The Preliminary Functional Studies Of MiR-106b On Radiaoresistant In Colorectal Cancer

Background and objectivesColorectal cancer (CRC) is one of the common malignancy. The incidence rate of CRC in china is increasing fast during the past decads.Surgery is the main treatment for patients with colorectal cancer. However,More than half of patients have found micro-metastasis before surgery and about50%postoperative patients occurred liver-metastasis, which is the main reason to death. Therefore, exploring the molecular mechanism of pathogenesis of CRC and searching for new therapeutic targets to enhance the efficacy of existing strageties is the urgent task of contemporary oncology investigation.Radioresistance is a challenge in the treatment of patients with colorectal cancer (CRC). Individuals display different therapeutic response for preoperative radiotherapy, Nonresponders who hardly benefit from preoperative radiotherapy just suffered more toxic effects, even maybe missing the best treatment opportunity. It suggests that sensitivity of CRC to radiotherapy is distinctly different.microRNA is an abundant class of small noncoding RNAs,17-25nucleotides (nt) in length, which is capable of degrading mRNA or suppressing translation of the targeted genes with high sequence complementarity. microRNAs are involved in suppression of oncogenes or tumor suppressor genes via complementary binding to their targeted gene transcripts, resulting in gene silencing and thus changes of cellular tumorigenecity in various cancers. There are mounting evidences suggesting that miRNAs have been shown to affect the pathogenesis classification, diagnosis, and prognosis and progression of cancer.MiRNAs have also been implicated to radiotherapy response of some tumors. Lin28-let7modulates radiosensitivity of human cancer cells with activation of K-Ras. miR-9and let-7g enhance the sensitivity to ionizing radiation by suppression of NFkappaB1. MicroRNA-181a sensitizes human malignant glioma U87MG cells to radiation by targeting Bcl-2. Up-regulating miR-101efficiently reduced the protein levels of DNA-PKcs and ATM in these tumor cells and most importantly, sensitized the tumor cells to radiation in vitro and in vivo through targeting DNA-PKcs and ATM.Accoding to the "cancer stem cell" hypothesis, to sustain their CSCs has many unique properties, including proliferation capacity, high self-renewal and aberrant differentiation potential, multidrug transporters, DNA damage repairment and detoxifying enzymes. Finally the CSCs survived the treatment and cause relapse ultimately and resulted in the resistance to chemotherapy and radiotherapy. Understanding the mechanisms back up these properties will facilitate the design of new therapeutic strategies targeting CSCs specifically and eradicating the total tumor cell mass, and thereby improving current clinical therapies against aggressive, metastatic, recurrent, and lethal cancers.Tumor-initiating cell is proposed to be responsible for the resistance to radiation and this characteristic is already evidenced in cancer.13,14MiR-106b, as a member of the miR-106b-25cluster, is known to promote cancer cell proliferation and survival in gastric cancer and hepatocellular carcinoma.15,16,17Jamie O. Brett and his collaborators have found miR106b-25regulates neural stem/progenitor cells (NSPC) through the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the neural stem cells(NSC) pool during aging.18But the relationship between miR-106b, sternness and radioresistance has been little reported.As far the roles and specific mechanism remain unclear in colorectal cancer. In this study, we aim to clarify the possible role of miR-106b in the stemness and radioresistance of CRC. It will be heleful to understand the molecular basis of CRC.Contents and Methods1.1Quantitive real-time PCR was used to detect the expression of miR-106b in colon cancer-derived cell lines (HT-29, SW480、LOVO、SW620), CRC tissues and paired normal tissues.2. The relationship between miR-106band irradiation sensitivity of colon cancer-derived cell2.1In vitro experiments:MTT\Clonogenic survival assay、Immunofluorescence staining.. WESTERN BLOT assay were performed to assess the sensitivity of cell to irradiation after enforced and reduced miR-106b expression by lentiviral transduction.2.2In vivo experiments:Athymic nude mice4to6weeks old (GuangDong Experimental Animal Center) were used for tumor implantation. All the animal experiments strictly adhered to the Regulations for the Administration of Affairs Concerning Experimental Animals, the Chinese national guide line for