNA. Overexpression of miR‐16 in CD19+ Tim‐1+ cells inhibited STAT3 transcription and its protein expression. Thus, the loss of miR‐15a/16 promoted induction of regulatory CD19+ Tim‐1+ cells in tumour microenvironment. These results confirmed that miR‐15a/16 could be used in tumour therapy due to its inhibition of tumour and regulatory B cells.

M1 macrophages are involved in inflammation by producing proinflammatory cytokines, whereas M2 macrophages are associated with wound healing and tissue regeneration by producing anti-inflammatory cytokines. MicroRNAs are involved in macrophage polarization. To evaluate whether miR-15a/16 is involved in macrophage polarization under tumour or inflammation microenvironments, we observed the growth of transplanted hepatic cancer (H22) cells or severity of dextran sulphate sodium (DSS)-induced colitis in 8-week-old miR-15a/16 knockout (KO) mice. Compared with littermate controls, the miR-15a/16 mice exhibited retarded tumour growth and increased sensibility to DSS-induced colitis. Meanwhile, the M1 cell frequencies were higher in tumour tissues and inflamed colons of KO mice than of littermate controls. Macrophages with miR-15a/16 deletion revealed an enhanced NF-κB transcription under the physiological state and lipopolysaccharide (LPS) or high mobility group box 1 (HMGB1) stimulation. STAT3 expression was also significantly increased in miR-15a/16 macrophages under LPS or HMGB1 stimulation. The polarization of M1 macrophages can be associated with the coactivation of NF-κB and STAT3. Results indicated that miR-15a/16 deficiency in the macrophages directs M1 polarization for tumour suppression and proinflammation. Thus, miR-15a/16 deletion in macrophages holds a distinct biological significance from that of the microRNA deficiency in tumour cells.