The Role And Mechanism Of MiR-454 In Colorectal Cancer

Colorectal cancer (CRC), occur in the junction of the rectum and colon, is a common occurrence in parts of the digestive tract colon cancer. 40 to 50 year age group had the highest incidence of male to female ratio of 2 to 3:1. Colorectal cancer is the third most commonly diagnosed carcinoma in males and the second in females. Colorectal cancer signs and symptoms depend on the location of tumor in the bowel as well as the extension of tumor in the body. Early colorectal cancer is difficult to diagnose because it has no symptoms or symptoms are not obvious.As the tumor progresses,its signs and symptoms emerges .Classic alarm symptoms include: persistent constipation, fecal occult blood, stool thinning, loss of appetite and weight loss etc. Bloody stools and anemia occurring in the people over 50 years old should be considered as high risk features.The main colon cancer types are adenocarcinoma, mucinous adenocarcinoma and undifferentiated carcinoma. The tumor morphology are generally polypoid, ulcer type etc. Colon cancer can develop along the intestinal wall ring, spread or deep infiltration to the intestinal wall and down along the longitudinal diameter of the bowel, except via lymphatics, blood transfer and local invasion, but also the transfer of planting or spread along the suture, the incision into the abdominal cavity surface. Patients with chronic colitis, colon polyps, male obesity as the susceptible population. Surgery is the main treatment for operable colorectal cancer, followed by chemotherapy、 radiation、biotherapy and Chinese medicine etc.However, recurrence and metastasis are very common. Thus, there is an urgent need to elucidate the underlying molecular mechanisms of CRC and find new molecular targets for treatment of this disease.Recently, microRNAs (miRNAs) were discovered to be the endogenous non-coding small RNA (19-22 nucleotides), which lead to mRNA degradation or inhibition of translation through imperfect hybridization to 3’-untranslated region (3’-UTR) in target mRNAs that directly regulate the expression of target genes. Among several candidate miRs regulating cell proliferation, miR-454 has been shown to be one of the important determinants of cell proliferation in several kinds of cancers. Thereafter, it has shown that miR-454 modulates a lot of target genes, among these genes, cylindromatosis (CYLD) has been shown to be involved in regulating the apoptosis of cancer cells. Cell proliferation, which is the most important hallmarks of cancer, is the leading lethal factors for malignant cancer, especially for CRC. However, the relationship between CRC and the expression of miR-454 has not yet been elucidated.In the present study, we report that upregulation of miR-454 in CRC is associated with development of CRC. Further investigations revealed that miR-454 directly targeted the 3’-UTR of CYLD to suppress the expression of this gene, which in turn promoted the proliferation of CRCs. Thus, we extrapolate to miR-454 is a molecular target for the treatment of colon cancer, can be for a new, effective treatment for colon cancer. This study is divided into five sections elaborate.Part1Objective: Find out miR-454 expression was in CRC tissues and CRC cell linesMethod:Eight paired human colorectal cancer tissues and the matched tumor adjacent tissues (TAT) were obtained from CRC patients and histopathologically diagnosed Tissue samples were collected at surgery, immediately frozen in liquid nitrogen and stored until total RNAs or proteins were extracted. Use human CRC cell lines SW403, HT-29, COLO320DM, COLO205, SW480, KM202L, SW620 and normal colonic cell line FHC. All CRC cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gbico, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA), and Normal colon FHC cells were grown in DMEM/F-12 medium with 10% FBS, lOng/mL cholera toxin,5μg/mL transferrin,5μg/mL insulin, 100ng/mL hydrocortisone and extra lOmM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Cell lines were cultured in a humidified incubator at 37℃ in an atmosphere of 5% CO2 and 95% air.Results:To investigate the role of miR-454 in CRC development, we evaluated the expression levels of miR-454 in CRC tissues and CRC cells by qRT-PCR. As showed in figure 1A, the results showed that the expression levels of miR-454 were consistently upregulated in the CRC tissues compared with the matched tumor adjacent tissues, and in all 8 tested CRC cell lines had significantly upregulated miR-454 levels than those in the normal colonic cell line FHC. These results showed that miR-454 played an important role in CRC. Together, these results suggest that miR-454 is significantly increased in CRC and may serve as a prognostic marker for patients with CRC.Conclusion:MiR-454 expression was upregulated in CRC tissues and CRC cell lines, indicate that miR-454 may play an important role in the human colon cancer paroxysm process.Part2Objective:miR-454’s influence to CRC proliferationMethod: carried by lipidosome, miR-454 promotor plasmid miR-454 mimics and suppress plasmid miR-454 inhibitor (miR-454-in) were transferred to human colon cancer cell SW480. And the result of transfection was confirmed by qRT-PCR. We compared the proliferation ability of these two kinds of colon cells by MTT, colony formation and anchorage-independent growth assay.Results:To investigate whether