animal experiment, issued in1988. All procedures involving animals and their care in this study were approved and performed by the Southern Medical University Institutional Animal Care and Use Committee (Permit Number:44007200000784). The cells were harvested by trypsinization, washed twice with cold serum-free medium, and re-suspended with200ul serum-free medium. For xenograft tumors assay,2×106cells were injected subcutaneously into the back of nude mice. After tumors were detected, tumor size was measured by a slide caliper and tumor volume was determined by the formula1/2×length×width2. Each group has six athymic nude mices and the mean tumor volume±SD of each group was calculated. Harvested tumors were imaged immediately after sacrifice. To evaluate tumor radioresistance in vivo, when tumors reached an average volume of about200mm3, the mices were exposed to8Gy X-ray alone. Tumor inhibition rate was recorded and calculated every4days. Inhibition rate=(1-irradiation treatment group/control group)×100%.2.3The expression of Bcl-2and Bax in xenograft tumor was detected by immunohistochemistry assay.3. To study the impact of miR-106b on stemness and its molecular mechanisms3.1The ability of colony spheres formation was evaluated after altering the expression of miR-106b.3.2Genes important for stem cell maintenance were screened by qRT-PCR and WESTERN BLOT after altering the expression of miR-106b.3.3The cells of colony spheres formation ability after altering the expression of miR-106b was evaluated when under irradiation conditions.3.4Genes important for stem cell maintenance were screened by qRT-PCR and WESTERN BLOT when cells were under irradiation conditions.4. Identification the target of miR-106b4.1We focused on targets of miR-106b and confirmed the genes of3’-UTR s contained regions matched to the seeds sequences of miR-106b through bioinformatics search Targetscan (http://www.targetscan.org)4.2PLuciferase reporter system was used to validate the direct binding of miR-106b and target genes.5Identification the function of target genes of miR-106b5.1constructed and transfected pcDNA3.1/PTEN and p21vector expressing PTEN and p21gene into SW620-Lenti-miR-106b cells and examnimed whether restoration of PTEN and p21in SW620-Lenti-miR-106b cells could increase sensitivity to irradiation.5.2transfected siRNA/PTEN and siRNA/p21into SW480-anti-miR-106b cells and examnimed whether decrease the expression of PTEN and p21in SW480-Lenti-miR-106b cells could decrease sensitivity to irradiation.5.3Blocking the PTEN/PI3K/AKT pathway with LY294002, and examnimed the change of sensitivity to irradiation.Results1. Identification of expression characteristics of miR-106b in CRC cell lines1.1We analyzed the expression pattern of miR-106b in CRC cell lines with different degrees of differentiation including LOVO (undifferentiated), HT-29(highly differentiation), SW620(poorly differentiation), SW480(highly differentiation). We found that the level of miR-106b was relatively higher in high differentiation cells lines HT-29and SW480.2. MiR-106b enhances colorectal cancer cells resistance to irradiation.2.1Radiosensitivities were assessed by MTT assays after CRC cells were exposed to X-rays. Data indicated that miR-106b could evidently enhance cells radioresistance (4Gy).2.2cells were irritated with various dose of X-ray (2,4,6and8Gy) and colony formation assays were performed to evaluate the survival fraction. Overexpression of miR-106b exhibited a stronger capacity of colony formation and higher survival fraction comparing with the control group, conversely decreased expression of miR-106b contributed to radiosensitivities.2.3Immunofluorescence staining suggested y-H2AX foci numbers was apparently decreased after upregulation of miR-106b, while increased by downregulating miR-106b expression.2.4The expression of y-H2AX was further verified by western blot and found γ-H2AX was apparently decreased after upregulation of miR-106b, while increased by downregulating miR-106b expression.2.5Up-regulation of miR-106b could remarkably lower caspase-3expression, while down-regulation got the converse result.2.6Apoptosis was assessed by Flow cytometric which showed Up-regulation of miR-106b could remarkably lower the percentage of apoptosis cells.2.7up-regulating miR-106b tumor inhibition ratio was relatively lower than the control group, while down-regulating the ratio was evidently higher.2.8Increased miR-106b expression could upregulate Bcl-2expression while downregulate Bax3. MiR-106b enhances tumor-initiating cell capacity3.1The ability of colony sphere formation was dramatically enhanced, a more numbers and larger volume of spheres, when enforced miR-106b expression. Conversely, decreased miR-106b expression could restrain the ability of colony sphere formation. 