The immunohistochemical staining of ATG12 and HMGB2 was performed as previously explained.49 The immunostaining results were scored as the percentage of cells staining positive as follows: 0 for 1% of cells, 1 for 1C25% of cells, 2 for 26C50% of cells, 3 for 51C75% of cells and 4 for 75% of cells. the clinical management of malignancy types, resulting in relapse and metastasis in most malignant tumors. Although much research into the mechanisms of chemoresistance, such as increased drug efflux, mutation of target genes, inactivation of detoxification enzymes, dysfunction of pro-apoptotic proteins or enhancement of DNA repair activity,21, 22 has been reported, the mechanisms involved in malignancy cell chemoresistance are still not clearly comprehended. Emerging evidence has exhibited that miRNAs are involved in chemoresistance in many tumors, such as hepatocellular malignancy, breast malignancy and ovarian malignancy.23, 24, 25, 26 However, you will find relatively few studies around the MDR of GC. According to the results of an miRNA array and high-throughput functional screening in our previous study,14, 15 we selected miR-23b-3p as a candidate gene for MDR in GC cells. Dysregulation of miR-23b-3p has been reported in many cancers, including colon cancer, prostate malignancy, renal malignancy and thymic lymphoma.16, 17, 18, 27 However, the functions of miR-23b-3p are not consistent in different cancers NSC348884 and are even conflicting within breast cancer,28, 29 and an association between miR-23b-3p and GC has not been previously reported. In the present study, the exact functions of miR-23b-3p in regulating MDR in GC cells were studied. The results showed that by targeting ATG12 and HMGB2, miR-23b-3p modulates the chemosensitivity of GC cells by mediating autophagy. To our knowledge, this statement identifies miR-23b-3p as a key regulator of the chemosensitivity of GC for the first time. miRNA usually has multiple target genes. To limit the range of candidate genes, we selected the target genes that were positive in all four prediction tools. The 10 candidate genes that were outlined are as follows: and and were more related to chemoresistance of malignancy. ATG12 is an important factor in autophagic vacuole formation.30, 31 Recent studies have reported that ATG12 is a novel determinant of chemoresistance or radioresistance.19, 32 Consistent with these reports, our results show that this expression of ATG12 was increased in MDR cells of GC. We also found that miR-23b-3p reduced ATG12 expression at both the mRNA and protein levels. Further knockdown of the expression of ATG12 by siRNA increased the sensitivity of MDR cells to chemotherapeutic brokers, which suggests that ATG12 is usually associated with chemosensitivity in GC. HMGB2 is usually a member of the HMGB protein family, which comprises ubiquitous, abundant nonhistone nuclear proteins with diverse functions in the cell.33 The HMGB family consists of HMGB1, HMGB2, HMGB3 and HMGB4. Overexpression of HMGB1 has been observed in several human cancers, such as breast malignancy and colon cancer.34, 35 Importantly, HMGB1 contributes to chemoresistance in many types of malignancy by activating autophagy.36, 37 HMGB2 is highly homologous to HMGB1, and NSC348884 it may have similar effects with regard to cancer development. However, compared with Rabbit polyclonal to ABCA13 HMGB1, relatively little is known regarding the biological function of HMGB2. Recently, it was reported that HMGB2 is overexpressed and promotes chemoresistance in glioblastoma and HCC.38, 39 In the present study, we found that the expression of HMGB2 was significantly higher in MDR GC cells than in the parental cells and that knockdown of HMGB2 significantly reversed MDR in NSC348884 GC. Similarly to ATG12, miR-23b-3p regulated HMGB2 by targeting its 3-UTR. Thus, these results suggest that overexpression of HMGB2 promoted drug resistance in GC. Emerging evidence indicates that autophagy is increased in several human cancers and contributes to chemoresistance.37, 40 ATG12 and HMGB2 were both overexpressed in MDR GC cells, which suggest that autophagy may be involved in MDR. To test this hypothesis, we detected the autophagic flux in our cell model. Consistent with the previous reports described above, our results indicated that MDR cells exhibited increased autophagy, which functions as a mechanism of chemoresistance. Reducing the expression of ATG12 or HMGB2 by administration of siRNA or CQ to MDR cells significantly decreased the level of autophagy, accompanied by increased sensitivity to drugs. Our data suggest that autophagy in MDR GC cells may be a survival mechanism that promotes chemoresistance and that inhibition of autophagy by interfering with ATG12 or HMGB2 has the potential to.
