MiR-124 Radiosensitizes Human Colorectal Cancer Cells By Targeting PRRX1

BACKGROUND AND OBJECTIVEColorectal Cancer(CRC) is one of the common Gastrointestinal Malignant Tumor. It is reported by the International Agency for Research on Cancer (IARC) of World Health Organization that CRC is the third leading cause which is after lung cancer and breast cancer of cancer deaths.The mortality rate ranks is fifth of cancer deaths. Epidemiological studies have found that the incidence of colorectal cancer and mortality of colorectal in our country is still an upward trend. The occurrence of colorectal cancer is the result of environmental and genetic interaction. There is more than half of colorectal cancer patients before the treatment appeared micro-metastases and 50% of clinical patients with liver metastases after surgery. Currently the main treatment for CRC liver metastases is still surgery,even if recurrence rate of colorectal cancer phase II-III period for 15 to 65%. After Total Mesorectal Excision (TME) treatment of stage III of colorectal cancer patients recurrence rate also high as 20 to 30%. To improve the local control rate, long-term survival and quality of life of patients, these patients should be standard adjuvant treatment of colorectal cancer at the same time.Radiation therapy as an important method of tumor therapy and adjuvant therapy in the clinical treatment has been widely used, but many problem such as radiotherapy side effects, complications, radioresistant, etc existence of this progression and is the main reason for the therapy failure. Different individuals obvious individual differences exhibited by radiotherapy. Radiation resistance resulting in patients not only did not respond to treatment but also may miss the best time for tumor therapy. Generating radiation resistance is a multi-factor and the complex mechanisms of gene interactions, mainly due to the occurrence of radiation sensitive cells is reduced. There are many factors related to radiation resistance such as cell cycle arrest, abnormally apoptosis, tumor micro-environment changes and other factors. Therefore, in the study of colorectal cancer in of Radiation Medicine, radiation resistance is a challenging project. Many researchers have tried to find information about the radiosensitivity biomarkers in colorectal cancer such as P53 and P21 for better patient selection. Elucidate the molecular mechanism of radiation-sensitive and radiation resistance will help solve this problem and bring light to treatment of colorectal cancer.MicroRNAs(miRNAs) is a class of non-coding small molecule RNA which encoded genome and participate the post-transcription regulation and length of 20-24nt.They can combination through base pairing with the 3’untranslated region or the coding region of the target gene mRNA sequence to regulate gene expression. MicroRNAs play a significant role in a variety of life activities, such as the space-time decision in development, cell proliferation and death, and tumorigenesis which found later are closely related. Some studies show that miRNA can increase or decrease tumor radiosensitivity, closely related to tumor radiotherapy resistance. miR-124 is closely associated with a variety of malignant tumors miRNA, such as liver cancer, breast cancer, bladder cancer, colorectal cancer, nasopharyngeal cancer, and play an important role in tumorigenesis. miR-124 was in the brain of mice was first extracted, it can act on the forkhead box protein Q1(FOXQ1), cycl in-dependent kinase 4 (CDK4), CDK6 etc. However, there is no reporter that announce relationship between miR-124 and radiation-sensitive or radiation resistance.This study intends to investigate the expression of miR-124 and relationship to radiosensitivity in colorectal cancer and colorectal cancer cells, further more elucidate its molecular mechanism may play a role. The research found that miR-124 in colorectal cancer cell lines and tissues to reduce,but overexpression of miR-124 can enhance the colorectal cancer cell radiosensitivity. Bioinformatics is found PRRX1 is downstream target genes directly regulated by miR-124, and knockout PRRX1 can enhance the colorectal cell radiosensitivity. but overexpression of PRRX1 could be reversed miR-124 on colorectal cancer cells radiosensitivity. This study provides new insights of the radiosensitivity molecular mechanisms and miR-124 in colorectal cancer cells. This paper not only provide a new theoretical basis for the clinical treatment of colorectal cancer targets but also provided new ideas and theories address the radiation resistance.METHODS:1. Detected miR-124 expression levels in the colorectal cancer cells and tissuesHuman colorectal cancer cells SW480、 LOVO、SW620、HCT-116、HT-29、 LST-174 and control cells FHC were purchased from Shanghai Institute of Cell Biology Cell Bank. All human colorectal cancer tissue samples were taken from Nanfang Hospital of Southern Medical University from 2010 to 2014 general surgical specimens, control above normal colorectal tissue away from the tumor were