The Effect Of MircoRNA-30B On Proliferation And Apoptosis Of Colorectal Cancer Cells And The Mechanism Involved

BackgroudColorectal cancer(CRC) is the third of the most common malignant tumors in China, and the incidence is increasing during the past decades. Up to90%of patients can be cured by surgery if the disease is detected at the early stage, but unfortunately it is often diagnosed only at an advanced stage and the prognosis is therefore poor. Therefore, to work out the mechanism of occurrence and progression of CRC and to find out the preventive and therapeutic methods is the main task in the field of colorectal cancer.MicroRNAs(miRNAs), an abundant class of17～25nucleotides small noncoding RNAs, posttranscriptionally regulate gene expression through directly binding to the3’untranslational region(3’UTR) of target mRNAs. Till now, about1000miRNA genes have been identified in mammals, but revealing their roles in physiology and pathology is still an ongoing process. The role of miRNAs in cell physiology and pathology is hard to understand due to their complex relation to biological function. The literature related to miRNAs showed us:1.miRNA can act as oncogenges or tumor-suppressor genes;2. miRNAs are involved in tumor progression and metastasis through their role in pathways that contribute to metastasis, including migration, invasion, cell proliferation, epithelial-to-mesenchymal transition(EMT), angiogenesis, and apoptosis;3. miRNAs might be useful as prognostic and predictive markers. As miRNAs involve in biological development, cell proliferation, differentiation and apoptosis, dysregulation of miRNAs appears to play a crucial role in cancer pathogenesis. Recently, emerging evidence hsa suggested that deregulated miRNAs are involved in the pathogenesis of CRC mainly by regulating the expressioin of oncogenes and tumor suppressors. Deregulation of miRNAs are encoded by oncogenes or tumor suppressors.MiRbase database showed us that miR-30b was21nt and its expression had no tissue specificity. Recent studies have demonstrated that the abnormal expression of miR-30b is involved in innate immune response, cell differentiation, initiation and progression of multiple solid tumors. Wszolek et.al showed us differential expression of miRNAs was identified from microarray analysis and included reduced expression associated with miR-30b, miR-31, miR-141, miR-200a, miR-200b, miR-200c, miR-205, miR-21in invasive lesions and elevated miR-99a in noninvasive UCB lesions. Reduced invasion potential was recorded in UM-UC-3, following pre-miR transfection, in all UCB cell lines with the exception of UM-UC-3/miR-30b transfectants. In addition, miR-30b can influence tumor invasive by targeting genes. Gaziel-Sovran et al, showed us miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. It can be seen, miR-30b played an important role in a variety types of tumor. However, there is no literature about the relation between miR-30b and colorectal cancer up to date.Based on the above findings, the aim of this study is to examine the expression of miR-30b in CRC tissues by real-time quantitative RT-PCR (qRT-PCR), identify its possible role in proliferation and apoptosis by up-regulate or down-regulate miR-30b in CRC cells. We hope that this study will improve the better understanding of CRC pathogenesis and the development of novel effective therapies for CRC.Materials and Methods1. Clinical specimensPrimary CRC biopsy specimens and their matched adjacent tissues were obtained from Nanfang Hospital(Southern Medical University, Guangzhou, China). Both tumor and normal tissues were histologically confirmed by H&E(hematoxylin and eosin) staining. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital.2. Cell cultureThe human CRC cell lines HCT116and SW480were cultured in RPMI-1640with10%FBS. HEK293FT cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with5%FBS. All cells were cultured in a humidified atmosphere of95%air and5%CO2at37℃.3. Vector construction(1)pLVTHM/miR-30b:A510bp fragment of miR-30b was amplified by PCR and cloned into pLVTHM. The vector was named pLVTHM/miR-30b. (2) PGL3-SOX43’UTR and PGL3-SOX43’UTR-Mut:SOX43’UTR was amplified by PCR and cloned downstream of the firefly luciferase gene in PGL3vetor. The vector was named PGL3-SOX43’UTR(wt3’UTR). Site-directed mutagenesis of the miR-30b binding site in SOX43’UTR was performed using PCR and named PGL3-SOX43’UTR-Mut(mut3’UTR).4. Production and purification of lentivirusThe transfer and the packaging plasmids were co-transfected into293FT cells using CaPO4precipitation.The supernatant was harvest48-72h posttransfection. Then, the viral particles were concentrated by ultracentrifugation, and the viral pellets were resuspended. The titration of lentiviral suspension was calculated by making a tenfold serial dilution.5. Cell transfection(1) miRNA transfection:The transfection was performed using Lipofectamine2000reagent.(2) Lentivirus infection:The packaged lentivirus were used to infect CRC cell lines. Following fluorescence-activated cell sorting, the GFP+cells were isolated, and the cell lines were established, which could stably overexpress miR-30b.