| Backgroud and ObjectiveIn 2013,American Cancer Society statistics show that the colorectal cancer incidence and mortality of Americans is accounting for about 9%of the total,ranking the third.The incidence is the second only to bronchial cancer and prostate cancer among men,and after lung,bronchial cancer and breast cancer in women.Colorectal cancer is also one of the common malignant tumors in our country.With the changes in diet and improvements of the living standards,the incidence of colorectal cancer showed an increasing trend in recent years.The most important reason of colorectal cancer mortality is invasion and metastasisMicroRNAs(miRNAs)which widely exist in eukaryotes are a class of evolutionary conserved endogenous non-coding small RNA family,and about 18-22 nucleotide sequences of RNA.It has been reported that MicroRNAs take an important role in the development of a variety of tumors.MiRNAs can regulate the expression of target gene causing its degradation or inhibited their translation through the incomplete complementary way binding to the 3’UTR of the targeting protein-coding gene mRNA on the post-transcriptional level.In the human genome it has been found nearly a thousand miRNAs.MiRNAs can regulate about 1/3 protein-coding genes which control of almost all cellular activities,including proliferation,differentiation,cycle,apoptosis,metabolism,individual development and so on.And they can aslo regulate tumor cells migration and invasion effect,which similar to the role of tumor suppressor or oncogene.Studies have shown that microRNAs are associated with a variety of tumors,such as leukemia,lung cancer,stomach cancer,breast cancer and colorectal cancer and so on.MiR-101 which is one of many members of the miRNA family and a common sequence of a mammal,have been detected in a variety of cells.The two precursors of RNA on miR-101 in human are miR-101-1 and miR-101-2 which are about 75bp and 79bp;respectively located on the first and the ninth of the human chromosome;and the mature miR-101 contains about 21 base pairs.It has been reported that miR-101 had downregulated in lung,breast,prostate,pituitary adenoma,gastric cancer,hepatocellular carcinoma and other tumors,and these findings explain that miR-101 may take part in metastasis,invasion and other processes of tumors.These researches indicate that miR-101 may play a role in suppressing tumors.And the target genes of miR-101 are also constantly being discovered,such as mTOR,DNA-PKcs,FOS and so on.The expression levels of some miRNAs in patients may not any contribute to the diagnosis and prognosis of tumor,but also affect the sensitivity to drug therapies.And some miRNAs have been attempted as new therapeutic target molecules in clinically.It is significance that deeply studied the function and expression of miRNA in human tumors,which is important for researching of the occurrence,development and the mechanism of tumor metastasis,and it is also beneficial to cancer diagnosis,treatment and prognosis.However,there are nearly few reserches about the effects of miR-101 on the radiosensitivity in human colorectal cancer.And it is worthy of us to do it.Therefore,in our study,we intends to identify miR-101 expression characteristics in colorectal cancer cell lines and alter the expression level of miR-101 in colorectal cancer so as to observe biological behavior of tumor cells as well as the radiosensitivity of colorectal cancer cell,which provide an important theoretical basis for investigating the occurrence,transfer mechanism and clinical treatment of colorectal cancer.Methods1.Identification of miR-101 in colorectal cancer cell linesReal-time RT-PCR was used to detect the expression level of miR-101 in 6 colorectal cancer(CRC)cell lines,including SW480,SW620,IHT29,HCT116,LOVO and LS174T.2.MiR-101 regulated the radiosensitivity in human colorectal cancer cells(1)Transfected the GV209-miR10I lentiviral vector into human colorectal cancer cells SW620 established a stable cell lines overexpressing the miR-101,LipofectamineTM2000-mediated transfection method was used to transfect the miR-101 inhibitors into colorectal cancer cell lines SW480,obstructed the expression of miR-101.