Methylation Mediated MiR-23b-3p Regulates The Chemoresistance Of Gastric Cancer Cells By Targeting ATG12 And HMGB2

【Background】 Gastric cancer(GC) has been the second leading cause of cancer-related deaths worldwide over the past century. Because of the lack of effective techniques for early diagnosis, most GC patients are diagnosed at an advanced stage of the disease. For these patients, chemotherapy is the first-line treatment. However, even though many novel chemotherapeutic drugs are used in clinical practice, chemotherapeutic approaches fail because of intrinsic or acquired drug resistance, particularly multidrug resistance(MDR). The mechanisms underlying MDR have been widely studied but the key determinants of this phenomenon in GC remain largely unclear. Recently micro RNAs(mi RNAs) have been reported to be involved in the MDRof various cancers through negtively regulating the expression of chemo-resistant related genes, however, only a few mi RNAs were reported to regulate chemoresistance by targeting apoptosis-associated pathways in GC up to now. Thus,the exact mechanism of mi RNAs in regulating GC drug resistance remains to be further studied. In our previousstudy, using an mi RNA array and a functional screening strategy, we found that mi R-23b-3p played a potential role in reversing drug resistance in GC. In this report, we intend to further investigate the exact role and mechanism of mi R-23b-3p in regulating of MDR in GC cells, and identify the relationship between mi R-23b-3p expression and clinicopathological parameters including prognosis in patients with gastric cancer.【Objectives】 1. To investigate the exact role and mechanism of mi R-23b-3p in regulating of MDR in GC cells in vitro and in vivo. 2. To identify the association among mi R-23b-3p expression, clinicopathological parameters and prognosis of GC patients.【Methods】 1. q RT-PCR was used to detect the expression of mi R-23b-3p in SGC7901 and SGC7901/VCR. In addition, gain or loss of function and MTT assays were performed to determine the potential roles of mi R-23b-3p in regulating MDR and proliferation of GC cells. 2. Multiple bioinformatics algorithms were used to predict the target genes of mi R-23b-3p, then q RT-PCR,western blot,CLSM and dual-luciferase reporter assay were used to verify the expression of target genes and confirm that whether the target genes were regulated by mi R-23b-3p. RNAi,q RT-PCR,Western blot and MTT assays were used to detect the potential roles of target genes, and confirm the relationship between ATG12 and HMGB2 simultaneously. 3. TEM、western blot and CLSM were used to compare the level of autophagy between SGC7901 and SGC7901/VCR. 4. RNAi,western blot,CQ were used to confirm whether the target genes could regulate autophagy of cells and investigate the relationship between autophagy and GC cells. 5. CLSM and western blot were used to confirm whether mi R-23b-3p could regulate autophagy,and then q RT-PCR,western blot were used to detect the expression of mi R-23b-3p, target genes and the level of autophagy before treating other GC cells with 5-Fu. In addition, using co-transfection method to confirm mi R-23b-3p regualtes autophagy by inhibiting target genes and then participate in MDR of GC.6. Stable transfectants overexpressing mi R-23b-3p or the negative control were generated using the p EZX-MR03 lentiviral transfer vector. Subcutaneous tumor experiment was used to determine mi R-23b-3p-mediated regulation of MDR in vivo. 7. Tissue microarray, immunohistochemistry, in situ hybridization were used to detect the expression of mi R-23b-3p and its target genes in GC tissue specimens, and then analyze the relationship between mi R-23b-3p and clinicopathological parameters or prognosis in patients with gastric cancer. 8. Using methylation databases to predict Cp G islands in upstream of mi R-23b-3p, and then detect expression of mi R-23b-3p before and after demethylation with 5-aza.