MicroRNA-501-5p Promotes Doxorubicin Resistance And Tumorigenesis Of Gastric Cancer Cell Through Targeting BLID

Research background:Gastric cancer(GC)is a high mortality rate digestive system malignancy which has the second highest risk of cancer-related deaths worldwide.Currently,surgical treatment and chemotherapy are the main strategies used to manage gastric cancer.Doxorubicin(Adriamycin,ADR),an anthracycline-based anti-tumor agent to inhibit synthesis of DNA or RNA,is effective for lymphoma,breast,lung and gastric cancers combined with other chemotherapeutic drugs including fluorouracil and mitomycin.However,the clinical evident shown that the treatment in cancer patients is still unsatisfactory because of the existence of resistance to frequent acquisition of ADR.Escaped from apoptosis is the most important factor of ADR resistance in cancer cells.MicroRNAs(miRNAs)are a class of small noncoding RNAs with 19~25nucleotides(~22 nt)that act as crucial regulators in a variety of biological processes such as cell proliferation,migration,invasions and drug resistance.The gene encoding hsa-miR-501-5p(miR-501)locates on chromosome X.Several recent studies demonstrate that mi R-501 is upregulated in the liver cancer,lung adenocarcinoma and gastric cancer.Moreover,high mi R-501 is associated with advanced lung adenocarcinoma and is a poor prognostic indicator of gastric cancer.But no explicit evidence points out the relationship between miR-501 and doxorubicin resistance.BLID(BH3-like motif containing protein,cell death inducer),also known as breast cancer cell 2(BRCC2),is a member of Bcl-2 family.The miRNA analysis software TargetScan predicted that BLID is the potential target of miR-501.A study reported that BLID induces the apoptosis either in a Caspase-dependent manner through activating Caspase-3,9 or interacting with Bcl-X_L.Another study found that BLID suppresses breast cancer cell growth and metastasis in vitro and in vivo via downregulating Akt pathway.Therefore,we hypothesized that miR-501 enhances the doxorubicin resistance in gastric cancer through suppressing apoptosis mediated by the downregulation of BLID.Objectives:(1)To determine whether BLID is a direct target of miR-501.(2)To investigate the role and mechanism of miR-501/BLID axis in doxorubicin resistance and tumorigenesis of gastric cancer cell.Methods:(1)In miR-501 upregulation experiment,non-specific miRNA(NC),miR-501 mimic(miR-501)and small interfering RNA against BLID(siBLID)were transfected into gastric cancer SGC7901 and BGC823 cells by lipofectamine 2000.In miR-501 downregulation experiment,non-specific miRNA inhibitor(NCi),miR-501inhibitor(501i)and co-transfection between miR-501 inhibitor and small interfering RNA(siRNA)against BLID(501i+si BLID)were transfected into doxorubicin resistance gastric cancer SGC7901/ADR and BGC823 cells by lipofectamine 2000.The endogenous levels of miR-501 in this paired gastric cancer cell lines were measured by Taqman probe real-time qRT-PCR.The SYBR Green qRT-PCR was used to detect the expression of BLID mRNA.Western blot was applied to measure BLID protein expression and downstream Caspase-3/-9?p-Akt protein expression.(2)Double-stranded oligonucleotides of BLID 3?UTR(wild-type or mutant)were synthesized and inserted into the XbaI site of the GV272 vector,the luciferase assay was performed to verify that BLID is a directly target of miR-501.The IC50 of gastric cancer cell line SGC7901/ADR and its parental cell line SGC7901 was investigate by CCK-8 assay.Annexin V-FITC/PI flow cytometry assay was determined the impact of miR-501 on the apoptpsis.Colony formation assay and transwell migratory/invasion assay was performed to assessed the influences of miR-501 on cell proliferation,migration and invasion.(3)Lentiviral expression vector Len-NC,Len-501 and shRNA against BLID(shBLID)infected SGC7901 cells.15 mice were randomly divided into 3 groups,totally 1×10~6each group infected cells in 100?l PBS/matrigel were subcutaneously injected into the nude mice respectively.When the volume of xenografts reached to approximate 100mm~3,the mice were administered by ADR(5 mg/kg)through tail vein three times a week for 2 weeks.The mice were sacrificed and the subcutaneous tumors were excised and frozen in liquid nitrogen until processing for RNA and protein isolation.The qRT-PCR was used to detect the expression of miR-501 and BLID mRNA.Western blot were applied to measure BLID,Caspase-3/-9?p-Akt protein expression.Results:(1)CCK-8 assay results showed that the IC50 of SGC7901/ADR cells was10.65-fold higher than SGC7901 cells(P<0.01).The expression of miR-501 in SGC7901/ADR cells was significantly higher than in the corresponding sensitive cells(P<0.05),whereas the expression level of BLID mRNA in SGC7901/ADR cells were dramatically lower and BLID protein was reduced in SGC7901/ADR cells compared to SGC7901 cells(P<0.01).