(2)Applying Q-PCR to detect the expression of target genes of miR-101 in different cell lines of colorectal cancer(3)CCK-8 method,colony formation assay,cell cycle analysis,apoptosis analysis carried out to detect the miR-101 influences the biological behaviors such as cell proliferation,invasion abilities and radiosensitivity of human colorectal cancer cells.(4)Western blots experiments3.Validation the target gene of miR-101 and the molecular mechanisms of radiosensitivity in human colorectal cancer cells(1)The three commonly used online bioinformatics websites including TargetScan,microRNA.org and Pictar were used to predict the target genes,screening of candidate target genes of miR-101.mRNAS with high predictive values(at least forecasted by three databases)were selected as candidates.(2)Recombined vector psiCHECK-2-RAC1 contained RAC1 3’UTR was constructed,and followed by site-directed mutagenesis vector named psiCHECK-2-RAC1-Mut was also established.Dual-luciferase reporter assay was utilized to validate the direct binding of RAC1 and miR-101.(3)Western blots experimentsWestern blot were used to detect the expression of RAC1,Cdc42 and RhoA,in SW620 overexpressed miR-101 and in SW480 obstructed miR-101.4.Statistical analysisSPSS 13.0 soRwarc was used for data statistical analysis.Data were expressed as Mean±SEM.And the experiments were repeated at least 3 independent times.Relative quantification value(2-△△Ct)of Real-time quantitative(qRT-PCR)was analyzed by One-way ANOVA.The results of CCK8 assay,colony formation assay,cell cycle analysis,apoptosis analysis were analyzed through Factorial design analysis of variance and One-way ANOVA.And the LSD,SNK,or Dunnett’s T3 tests were used for multiple comparisons.Two-tailed Student’S t test was applied for comparisons of relative luciferase activities of dual-luciferase reporter assay.P<0.05 were considered statistically significant.Result1.Identification of the expression levels of miR-101 in CRC(Colorectal cancer)cell linesQuantitive real-time PCR was applied to detect the expression levels of miR-101 in 6 CRC cell lines.Used the HT29 cells as a reference,the results of One-Way-ANOVA ananlysis showed that the expression of miR-101 in the 5 cell lines was different from each other,and the difference was statistically significant(F=4.647,P<0.001).Dunnett T3 multiple comparison statistical results showed that the expression of miR-101 in Lovo cell lines are different from in SW480 and SW620 cell lines,and expression differences were statistically ’significant(P<0.05),whereas there were no significant differences of expressions of miR-101 between LS174T and HCT116(P>0.05).2.MiR-101 regulated the radiosensitivity in human colorectal cancer cells(1).Using the GV209-miR101 lentiviral vector;established a stable cell sublines of SW620 which is overexpression of miR-101.Real-time PCR results showed that the expression of miR-101 in miR101-SW620 cells were significantly higher than GV209-SW620 and blank group(t =-12.775,P = 0.012).The cell sublines of SW620 which was overexpression of miR-101 was successfully constructed.The Expression of RAC 1 between the two cells was difference,and the difference was statistically significant(t = 13.545,P = 0.000);the expression of RAC1 in miR101-SW620 cell lines was significantly lower than it in GV209-SW620.(2).LipofectamineTM2000-mediated transfection method was used to transfect the miR-101 inhibitor into colorectal cancer cell lines SW480,obstructed the expression of miR-101.Real-time PCR was used to detect the expression of miR-101 inhibitors;the expression levels of miR-101 were differences between SW480-NC and SW480-miR101 inhibitors,and the difference was statistically significant(t =-3.647,P = 0.022).Compared with SW480-NC,SW480-miR101 inhibitors reduced the expression level of miR-101,which meaned transfection successful.The Expression of RAC1 between the two cells was difference,and the difference was statistically significant(t=-20.777,P=0.000);the expression of RAC1 in SW480-miR101 inhibitors cell lines was significantly higer than it in SW480-NC.