CRC cell proliferation was regulated by miR-454, we investigated whether miR-454 plays a tumor positive role in cell proliferation of CRC. We transfected the SW480 cells with miR-454 mimics, miR-454 inhibitor or the respective controls. Relative miR-454 expression was verified using qRT-PCR. We first evaluated the effect of miR-454 on cell proliferation using MTT, colony formation and anchorage-independent growth, respectively. To investigate the role of miR-454 upregulation in the development and progression of CRC, we next examined its effect on cellular proliferation. MTT assay showed that miR-454 upregulation significantly increased the proliferation rate of SW480 cells, and this was further confirmed by a colony formation assay. Strikingly, we found that enforced expression of miR-454 in SW480 cells drastically enhanced their anchorage-independent growth ability. In contrast, the cell growth rates and colony numbers of SW480 cells transfected with miR-454 inhibition (miR-454-in) were significantly inhibited the cell growth rate than those transfected with NC. In addition, the anchorage-independent growth ability of SW480 cells was significantly decreased in response to miR-454-in. These results showed that miR-454 increased CRC cell tumorigenicity in vitro.Conclusion: MiR-454 promoted CRC cell proliferationPart3Objective: to find out how miR-454 suppress CYLD expressionMethod:It is generally accepted that miRNAs exert their functions by regulating their downstream target genes expression. Potential targets of miR-454 were predicted using bioinformatics methods. CYLD, a tumor suppressor gene containing a binding site 3’-UTR for miR-454, was selected as the target for further analysis.To determine whether miR-454 affects CYLD expression, expression of CYLD were detected by western blot in the SW480 cells, which were transfected with miR-454 mimics, miR-454-in or the respective controls. To verify the effect of miR-454 on the inhibition of CYLD expression, we examined whether CYLD is regulated by miR-454 through direct binding to its 3’UTR. CYLD 3’-UTR (wild or mut) vector were cotransfected in SW480 cells with miR-454 mimic, miR-454-in or miR-NC, followed by measurement of luciferase activity.Results:Western blotting analysis showed that miR-454 mimics markedly suppressed CYLD protein levels in SW480 cells (Fig.4B), while miR-454-in clearly promoted CYLD protein expression. As shown in figure 4C, a consistent and dose-dependent reduction of luciferase activity was observed in SW480 cells transfected with miR-454 mimic, whereas the repressive effect of miR-454-in increased wild-type CYLD luciferase activity. Meanwhile, overexpressing miR-454 had no effect on the luciferase activity of CYLD 3’-UTR mut type. These results, taken together, demonstrated that CYLD is a bona fide target of miR-454.Conclusion:MiR-454 directly targets CYLD by binding to its 3’-UTR.Part4:Objective: besides CYLD there is any protein that related to proliferation regulated by miR-454Method:We had resulted that miR-454 could promote the growth and proliferation of CRCs and CYLD was a direct target of miR-454. It was reported previously that CYLD is closely correlated with cell proliferation. The expression levels of a number of critical cell-proliferation regulators were also detected. We also detected the expression of two important cell proliferation regulator factors, c-Myc and Cyclin D1. c-Myc is a nuclear transcriptional factor and most of its targeting genes including Cyclin D1 are crucial in the regulation of cell proliferation, differentiation, apoptosis and other cellular functions. By western blot we detected the expression level of c-Myc and Cyclin D1 in SW480 transfected by miR-454 mimics, miR-454-in and miR-NC.Results:Furthermore, the expression of Cyclin D1 and c-Myc were upregulated in SW480 cells transfected with miR-454 mimic, but decreased in the cells transfected with miR-454-in, relative to control cells (Fig. 4 E). Thus providing further evidence that miR-454 plays an important role in CRC cell proliferation. Altogether, our results indicated that miR-454 functionally modulates cellular proliferation regulators, Cyclin Dl and C-Myc, thus relevant to cell proliferation.Conclusion:miR-454 could significantly upregulate the level of c-myc and Cyclin D1.Part5Objective:to know if miR-454 down regulates CYLD to realize the proliferation of CRCMethod:To further investigate the role of CYLD reduction in miR-454-induced CRC proliferation, we first examined the effects of CYLD downregulation on CRC cell proliferation. We transfected two artificial siRNA (Small interfering RNA) of CYLD to SW480-miR-454-in cells. Using western blot to test the CYLD expression level, we detected the cell proliferation ability these cells by colony formation and anchorage-independent growth assay.Results:As predicted, Western blot analysis verified that CYLD-siRNA#1 and CYLD-siRNA#2 both effectively decreased the expression of CYLD in miR-454-in-transfected SW480 cells (Fig. 5A). Colony formation and anchorage-independent growth assays showed that CYLD-silenced in miR-454-in-transfected SW480 cells have an additive effect on cells proliferation (Fig. 5 B and C). Taken together, these results demonstrate that direct CYLD downregulation is required for miR-454-induced CRC cell proliferation.Conclusion:CYLD down regulation is required for miR-454-induced proliferation of CRC cell.