【Results】 1. mi R-23b-3p regulates the sensitivity of GC cells to chemotherapeutic agents in vitro. q RT-PCR showed that compared to SGC7901 cells, mi R-23b-3p was significantly down-regulated in SGC7901/VCR cells. Overexpression of mi R-23b-3p dramatically enhanced the sensitivity of SGC7901/VCR cells to 5-FU, VCR, and CDDP. Transfection of SGC7901 cells with a specific inhibitor of mi R-23b-3p increased the IC50 values for these 3 chemotherapeutic agents. However, the proliferatin of GC cells were not influenced by the expression of mi R-23b-3p. 2. ATG12 and HMGB2 are direct targets of mi R-23b-3p. ATG12 and HMGB2 were up-regulated in SGC7901/VCR cells compared to SGC7901 cells. In addition, mi R-23b-3p negtively regulated ATG12 and HMGB2 by targeting their 3’UTR. Silencing of either ATG12 or HMGB2 sensitized SGC7901/VCR cells to chemotherapeutic agents. Interestingly, we found that the silencing of HMGB2 reduced the expression level of ATG12, whereas the expression of HMGB2 was not markedly altered by a si RNA targeting ATG12. 3. Chemo-resistant GC cells exhibit increased autophagy. TEM and CLSM revealed a marked accumulation of autophagosomes in the cytoplasm of SGC7901/VCR cells compared to SGC7901 cells. Western blot revealed that increased LC3-II expression and an accompanying decrease in p62 expression were clearly detected in SGC7901/VCR cells compared to SGC7901 cells.4. Inhibition of autophagy restores the chemo-sensitivity of chemo-resistant cells. Autophagy was markedly decreased in SGC7901/VCR cells that were transfected with si RNAs targeting ATG12 or HMGB2. Increased expression of LC3 II was observed in SGC7901/VCR cells after 24 h of treatment with CQ. In addition, MTT assays showed that CQ greatly enhanced the sensitivity of SGC7901/VCR cells to chemotherapeutic agents. 5. mi R-23b-3p inhibits autophagy by regulating ATG12 and HMGB2. overexpression of mi R-23b-3p in SGC7901/VCR cells significantly decreased the accumulation of autophagosomes. In contrast, inhibition of endogenous mi R-23b-3p in SGC7901 cells showed the opposite effects. In addition, q RT-PCR and western blot indicated that at 24 h after treating with low concentration of 5-Fu, mi R-23b-3p expression in AGS and BGC823 cells was decreased, while the expression of ATG12, HMGB2 and LC3-II were up-regulated. We further cotransfected SGC7901 cells with mi R-23b-3p inhibitors and si RNAs targeting ATG12 or HMGB2; the si RNAs targeting ATG12 and HMGB2 attenuated the effect of mi R-23b-3p inhibitors. 6. Restoration of mi R-23b-3p increased the drug sensitivity of gastric cancer cells in vivo. q RT-PCR showed that compared with Lenti-NC, the expression of Lenti-mi R-23b-3p in SGC7901/VCR was significantly increased. We then transplanted SGC7901/VCR-Lenti-NC or SGC7901/VCR-Lenti-mi R-23b-3p cells into nude mice. The volumes of tumors were not significant changes before intraperitoneal injection of drugs. However, the volumes of the mi R-23b-3p transfected tumours were markedly decreased after chemotherapy. 7. mi R-23b-3p expression is down-regulated in chemo-resistant patients and correlated with overall survival in patients with gastric cancer. The expression levels of mi R-23b-3p were decreased in 24 chemo-resistant patients, while the expression of ATG12 and HMGB2 were significantly up-regulated in these patients compared with chemo-sensitive patients. In addition, we found that expression of mi R-23b-3p did not differ in patients with different factors, including age, gender, differentiation, depth of invasion and the TNM staging. However, the expression of mi R-23b-3p correlated with the distant organ metastasis and the patients in the low mi R-23b-3p expression group hada signi?cantly poorer prognosis than those in the high mi R-23b-3p expression group. 8. The expression of mi R-23b-3p was regulated by DNA methylation. According to the methylation softwares, the upstream of mi R-23b-3p exists Cp G island. After treating SGC7901/VCR and SGC7901 cells with 5-azacytidine(5-Aza), mi R-23b-3p expression was significantly increased in SGC7901/VCR but not in SGC7901.【Conclusion】 In summary, our data confirm that mi R-23b-3p is a novel mi RNA that regulates MDR in GC. Methylation mediated mi R-23b-3p inhibits autophagy in MDR cells by targeting ATG12 and HMGB2 and increases the sensitivity of MDR cells to chemotherapeutics. mi R-23b-3p expression positively correlates with the chemosensitivity and overall survival in patients with gastric cancer. Thus, the expression of mi R-23b-3p may be a marker to predict MDR and a potential therapeutic strategy for the treatment of MDR in GC.