at least 5cm. All the samples had not received radiotherapy, chemotherapy and other anti-tumor therapy, eventually diagnosed as pathological diagnosis of colorectal cancer.Total RNA was extracted from the cells using TRIzol reagent manufacturer’s protocol. By TRIzoI pyrolysis, chloroform extraction, isopropanol precipitation method extraction in colorectal cancer cells and tissue samples of total RNA. According to Thermo Fisher Scientific reverse transcription kit instructions, reverse transcription synthesis of cDNA, using real-time quantitative PCR kit, varying levels of real-time detection of miR-124 expression in different cells and tissues of colorectal cancer, and then analyzed statistically.2. Detection of cell radiosensitivity2.1. Stable over-expression of miR-124 colorectal cancer cell lines establishedLV-miR-124 and LV-con were infected with lentivirus supernatant to cell line SW480, LOVO, followed by flow cytometry sorting GFP+cells, stable expression of miR-124 in colorectal cancer cell lines.2.2. Detect different doses of radiation Overexpression of miR-124 cell survival fractionAt logarithmic growth phase untreated colorectal cancer cell lines SW480, LOVO and stable overexpression miR-124 cell seeded in 6-well plates, when cell adherent were treated with 0,2,4,6,8,10 Gy of X rays (using Varian 600C accelerator 6MV-X line, SSD of 100 cm). Each dose point 3 parallel samples, conventional culture two weeks. After culture irradiation with 4% paraformaldehyde, stained with Giemsa. The plates were placed under a low magnification microscope calculated for each cell clones (count more than50 cell is an efficient cloning). The surviving fraction was calculated according to the mufti-target single-hit model.2.3 Detection apoptosis-related protein in the overexpression miR-124 cell lines with different doses of radiationTaking at logarithmic growth phase untreated colorectal cancer cell lines SW480, LOVO and stable overexpression miR-124 cell after 6h radiation extract total cellular protein. Westernblot detection above cells apoptotic-associated protein such as Bcl-2, caspase-3 and phosphorylation of γ-H2AX changes.3. MiR-124 regulation of colorectal cancer cell radiosensitivity molecular mechanisms3.1 Bioinformatics prediction miR-124 regulation of downstream target genesBioinformatics prediction software targetscanhuman6.0 (http://www.targetsca-n.org/) and common miRNA database TargetScan and Pictar were used to miR-124 target gene predictive analysis. According to the software operation flow forecast downstream regulation of miR-124 target genes and literature screening, and ultimately selected as the target PRRX1 further research.3.2 pairs of luciferase reporter system verification miR-124 target genes PRRX1Construction of miR-124 binding sites PRRX1 assumed gene 3’untranslated region (3’UTR) and mutant PRRX1 of 3’UTR luciferase reporter plasmid. The pLUC-3’UTR-PRRX1 and pLUC-3’UTR-mut-PRRX 1 luciferase reporter plasmid for co-transfection and rear dual luciferase reporter gene system detects. The pLUC-3’UTR-PRRX1 and pLUC-3’UTR-mut-PRRX 1 luciferase report plasmid and miR-124mimics and inhibitors were co-transfected using the Promega Dual-luciferase reporter assay system luciferase activity is detected, all experiments were repeated three times.3.3 Interference PRRX1 on colorectal cancer cells radiosensitivityLV-PRRX1 and control LV-shcon lentivirus suspensions were infected with colorectal colorectal cancer cells, then sorted by flow cytometry GFP+cells obtained stable interference PRRX1 cells. The cells are divided many teams into different irradiation doses calculated cloning efficiency and survival fractions.The surviving fraction was calculated according to the mufti-target single-hit model.3.4 Overexpression PRRX1 can reverse radiation sensitivity of miR-124 in colorectal cancer cellsBased cationic liposome plasmid pCDNA3.1-PRXX1 to the constructed stabilized expressing miR-124 colorectal cancer cells. After 48h transfection efficiency was detected by Western blot, cell cloning forming experiments detection of changes in radiation sensitive. The cells were divided into different groups with different doses irradiation, cloning experiment to calculate the change rate and clonogenic survival fraction, using GraphPad Prism 5.0 software "multi-target model click" curve fitting, and Western blot analysis of apoptosis-related proteins.3.5 Animal experimentsObserve the effects of miR-124 in colorectal cancer radiation sensitivity in vivo environment. Measuring the size of tumor tissue removed, rendering the radiation under conditions of tumor volume growth curve, statistical analysis.4. Statistical AnalysisUsing SPSS 13.0 software for statistical analysis, two samples were compared using independent samples t-test, more than two samples were compared using data completely randomized design analysis of variance, homogeneity of variance using Levene’s Test. Data comparison method with single factor analysis of variance, P <0.05 was considered statistically significant.RESULT:1. miR-124 