| 【Background】Colorectal cancer (CRC), both in worldwide and in China, is one of the mostcommon malignant tumors. The main therapy treatments on CRC areneoadjuvant chemotherapy, radiotherapy, surgery, and palliative treatment.Although considerable advances in recent years in the early diagnosis, radiationtherapy, chemotherapy, and radical surgical procedures of colorectal cancer, butthe overall5-year survival rate of CRC patients has not been significantlyimproved. It is of great importance to find new tumor molecular markers andtranslate the study data into patient care which may open the door to elucidate thepathogenesis of colorectal cancer and improve the quality of life and prognosis ofCRC patients.Ran (Ras-related nuclear) also known as TC4, was originally discovered as ahuman cDNA encoding a translation box, and as a purified protein, first isolatedfrom the human teratoma cell lines. Ran play important roles in a series ofbiological processes of eukaryotic cells, such as the replication of DNA, the transcription and processing of RNA, mitosis and meiosis control, especially inthe spindle assembly, the correct distribution of chromosomes, the breakdownand reorganization of nuclear envelope, and the regulation of spindle checkpoint.However, the most popular function of Ran is the role in DNAreplication as wellas the nucleoplasm transport of RNA and proteins. These studies in mechanismsreveals that Ran may serve as an important oncogene involved in tumormalignant behavior. In renal clear cell carcinoma, high expression of Ranindicates a poor prognosis. The expression level of Ran increased in ovariancancer and relates closely to cell mitosis. Studies in other tumors such as lung,liver, stomach, colorectal cancer, prostate cancer, and pancreatic cancer are onlylimited to the cellular level. The expression patterns of Ran in colorectal cancerand its function on the malignant phenotypes of cancer cells has not been studied.【Aims】1. To evaluate the expression of Ran in colorectal cancer tissues and celllines and its clinicopathological significance.2. To explore the relationships between Ran expression levels and theprognosis of colorectal cancer patients.3. To observe the effect of Ran on colorectal cancer cell proliferation.【Methods】1. Immunohistochemistry and western blot were performed to detect theexpression levels of Ran in colorectal cancer and the matched normaltissues.2. The subcellular localization of Ran in cells was performed byimmunofluorescence staining using a laser scanning confocal microscope (LSCM).3. The relationships between Ran expression and the clinicopathologicaldata were analyze by statistical methods.4. The factors which may affect the overall survival of CRC patients werescreened by the Kaplan-Meier method and Cox’s proportional hazardsregression model through the follow-up data.5. The Ran-specific siRNAs was transfected into colorectal cancer cells. Toscreen the siRNAwhose effectiveness of interference is best.6. Lentivirus which may stable down-regulated the expression of Ran wasconstructed.7. MTT assay, flat colony formation experiment, and flow cytometry wereused to observe the effect of Ran on colorectal cancer cell proliferationin vitro.And subcutaneous tumor formative assay was used to observethe effect of Ran on proliferative ability in vivo.【Results】1. The positive rate of Ran expression was91.29%in287cases ofcolorectal cancer tissue specimens.Of which25cases showed negativestaining (-),76cases expressed as weakly positive staining (+),118cases expressed as moderately positive staining (++), and68casesexpressed as strongly positive staining (+++). Ran expression wascorrelated with tumor differentiation (P=0.005), depth of invasion (P=0.001), lymph node metastasis (P <0.001), and distant metastasis (P=0.001). There was no significant differences between Ran expressionand patient’s age or gender (P=0.984and0.592, respectively).There wasno difference of the level of Ran expression in the total proteins of various colorectal cancer cell lines including SW480, SW620, LoVo,and HT-29by western blot experiments. When the total proteins werereplaced by the extraction of nucleoprotein of the same cell lines, wefound the level of Ran expressed higher in SW620and HT-29and weakor almost no expression in LoVo.2. Ran is located both in the cytoplasm and nucleus, but mainly in thenucleus. Immunofluorescence staining revealed that Ran expression waslocated primarily in the nuclei in SW480, SW620, and HT-29cells, andin the cytoplasm in LoVo cells. These results are consistent with thewestern blot data.3. Kaplan-Meier analysis revealed that with the increased expression levelof Ran, the overall survival time of colorectal cancer patients wasshortened along with the increasing level of Ran expression (log-ranktest: P <0.001).The postoperative mean and median survival time of allCRC patients were33.664±1.073months and28months, respectively.Of287CRC cases patients, the mean survival time of Ran-negativeexpression group (-) was52.280±2.855months. The mean survivaltime of Ran expression weakly positive group(+), moderately positivegroup(++) and strongly positive group(+++) were38.623±1.736months,31.335±1.440, and21.245±1.073months, respectively.Univariate analysis showed that the expression level of Ran,differentiation and clinical pathological staging were significantlyassociated with overall survival time of CRC patients. In multivariateanalysis, the adjusted HR presumed to be1.00(as a reference) in thenegative group (-) of Ran, and the adjusted HR values of the weakpositive group (+), the moderately positive group (++) and strongly positive group (+++) were1.724(P=0.161),2.475(P=0.018) and5.374(P <0.001). Both the univariate and multivariate survival analysisfound that the expression level of Ran can be used as independentprognostic factor in CRC patients.4. The result of western blot reveals that among the three siRNAs,siRNA-1has the most efficient interference, especially at48h aftertransfected.5. The constructed stable cell lines are checked successfully by westernblot. Compared with the control group, SW620cells transfected with thelentivirus enclosed with Ran-specific siRNAshowed slower growth rate,a diminished capacity in the single-cell colony formation, and reducedratio of G1phase to S phase cells. Inoculate the stable cell linestransfected by destinative lentivirus, the tumorigenesis ability wasdecreased.【Conclusions】1. The expression of Ran in colorectal cancer tissues was significantly higherthan that in normal tissues.2. Ran may predict the prognosis of CRC patients, and serves as a potentialprognostic biomarker.3. Ran can promote the proliferation of colorectal cancer cells both in vitro andin vivo. |
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