| Objective:(1)To investigate the relationship between the therapeutic effect of resveratrol(Res.)on treatment of colon colitis and colitis-associated cancer and miR-31-5p.(2)The hsa-miR-31-5p is homologous to muu-miR-31 and they have the same sequence.Using transgenic mice with the overexpression of miR-31 to develop DSS(Dextran Sulfate Sodium)-induced colitis model,observe the role of miR-31 in the development of ulcerative colitis,and subsequently further to clarify the specific mechanism of resveratrol.Methods:(1)Human colon cancer cell line,HCT 116 cells,were divided into 6 groups: control group,24 h resveratrol group,mi R-31-5p mimic group,miR-31-5p mimic plus 24 h administration group,miR-31-5p inhibitor group,mi R-31-5p inhibitor plus 24 h administration group.Using liposomes to transfect miR-31-5p mimic and miR-31-5p inhibitor into HCT 116 cells followed by incubation with 10 ?g/mL resveratrol.qRT-PCR was used to validate the transient transfection efficiency of miR-31-5p mimic and inhibitor.The role of miR-31-5p mimic is to cause overexpression of miR-31-5p and miR-31-5p inhibitor is mainly responsible for knockdown of miR-31-5p expression.(2)Clonogenic assay was used to examine the number and colony size of cells in each group after 14 days of culture to determine the effect of resveratrol on the proliferation of HCT 116 cells;wound healing assay was used to detect the the healing ability of cells in each group after the scratches at 24 hours,in order to determine the effect of resveratrol on cell migration;the proliferation and division of the control group and the 24 h resveratrol group cells were dynamically monitored for up to 16 hours using a new Cytation5 cell imaging microplate reader to further confirm the effect of resveratrol on cell proliferation in each group.(3)Flow cytometry was used to detect the apoptosis and cell cycle of HCT 116 cells in each group.The effect of resveratrol on the apoptosis and cell cycle of each group of cells was observed.(4)Using mi RNA target prediction sites such as Targetscan and mi RWalk and the results of sequencing to predict the potential mRNA targets of miR-31-5p.qRT-PCR was used to observe the change of expression of possible targets of miR-31-5p in control group,miR-31-5p mimic group,miR-31-5p mimic plus 24 h group and miR-31-5p inhibitor group.(5)Western Blot was used to detect the expression of NF-?B,ERK1/2,Bcl-2 and Bax protein in six groups.(6)Thirty mice were randomly divided into 4 groups: control group,control + DSS group,miR-31 over-expressed control group,mi R-31 over-expressed + DSS group.The hsa-miR-31-5p is homologous to muu-miR-31.C57BL/6 homologous transgene mice with the overexpression of miR-31,FVB mice,were induced colitis by 1% DSS,observing the role of miR-31 in colitis.The cycle of establishing model was 2 and a half cycle with 1% DSS,weighing every two days and daily observing inflammatory signs and mouse activity status.After the end of the experiment,the colon tissues were fixed in formalin and paraffin-embedded for H&E staining to observe the state of colitis.Results:(1)In our previous experiments,we used gene chip technology to screen differentially expressed miRNAs of tissues in patients with ulcerative colitis,and we found that the less studied miRNA,hsa-miR-31-5p,showed the most significant expressed difference.In addition,the results of H&E staining from animal colitis showed that the incidence of DSS induced colitis from miR-31 over-expressed mice was more severe than normal control mice.Infiltration of inflammatory cells was obvious under the microscope.Lesions could reach the mucosal layer,muscle layer,and even pulp.In the membrane layer,most of the crypts were destroyed,deformed,and disordered,and the glands were heterogeneously proliferated.In combination with the above results we focused on the study of miR-31-5p.(2)The results of qRT-PCR demonstrated that resveratrol can down-regulate the expression of miR-31-5p in HCT 116 cells in a time-dependent manner.After miR-31-5p mimic and miR-31-5p inhibitor were transfected into HCT 116 cells,the expression of miR-31-5p increased approximately 80-fold(P<0.01),and the latter decreased approximately 1-fold(P<0.01),demonstrating that transfection was effective.(3)The results of wound healing and clonogenic assay showed that resveratrol can reduce the number of colonies and inhibit the migration of HCT 116 cells.After transfection of miR-31-5p mimic and inhibitor,the area of cell migration of transfecting miR-31-5p mimic increased(P<0.01)and the number of colonies increased(P<0.01);compared with miR-31-5p mimic group,the migration area was decreased(P<0.01)and the number of colonies decreased after resveratrol treatment(P<0.01).When miR-31-5p inhibitor group was given resveratrol,the migration area(P<0.01)and colony number were decreased(P<0.01).(4)The results of flow cytometry indicated that resveratrol promoted apoptosis of HCT 116 cells(P<0.01)and arrested cell cycle at G2 phase.The apoptosis rate of miR-31-5p mimic group was not significantly different from that of control group,but apoptosis was increased after resveratrol treatment(P<0.01).The apoptosis rate of miR-31-5p inhibitor group was significantly higher than that of control group(P<0.05),and the increase of apoptosis after resveratrol was significant(P<0.01).The results of cell cycle showed that after overexpression of miR-31-5p,the proportion of HCT 116 cells in S phase increased,but after giving resveratrol,the proportion of S phase decreased;after knocking down miR-31-5p,the proportion of cell cycle had not have obvious change,but the proportion of cell cycle in S phase increased after administration of resveratrol.(5)The results of qRT-PCR revealed that when miR-31-5p was overexpressed,the expression of SATB2 was lower than control group(P<0.05),and the expression was increased after resveratrol treatment(P<0.05);when miR-31-5p was knocked down,the expression level was higher than that of mi R-31-5p mimic group(P<0.01).The knockdown of miR-31-5p expression increased the expression of PPP2R2 A compared with the control group(P<0.05).(6)Resveratrol inhibited the expression of NF-?B(P<0.05).Compared with the control group,the expression of NF-?B was increased when mi R-31-5p was overexpressed(P<0.05)and after the treatment of resveratrol,the expression of NF-?B was lower than that of miR-31-5p mimic group(P<0.05);when knocked down miR-31-5p and given resveratrol,the expression of NF-?B was lower than control group(P<0.01).In addition,resveratrol also down-regulated the protein expression ratio of Bcl-2 and Bax(P<0.05).When overexpressed miR-31-5p,the ratio of Bcl-2 and Bax was higher than that of control group(P<0.05)and the ratio was decreased(P<0.01)when resveratrol was given,however,the level could not reach to the level of resveratrol given alone;when knocked down miR-31-5p and given resveratrol,the Bcl-2 and Bax ratio decreased relative to the control group(P<0.05).There was no significant change in ERK1/2 protein expression in each group.Conclusion:Resveratrol exert its anti-proliferative and pro-apoptotic effects in HCT 116 cells through targeting miR-31-5p.Overexpression of miR-31 aggravate the development of ulcerative colitis. |
PDF Full Text Request