(3) miRNA and plasmid co-transfection:The co-transfection was performed using Lipofectamine2000reagent. A working concentration of miRNA was50nM, a working concentration of wt3’UTR and mut3’UTR mut was lug, and a working concentration of an internal control pRL-SV40vector was0.05μg in a24-well plate.6. Real-time PCR Total RNA was extracted from cells or tissues. The expression of miR-30b was detected by All-in-OneTM First-Strand cDNA Synthesis Kit and All-in-OneTM qPCR Mix. The SOX4mRNA was detected by Reverse Transcription System (TAKARA) and SYBR(?) Green Realtime PCR Master Mix(TOYOBO). All samples were normalized to internal controls and fold changes were calculated through relative quantification(2-ΔΔCt).7. Cell biology experimentsMTT assay, colony formation assay and soft argar assay were performed to detect the effect of miR-30b on cell growth of colorectal cancer cells. Flow cytometry with annexin V FITC/PI double staining was used to detect the effect of miR-30b on apoptosis of colorectal cancer cells(Annexin V Flous Staining Kit (Roche)).8. Western blotTotal protein was isolated and quantitated using BCA assay. The protein lysates were separated by10%SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies and detected by chemiluminescence.9. Luciferase report assayAccording to the instructions of Dual-Luciferase(?) Reporter Assay System (Promega). At48h post-transfection, Relative luciferase activity was measured by the activity of firefly luciferase and renilla luciferase.10. Statistical analysis SPSS13.0software was used for statistical analysis. Levels of miR-30b in43primary colorectal tumors compared with their adjacent normal tissues were determined by the Wilcoxon matched pair test. The results of MTT were analyzed by Factorial design analysis of variance. Differences between two groups in colony formation assay, soft argar assay,Flow cytometry with annexin V FITC/PI double staining and Luciferase report assay were determined by two independent t test. P<0.05was considered statistically significant.Result1. The expression level of miR-30b in CRC tissues.The results of qRT-PCR showed that the expression of miR-30b was decreased in cancer tissues, compared with their adjacent normal tissues(P=0.001). The media of relative expression of miR-30b was0.149(25th-75th percentile,0.030-0.309) in tumor samples, with that in non-tumor control samples was0.252(25th-75th percentile,0.078-0.630)2. The effect of miR-30b on CRC cells in vitro.To explore the effect of miR-30b on cell growth, HCT116and SW480were transfected with miR-30b mimics or inhibitor, respectively. The results showed that the expression of miR-30b was up-regulated by4.774-fold(P=0.001) in HCT116cells and7.107-fold(P=0.001) in SW480cells. However, the expression of miR-30b was reduced by83%(P=0.001) in HCT116cells and78%(P=0.001). All these data demonstrated that the transient transfection could effectively upregulate or downregulate the expression of miR-30b. The result of MTT assay displayed that miR-30b overexpression inhibited cell growth by34.4%in HCT116cells and by22.5%in SW480cells at5day post-transfection, and miR-30b down-regulation promoted cell growth by34.3%and33.8%, respectively. Lentiviral vectors were used to restore the expression of miR-30b in HCT116and SW480cells. Following fluorescence activated cell sorting, and cell lines HCT116/miR-30b and SW480/miR-30b were established to stable overexpress miR-30b. As demonstrated in colony formation assay, the colony number was11.7±1.1in HCT116/miR-30b group and12.5±0.8in SW480/miR-30b group, less than their control group(t=8.652, P=0.001; t=7.917, P=0.001); Soft argar assay showed that the colony number was10.6±1.2in HCT116/miR-30b group and14.8±1.0in SW480/miR-30b group, less than their control group(t=9.133, P=0.001; t=9.263, P=0.001). Silencing miR-30b expression can enhance the ability of colony of CRC cells.To identify the potential mechanism responsible for the observed effects of miR-30b on cell growth in CRC cells, we evaluated apoptosis rates in HCT116cells transduced with the miR-30b mimics or inhibitor. Apoptosis rates were evaluated via double staining of cells with Annexin V FITC/PI, distinguishing between apoptotic cells and vital cells. Transduction of miR-30b resulted in a significant increase in apoptotic cells compared with transduction of miR-control in HCT116cells(t=-6.944, P=0.002). Transduction of miR-30b inhibitor resulted in a significant decrease in apoptotic cells compared with transduction of miR-control in HCT116cells(t=4.922, P=0.008)All these results demonstrated that the abnormal expression of miR-30b is associated with cell proliferation and apoptosis of CRC cells, and it functions as a potential tumor suppressor in CRC pathogenesis. 3. miR-30b inhibited the expression of SOX4via binding to its3’UTROn the basis of three major prediction softwares, miRBase (http://microrna. sanger.ac.uk/targets/v51), TargetScan (http://www.targetscan.org/) and miRanda(http://www.microrna.org/microrna/home.do), potential binding sites of miR-30b in the3’UTR of SOX4were predicted. Then we constructed the recombinant plasmid pGL3-SOX43’UTR and mutant vector pGL3-SOX43’UTR-Mut, and performed dual luciferase reporter assay. The result showed a significant decrease of luciferase activity when miR-30b mimics and pGL3-SOX43’UTR were co-transfected in HCT116cells(t=10.815, P<0.001), suggesting miR-30b could directly target the3’UTR of SOX4in CRC cells. In addition, a significant increase of luciferase activity was examined when miR-30b inhibitor and pGL3-SOX43’UTR vector were co-transfected in HCT116cells(t=-7.920, P=0.001).SOX4mRNA and protein levels in HCT116/pLVTHM and HCT116/miR-30b were detected by qRT-PCR and western blot. The results showed that ectopic expression of miR-30b lead to significant decrease of SOX4mRNA and protein levels in HCT116cells. Moreover, silencing miR-30b could upregulate the expression of SOX4mRNA and protein. These results suggested SOX4was a target of miR-30b in CRC cells.Conclusions1. The expression level of miR-30b is downregulated in CRC tissues.2. The abnormal expression of miR-30b is associated with cell proliferation and apoptosis of CRC cells, which functions as a potential tumor suppressor in CRC pathogenesis. 3. SOX4is a target of miR-30b in CRC cells.