Experiment Study On The Function And Mechanisms Of MiR-423-5p In Regulating Radiosensitivity Of Rectal Cancer

Neoadjuvant(chemo)radiotherapy(nCRT)remains a standard treatment for locally advanced rectal cancer(LARC),which could reduce local recurrence and increase anus sphincter preservation rate in obout two thirds of patients.However,unfortunately about one third of the patients,due to the tumor hypo-radiosensitivity or radioresistance,even though spent a lot of money,delayed the proper operation time and got the side effects of radiotherapy,didn’t get the benefits.So searching of potential biomarkers and therapeutic targets for radiosensitization is urgently needed.MicroRNAs(miRNAs),a class of small(20-24 nt)noncoding RNA molecules,regulate the expression of target genes post-transcriptionally that play a pivotal role in both physiological and pathophysiological processes.Recently,miRNAs have been demonstrated to exert effects on the response of cancer cells to radiotherapy.The aim of our study was to elucidate the altered miRNA expression patterns and their exact role involved in radioresistance in LARC,and to develop a theoretical foundation for the clinical application.In the first part,two acquired radioresistant colorectal cancer(CRC)cell lines were established,named HCT116-R and RKO-R,using the strategy of fractioned irradiation of HCT116 and RKO cells by our group previously.The changes of radioresistant phenotype were evaluated by CCK-8,colony formation assays and flow cytometry assays exposing cells to various doses of X-rays.The results illustrated that HCT116R and RKO-R cells exhibited enhanced proliferation,elevated survival fraction,and decreased apoptotic capacities,which suggested that the acquired radioresistant cells displayed more resistant to radiotherapy compared with their parental counterparts.Subsequently,the differentially expressed miRNA candidates in HCT116-R and RKOR cells were determined using miRNA sequencing analysis and further confirmed by quantitive real-time PCR.Multiple miRNAs,including miR-423-5p,miR-7-5p,miR522-3p,miR-3184-3p,and miR-3529-3p,were identified with altered expression in both of the radiotherapy-resistant cells compared with the parental cells.Meanwhile,the miRNA expression levels were investigated in the microarray data of two different independent cohorts from the GEO database(GSE29298 and GSE68204).Consistent with our results in colorectal cancer cells,miR-423-5p expression level was significantly lower in non-responders with TRG 3-5 compared to responders with TRG 1-2,suggesting its potential role in regulating radiosensitivity in CRC.ROC analysis revealed that the AUC values for miR-423-5p were 0.695(GSE29298)and 0.678(GSE68204).The results lent a degree of credibility for the predictive value of miR423-5p in discriminating between radiosensitive and radioresistant CRC patients.Additionally,the downregulation of miR-423-5p was further verified in the fresh biopsies of LARC patients before(chemo)radiotherapy.Thus,miR-423-5p was selected as a final target.In the second part,we preliminary investigated the potential role of miR-423-5p in radioresistance in colorectal cancer cells.After knockdown of miR-423-5p in relatively radiosensitive cells(HCT116 and RKO),the proliferation rates increased within 48 h after 4 Gy irradiation.Meanwhile,after overexpressing miR-423-5p in the acquired radioresistant cells(HCT116-R and RKO-R),the proliferation rates significantly decreased.In the further investigation,parental colorectal cancer cells exhibited an enhanced colony formation ability and survival fractions after knockdown of miR-423-5p with different doses of X-rays,while overexpressing miR-423-5p resensitized the acquired radioresistant colorectal cancer cells,which exhibited decreased colony formation ability and survival fractions.These results demonstrated that knockdown of miR-423-5p in colorectal cancer cells decreased their sensitivity to radiation,and overexpression of miR-423-5p in radioresistant colorectal cancer cells rescued their radiation sensitivity.Furthermore,our results identified that miR-423-5p could enhance radiation sensitivity of CRC cells by enhancing radiation-induced apoptosis through upregulating caspase 3 expression and downregulating the expression of Bcl-2 and Bcl-xL.MiR-423-5p may be a pro-apoptosis factor in the presence of radiation.Based on the above results in vitro,we hypothesized that miR423-5p may be a key target for radiation therapy in colorectal cancer.Then we proceeded to study in vivo whether treatment with lentiviral vectors to stably overexpress miR-423-5p could enhance tumor response to radiotherapy.The results showed that overexpressing miR-423-5p dramatically inhibited the growth of xenograft tumors in nude mice and suppressed the expression of anti-apoptosis proteins,Bcl-2 and Bcl-xL.Above all,these results indicated that miR-423-5p overexpression could radiosensitize colorectal cancer cells through promoting cell apoptosis via regulating apoptosis proteins.In the third part,to determine the potential mechanisms of miR-423-5p in radioresistance of CRC,the target gene of miR-423-5p related to apoptosis was predicted using the online tools(TargetScan and Starbase)and luciferase reporter assay.The results confirmed that Bcl-xL is a direct target gene for miR-423-5p.Rescue experiments were further carried out to investigate whether Bcl-xL could functionally reverse the decreased radioresistance induced by miR-423-5p.HCT116-R and RKO-R cells were transfected with miR-control or miR-423-5p mimics or co-transfected miR423-5p mimics with pcDNA3.1-Bcl-XL.Transfected cells were irradiated exposing to 4 Gy X-rays for the CCK-8,colony formation assays and flow cytometry analysis.The results demonstrated that ectopic expression of Bcl-xL by pcDNA3.1-Bcl-XL significantly relieved the reduced survival fractions of cellular colony and the increased apoptosis triggered by miR-423-5p in HCT116-R and RKO-R cells.Taken together,these results indicated that miR-423-5p enhanced radiosensitivity of CRC cells by targeting Bcl-xL.It is concluded that miR-423-5p was significantly downregulated in the acquired radioresistant CRC cells and also in pretreatment biopsy tissue samples of(chemo)radiotherapy resistant patients with rectal cancer.Bcl-xL is a direct target gene of miR-423-5p and miR-423-5p can be a critical mediator of radiosensitivity in colorectal cancer cells by targeting Bcl-xL.