3.2Overexpressed of miR-106b significantly increased expression of CD133、 Sox2、 Oct4and Bmil, and which were decrease when knock down miR-106b. Additionally, CD133and Sox2were further verified by western blot and also got the similar results to qRT-PCR, however the expression of Oct4and Bmi1did not show any significant change (data not shown).3.3MiR-106b could also enhance the ability of colony spheres formation under irradiation conditions. Overexpression of miR-106b showed a stronger colony spheres formation capacity, while inhibition of miR-106b emerged an opposite effect.3.4MiR-106b could upregulate stemness-relative genes under irradiation conditions. Overexpression of miR-106b increased CD133protein level, while inhibition of miR-106b emerged an opposite effect. However, Sox2expression was reduced conspicuously when miR-106b downregulated expression but hardly verified after miR-106b upregulation.4. The miR-106b targets PTEN and p21for repression4.1We focused on targets of miR-106b and found3’-UTR s of human PTEN and p21contained regions that matched to the seeds sequences of miR-106b through bioinformatics search Targetscan (http://www.targetscan.org).4.2PTEN3’-UTR, the sequence containing the miR-106b binding sites, was cloned into the downstream of pLuciferase open reading frame. Cotransfection of miR-106b mimics and PTEN-3’-UTR-wild vector into SW620(PLuc-PTEN-3’-UTR) significantly decreased the pLuciferase activity compared with miR-NC mimics. In contrast, transfection inhibitors of miR-106b into SW480evidently increased the pLuciferase activity. However, transfection of mimics or inhibitors of miR-106b with the mutant3’-UTR vector (PLuc-PTEN-mut3’-UTR) had no effect on pLuciferase activity. We also have successfully constructed a reporter vector including the p213’UTR-wild (PLuc-p21-3’UTR). Cotransfection miR-106b mimics and p21-3’UTR-wild vector into SW620(PLuc-p21-3’UTR) significantly decreased the pLuciferase activity compared with miR-NC mimics. In contrast, transfection inhibitors of miR-106b into SW480significantly increased the pLuciferase activity. The results confirmed PTEN and p21is direct target of miR-106b.5. MiR-106b mediated-radioresistance can be reversed by PTEN/PI3K/AKT and p21pathway.5.1overexpression of the miR-106b improved p-AKT1/2expression, while downregulation of miR-106b inhibited the expression of p-AKT1/2.5.2The pcDNA3.1/PTEN vector expressing PTEN gene successfully was constructed and transfected into SW620-Lenti-miR-106b cells, then PTEN expression was upregulated obiviously campared to the SW620-Lenti-106b/pcDNA3.1/NC.5.3MTT assays showed that PTEN upregulation resulted from transfecting pcDNA3.1/PTEN vector in SW620-Lenti-miR-106b cells could undoubtedly enhance the sensitivity to irradiation.5.4The immunofluorescence results confirmed that overexpression of the PTEN showed a significantly higher y-H2AX foci numbers compared with control. Additionally, we also found downregulated PTEN expression by transfecting siRNA/PTEN into SW480-Lenti-anti-106b could restored the cells resistance to irradiation which was confirmed by MTT assays. The immunofluorescence results showed a relatively fewer y-H2AX foci numbers after knocking down the expression of PTEN.5.5p21was knocked down by transfecting siRNA into SW620and then exposed to irradiation (4Gy), the y-H2AX foci numbers and the y-H2AX expression were both much higher than the control.5.6pcDNA3.1/p21vector was transfected into SW620-Lenti-106b cells, which overexpressed miR-106b. We found y-H2AX foci numbers and y-H2AX expression were both dramatically higher compared with the control.5.7We transfected siRNA/p21into SW480-Lenti-anti-miR-106b and evaluated restoration p21expression whether SW480-Lenti-anti-miR-106b cells could regain the radioresistance potential, the results showed the y-H2AX foci numbers showed no significant change compared with the control.5.8We treated SW620-Lenti-106b cells with LY294002, which is a highly selective inhibitor of Akt, and then cells were exposed to irradiation (4Gy). Data showed that the radisensitivity was increased after blocking the PTEN/PI3K/AKT pathway.Conclusions1. MiR-106b enhances colorectal cancer cells resistance to irradiation2. MiR-106b enhances tumor-initiating cell capacity3. The miR-106b targets PTEN and p21for repression4. MiR-106b mediated-radioresistance can be reversed by PTEN/PI3K/AKT and p21pathway.