expression in colorectal cancer cell lines and tissues decreaseQuantitative PCR showed that miR-124 expression decreased in colorectal cancer cell lines. Compared with adjacent normal colorectal samples, miR-124 expression in cancer tissues was significantly reduced.2. Overexpression of miR-124 enhanced radiosensitivity in colorectal cancer cells2.1. Overexpression of miR-124 at different doses of radiation cell survival fractionClonogenic assay results confirmed that Compared with the control group, the over-expression of miR-124 in the radiation (different doses) can reduce cell survival fraction, increase the radiation sensitivity.2.2. Overexpression of miR-124 detection of different doses of radiation apoptosis-related proteinsAfter 6h radiation detector cell protein level, Westernblot results show:when radiation overexpression miR-124 can reduced Bcl-2 expression, and increases caspase-3 and phosphorylated γ-H2AX. Description overexpression of miR-124 in the radiation can enhance the efficiency of cell apoptosis in colorectal cancer, result in miR-124 can enhance colorectal cell radiosensitivity.3. PRRX1 is a downstream target genes of miR-124 directly regulated3.1 Bioinformatics prediction miR-124 regulation of downstream target genes is PRRX1By TargetScan online software to predict miR-124 and PRRX1 the 3’UTR region has binding sites, and has been preliminarily verified in conventional miRNA database TargetScan and Pictar in.3.2 pairs of luciferase reporter system validation PRRX1 is miR-124 target genesDual luciferase reporter system showed that compared to the group miR-124 and miR-con, luciferase activity decreased in the transfected plasmid pLUC-3’UTR-PRRXl experimental group; while compared to anti-miR-124 group and anti-miRcon group the luciferase activity increased, and the differences were statistically significant, P＜0.05. Transfected pLUC-3’UTR-mut-PRRX1 plasmid experimental group, miR-124 and miR-con compared luciferase activity was no significant difference; anti-miR-124 group compared with the group of anti-miRcon, luciferase activity was also no significant difference.Evident prove that miR-124 can bind to gene PRRX1 3’UTR region, thus inhibiting the luciferase activity, suggesting that PRRX1 direct downstream target genes of miR-124.3.3 Interference PRRXl on colorectal cancer cells radiosensitivityWesternblot results showed that in colorectal cancer cells LOVO and SW480, the PRRX1 interference is stable and effective, reduce cell survival fraction after radiation, the difference was statistically significant. Then Westernblot apoptosis-related proteins showed that after interference PRRX1 radiation, compared with the control group,Y-H2AX and activity of caspase-3 increased expression levels, lower levels of expression of Bcl-2. Those evident certification apoptosis enhancement, indicating interference PRRX1 on colorectal cancer cells enhanced.3.4 overexpression PRRXl can reverse radiation sensitivity of miR-124 in colorectal cancer cellsIn order to further clarify PRRX1 as miR-124 regulate the radiation sensitivity target gene in colorectal cancer, in stable overexpression of miR-124 colorectal cancer cells lines and SW480 and LOVO transfected plasmid pCDNA3.1-PRXX1 to recovery PRRX1 expression, westernblot and clone experimental results show that compared with the control group, not only cell cloning efficiency and surviving fraction increased but also cell resistance to radiation enhancement. This suggested that PRRX1 can reverse miR-124 caused radiation-sensitive enhanced in certain extent, Meanwhile Westernblot results found, and PRRX1 can reverse the expression of miR-124 overexpression induced apoptosis-related genes. These results indicate overexpression PRRX1 can reverse radiation sensitivity increase of miR-124 in colorectal cancer cells and been a target gene of miR-124 enhanced radiation sensitivity.3.5 miR-124 can enhance the radiation sensitivity in vivoThe results show unradiotherapy LV-con group compared with LV-miR-124 group, no significant difference in tumor size, but radiotherapy LV-con+IR group compared with the LV-miR-124+IR, tumor volume was significantly reduced. We mapped the change in tumor volume and the number of days of radiotherapy diagram showed that:miR-12+IR group compared with the LV-con+IR group, tumor volume decreased with increasing number of days of radiotherapy, tumor volume growth efficiency becomes small tumor growth delay. In summary explanation, we have developed a new approach to sensitizing radioresistant cancers by targeting miR-124.CONCLUSION:1. The miR-124 is reduced in colorectal cancer cells and tissues.2. Overexpression of miR-124 enhanced radiosensitivity in colorectal cancer cells.3. The miR-124 directly regulated downstream target genes is PRRX1.4. Interference PRRX1 can enhance colorectal cell radiosensitivity.5. PRRX1 can reverse the effects of overexpression of miR-124 in colorectal cancer cell radiosensitivity.6. The nude mice experiments show that miR-124 in vivo can enhance the sensitivity of radiotherapy.