(2)Luciferase assay showed that the co-transfection of mi R-501 mimic and BLID-3'UTR vector into SGC7901 and 293T cells evidently suppressed the luciferase activity compared to the co-transfection of negative mi RNA and wild-type 3'UTR of BLID mRNA.The real-time qRT-PCR revealed that SGC7901 and BGC823 cells transfected with miR-501 mimics and BLID mRNA level was significant decrease(P<0.01),whereas siBLID group had no significant compared with NC group(P>0.01).Western blot analyses revealed that BLID protein level was significant decrease,whereas siBLID group had no significant compared with NC group.In addition,the real-time qRT-PCR and Western blot analyses revealed that transfected with mi R-501inhibitor restored the expressions of BLID mRNA and protein compared with the NC group and 501i+siBLID group in SGC7901/ADR and BGC823 cells(P<0.01).(3)The CCK8 assay revealed that mi R-501 overexpression and BLID knockdown significantly enhanced the cell viability to doxorubicin treatment compared to the negative control in SGC7901 and BGC823 cells.On the contrary,miR-501 inhibitor transfected SGC7901/ADR and BGC823 cells were more sensitive to doxorubicin as compared with the two negative controls(P<0.01).(4)Annexin V-FITC/PI flow cytometry assay shown that a significant inhibition of apoptotic rate was observed in miR-501 and si BLID group compared with the NC group in SGC7901 and BGC823 cells(P<0.01).Western blot shown that cleaved Caspase-9 and Caspase-3 was inhibited in SGC7901 and BGC823 cells transiently transfected with miR-501 mimic or BLID siRNA.In contrast,SGC7901/ADR and BGC823 cells transfected with miR-501 inhibitor had a higher apoptotic rate than those transfected with scrambled mi RNA inhibitor(P<0.01).Cleaved Caspase-9 and-3 expressions were also increased by knockdown of miR-501.(5)Colony formation assay shown that miR-501 overexpression and BLID downregulation dramatically increased the colony formation efficiency compared to the negative control in transfected SGC7901 and BGC823 cells(P<0.01).The transwell migratory/invasion assay shown that miR-501 overexpression significantly increased SGC7901 and BGC823 cells passing through the chamber compared with the negative control(P<0.01).Western blot shown that upregulation of miR-501 and knockdown of BLID dramatically activated the phosphorylation of Akt at Ser 473compared with the NC group.Conversely,the knockdown of miR-501 inhibited the potentials of proliferation,migration and invasion in SGC7901/ADR and BGC823cells(P<0.01).(6)In vivo experiment shown that the tumors of Len-501 group grew faster than those of Len-NC group,and silenced BLID by shRNA appeared as a similar trend with Len-501 group and both Len-501 group and shRNA group had significantly larger tumor volume than Len-NC group after 2 weeks of ADR treatment(P<0.05).The expressions of miR-501 was upregulated and its target BLID at both mRNA and protein levels was downregulated in tumor tissues of Len-501 group compared to Len-NC group,whereas BLID was markedly downregulated at both mRNA and protein levels in Len-501 group and shBLID group compared to Len-NC group.The protein levels of cleaved Caspase-9 and-3 were decreased and the phosphorylation of Akt was increased dramatically in the Len-501 injected xenograft tumor tissues compared to len-NC by western blot analysis.Conclusions:(1)BLID is a direct target of miR-501 and it is directly regulated by miR-501 at transcriptional level.(2)miR-501 enhances the proliferation,migration,invasion and resistance to doxorubicin of gastric cancer both in vivo and in vitro.(3)miR-501 enhances resistance to doxorubicin through depressing apoptosis mediated by Caspase pathway inactivation via directly targeting BLID expression on onehand,and on the other hand,it accelerates gastric cancer cell proliferation,migration and invasion via phosphorylation of Akt.As a result,miR-501 might be a potential target for doxorubicin resistance and gastric cancer therapeutics.