(3).Used the CCK-8 assay to detect the survival rate of overexpression and disturbances of miR-101 which were suffered different doses of X-ray treatment,and plotted survival curves.The cell survival fraction in SW620,GV209-SW620 and miR101-SW620 which were treated with different radiation doses was statistically significant(F = 1262,P = 0.000;F = 555,711,P = 0.000;F = 70.699,P = 0.000).The cell viability which respectively treated SW620,GV209-SW620 and miR101-SW620 with 2Gy,4Gy,6Gy and 8Gy was statistically significant difference(F = 26.454,P =0.001;F = 57.612,P = 0.000;F = 262.282,P = 0.000;F ＝ 117.05,P = 0.000).The results suggested that as the dose of X-ray irradiation increased the survival rate of cells descreased and in the same dose of X-ray irradiation the viability of miR101-SW620 cell was significantly reduced compared with the blank groups and control groups,which indicated that miR101-SW620 cells irradiated with a faster decline in the survival curves.This shows that over expression of miR-101 in SW620 cells may enhance the sensitivity of SW620 cells to radiotherapy.The survival rate in SW480,SW480-NC and SW480-miR101 inhibitors cell lines which was treated with different doses of X-ray were significant differences(F = 1221,P = 0.000;F ＝492.815,P = 0.000;F = 4545,P = 0.000).The cell survival rate which respectively treated with 2Gy,4Gy,6Gy and 8Gy was statistically significant difference(F =129.202,P = 0.000;F = 8.76,P = 0.017;F = 31.635,P = 0.001;F = 96.548,P ＝0.001).The results suggested that as the X-ray irradiation dose increased the survival rate of cells decreased,and in the same X-ray irradiation dose,the survival rate of SW480-miR101 inhibitors cell was significantly higher than the control group and the blank group,which means the SW480-miR101 inhibitors cells were irradiated slow decline in the survival curves.This shows that after disturbed the expression of miR-101 may decrease the sensitivity of SW480 cells to radiotherapy.(4).Used the CCK-8 assay to detect the cell proliferative capacity of miR-101 overexpression groups and inhibition groups which were treated with 0GY/4GY X-ray irradiation and plot growth curve.Factorial analysis of variance showed that the growth time level in each groups of SW620 cells was a significant difference(F =3962,P = 0.000),the cell proliferative capacity was a significant difference between groups(F = 1250,P = 0.000),cell proliferation with X-ray irradiation treatment was a significant difference(F = 6245,P = 0·000),the interaction between time and groups was a significant difference(F = 1433,P = 0.000),time and X-ray irradiation dose interaction was significant difference(F = 829.946,P = 0.000),the interaction between X-ray dose irradiation and groups was significant difference,(F = 2820,P =0.000),the interaction among time,between groups and X-ray irradiation dose was significant difference(F = 433.170,P = 0.000).The results suggested that without X-ray irradiation miR101-SW620 proliferation rate was significantly reduced,compared with the blank group and control group;compared with no X-ray irradiation treatment,the cell proliferation capacity of blank group,control group and miR101-SW620 group which were given 4GY X-ray irradiation treatment were all significantly reduced,and cell proliferation capacity of miR101-SW620(4GY)was the slowest of the three groups.This indicates that the over expression.of miR-101 may inhibit the proliferation of SW620 cells in vitro,and up-regulated miR-101 may enhance the sensitivity of SW620 cells to radiotherapy.The growth time level in each groups of SW480 cells was a significant difference(F = 23600,P = 0.000),the cell proliferative capacity was a significant difference between groups(F = 1727,P =0.000),cell proliferation with X-ray irradiation treatment was a significant difference(F = 2211,P = 0.000),the interaction between time and group was a significant difference(F = 243.141,P = 0.000);the interaction of time and X-ray irradiation dose was significant difference(F = 251.555,P = 0.000);the interaction between X-ray dose irradiation and groups was significant difference(F = 180.773,P = 0.000),the interaction among time,between groups and X-ray irradiation dose was significant difference(F = 42.313,P = 0.000).The results suggest that without X-ray irradiation cell proliferation capacity was significantly increased in SW480-miR101 inhibitors compared with the blank group and control group;compared with no X-ray irradiation treatment,the cell proliferation capacity of blank group,control group and SW480-miR101 inhibitors group which were given 4GY X-ray irradiation treatment were all significantly reduced,and the cell proliferation capacity of SW480-miR101 inhibitors(4GY)was the was significantly more faster than the other two groups with 4GY X-ray irradiation treatment.This suggests that disturbed miR-101 may promote the proliferation capacity of SW480 cells in vitro;down-regulated miR-101 might weaken the sensitivity of SW480 cells to radiotherapy.(5)Colony formation assayWithout no X-ray irradiation(OGY)the cloning efficiency of SW620 blank group,GV209-SW620 group and miR101-SW620 group were 44.33%,48.17%and 35.17%respectively;with 2GY X-ray irradiation were 8.3%,9.5%and 4.5%respectively;with 4GY X-ray irradiation were 2.5%,2.9%,1.28%respectively;with 6GY X-ray irradiation were 0.60%,0.63%and 0.27%respectively;and with 8GY X-ray irradiation were 0.068%,0.085%and 0.023%respectively.Factorial analysis of variance showed that cell cloning efficiency levels in SW620 cell sublines of each groups were significant differences(F = 166.375,P = 0.000).The results showed that in SW620 cell sublines compared with blank group,control group,the cell colony formation rate in overexpression of miR-101 was significantly reduced.With X-ray irradiation treatment of SW620 cell sublines,colony formation rate of each groups was reduced,and with the doses of 4 GY X-ray irradiation increment,the colony formation rate gradually decreased,while the colony formation rate in overexpression of miR-101 group cell lines reduced the most obviously than the other two groups.The cell viability of SW620 cell sublines including blank group,GV209-SW620 group and miR-101 overexpression group were respectively 1,1 and 1 with OGY X-ray irradiation treatment;with 2GY X-ray irradiation were respectively 18.72%,19.72%and 12.94%;with 4GY X-ray irradiation were respectively 5.64%,6.03%and 3.66%;with 6GY X-ray irradiation were respectively 1.35%,1.31%and 0.77%;with 8GY X-ray irradiation were respectively 0.154%,0.177%and 0.067%.Factorial analysis of variance showed that the radiation dose levels of cell viability in SW620 cell sublines was a significant difference(F = 27300,P = 0.000).The results showed that with X-ray irradiation treated to SW620 cell sublines of each groups,the cell viability of each groups reduced obviously,and with incremental doses of X-ray irradiation,the survival rate of each groups gradually decreased.Compared with blank-group and GV209-SW620 group,the survival rate of over-expression of miR-101 decreased the most obviously.GraphpadPrism5 software was used to do click multiple target model fitting curve of cell survival with linear quadratic model fitting curve of cell survival respectively.The results indicated that over-expression of the miR-101 of SW620 may enhance its sensitivity to radiation,compare to the SW620 blank group and GV209-SW620 group,and it was statistically significant(p<0.05).(6)Cell cycle experimentsFow cytometry was used to detect cancer cell cycle processes which were affected in overexpression and interference of miR-101 in colorectal cancer cell lines.The results showed that each group of SW620 cell sublines in cell cycle distribution was statistically significant differeces(F=33.329,P=0.000,F=474.450,P=0.000;F=772.063,P=0.000).Compared with SW620 blank group and GV209-SW620 group,miRl 01-SW620 group appeared in G2/M and G1 phase cycle arrested(P<0.05).Compared with SW620 blank group and GV209-SW620 group,miR101-SW620 group appeared in G2/M and G1 phase cycle arrest which were given 4GY X-ray irradiation treatment(P<0.05).Compared with the previous radiation treatment of each groups,the same group of cells appeared in G2/M phase cycle arrest after radiotherapy(P<0.05).Each group of SW480 cell sublines in cell cycle distribution was statistically significant differeces(F=176.010,P=0.000;F=233.817,P=0.000;F=1403,P=0.000).Compared with the blank group and the control group,SW480-miR101 inhibitors group arrested in S phase of cell cycle(P<0.05).After 4GY X-ray irradiation treatment,SW480-miR101 inhibitors group displayed arrested in S phase of cell cycle compared with the blank group and the control group(P<0.05).Compared with no radiation treatment,the same group cells occurred the G2/M and S phase of the cell cycle arrest with radiotherapy(P<0.05).(7)Apoptosis experimentsThe results which used flow cytometry to detect apoptotic cells show that apoptosis rate of cells of SW620 cell sublines in each group was statistically significant difference(F=6115,P=0.000).Compared with SW620 group and GV209-SW620 group,miR-101-SW620 group increased the rate of cell apoptosis.Compared with no radiotherapy treatment of each group,the same groups increased apoptosis rate after radiotherapy.With 4GY X-ray irradiation treatment,compared with SW620 blank group and GV209-SW620 group,miR101-SW620 group significantly increased the apoptosis rate.The apoptosis rate of cells in each group of SW480 cell sublines was statistically significant difference(F=5419,P=0.000).Contrasted with SW480 blank group and SW480-NC,SW480-miR101 inhibitors group reduced the rate of apoptosis.Compared with the previous radiation treatment of each group of SW480,the apoptosis rate of the same group increased with 4GY X-ray irradiation treatment.Contrasted with SW480 blank group,SW480-NC,the apoptosis rate of SW480-miR101 inhibitors increased the most obviously.(8).Western blotIn each groups of SW620,the protein level of Y-H2AX and Caspase-3 up-regulated with the radiation dose increased,moreover the the protein level ofγ-H2AX and Caspase-3 in miR101-SW620 increased more obviously than these in the GV209-SW620.3.Validation the target gene of miR-101 and the molecular mechanisms of radiosensitivity in human colorectal cancer cells(1).The three commonly used online bioinformatics websites which were used to forecast miR-101 target sites were TargetScan,miRBase and Pictar.A lot of genes are get from the intersection of the three websites,such as EZH2,FOS,Cox-2,Mcl-1,RHOA,RAC1,BMI-1 and so on.Through reading literature,RAC1 gene was identified as a target gend of miR-101 finally.(2).Recombined vector psiCHECK-2-RAC1 containing RAC 1 3’UTR was constructed,and following by established the mutation expression vector psiCHECK-2-RAC1-Mut.In HEK293A cells,the results of dual luciferase report experimental show that miR101-inhibitors groups can increase about 30.50%on the relative luciferase activity of Rac1 3’UTR(t = 9.725,P = 0.001),while the miR101-NC group showed no changed on the relative luciferase activity of Racl 3’UTR;and the relative luciferase activity have no differences between miR101-inhibitors group and miR101-NC group for Racl 3’UTR Mut(t =-0.6580,P=0.3240).In SW480 cells cells,miR101-inhibitors can increase about 29.34%on the relative luciferase activity of Racl 3’UTR(t = 4.9410,P = 0.008),while the miR101-NC group showed no changed on relative luciferase activity of Racl 3’UTR,and the relative luciferase activity between the miR101-inhibitors group and miR101-NC to Rac1 3’UTR Mut were no differences(t =-0.8500,P = 0.5460).(3)Western blot results showed that miR101-SW620 decreased the protein level of RAC1,contrasted with SW620 and GV209-SW620.Compared with SW620 and GV209-SW620,miR101-SW620 down regulated the protein level of Cdc42 and RhoA.Compared with SW480,SW480-NC,SW480-miR101 inhibitors upregulated the target protein genes of RAC1.Compared with SW480 and SW480-NC,SW480-miR101 inhibitors group upregulated Cdc42 and RHOA protein.In each groups of SW620,the protein level of RAC1,Ccd42 and RhoA down-regulated with the radiation dose increased,moreover the the protein level of RAC1,Ccd42 and RhoA in miR101-SW620 decreased more obviously than these in the GV209-SW620.Conclusions1.We initially confirmed that RAC1 is the direct target gene of miR-101 in colorectal cancer.2.Overexpression of miR-101 can inhibit cell proliferation,colony formation,promote apoptosis and G2/M and G1 of cell cycle arrest in colorectal cancer of SW620.And interference miR-101 can promote cell proliferation,inhibition of apoptosis and arrested S phase of cell cycle in colorectal cancer cell of SW480.These means miR101 can inhibit cell proliferation,colony formation,promote apoptosis and arrested G2/M and G1 phase of cell cycle in colorectal cancer.3.Over-expression of miR-101 can enhance colorectal cancer to radio sensitivity.And the specific mechanism of which may directly down regulate its target genes RAC I,down regulate the RhoA and other genes,and regulate the RAC1/Cdc42/RhoA pathway,to arrest cell cycle arrest at G2/M phase and promote cell apoptosis. |
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