| Background:Gastric cancer is a serious threat to human health,however,its specific pathogenesis still remains unclear.To elucidate its mechanism may provide new ideasand stratage for clinical treatment of gastric cancer.It has been found that miR-130a-5p is closely related to the development of cancer,and its role in the development of cancer is closely associated with the types of cancer.At present,the role and mechanism of miR-13 0a-5p in the development of gastric cancer arestill not clear.In addition,CRMP4a and CRMP4b have been reported as potential downstream targets of miR-130a-5p,and CRMP4a and CRMP4b play opposite roles in regulating various life activities.However,there is no literature on the roles of CRMP4a and CRMP4b in the development of cancer,and it is not clear whether these two target genes have regulatory roles.RhoA plays a role in the development of gastric cancer,and is also a potential down stream target gene of CRMP4a.Based all the above,we speculate that miR-130a-5p may participate in the pathogenesis of gastric cancer by regulating the molecular axis of CRMP4a/CRMP4b/RhoA.Objective:To confirme whether miR-130a-5p might be involved in the specific molecular mechanism of gastric cancer through regulating the molecular axis of CRMP4a/CRMP4b/RhoA.Methods:(1)From June 2015 to June 2016,53 cases with gastric cancer were collected from Yan'an Hospital Afiliated to Kunming Medical University,including 33 male patients,20 female patients and 35 patients aged over 55 years old.Twenty-two patients were in TNM ?-? phase and 31 patients in TNM ?-? phase.RT-qPCR was used to detect the expression of microRNA-130a-5p in tumors and adjacent tissues of patients.Human normal gastric epidermal cell lines GES-1 and human gastric cancer cell lines BGC823,GC9811,HGC-27,MGC-803 and NCL-N87 were cultured in vitro,respectively.RT-qPCR was also used to detect the expression of miR-130a-5p in the above cells.Mimics and inhibitors of miR-130a-5p were designed be transfected to human gastric cancer cell line BGC823.The transfection efficiency was determined by use of RT-qPCR.Cell proliferation,apoptosis,and migration were detected respectively by use of MTT assay.Flow cytometry and transwell invasion test and Western Blot were used to detect the expression of apoptosis-related proteins(Caspase 3,Bcl-2,and Bax).(2)Cancer tissues(experimental group)and adjacent tissues(control group)were collected from the above patients.The expression levels of CRMP4a and CRMP4b were detected by RT-qPCR and Western Blot respectively.CRMP4a and CRMP4b lentiviral super expression vectors(pLVX-CRMP4a and pLVX-CRMP4b)and knockdown vectors(pLVX-sh-CRMP4a and pLVX-sh-CRMP4b)were designed to be transfected to human gastric cancer cell lines BGC823,and RT-qPCR was used to determine transfection efficiency.MTT assay,flow cytometry,and transwell invasion as well as clone formation assaywere used to detect cell proliferation,apoptosis,cycle andinvasion ability,respectively.(3)RT-qPCR and Western Blot were used to detect the expression of RhoA in gastric cancer tissues and cell lines at transcriptional and translation levels respectively.BGC823cellswerecultured in vitro and transfected with CRMP4a and CRMP4b lentiviral overexpression and knockdown vectors respectively.The expression of RhoA in gastric cancer cells was detected by RT-qPCR and Western Blot at transcription and translation levels respectively.The correlation between the expression of RhoA and the levels of miR-130a-5p,CRMP4a and CRMP4b was analyzed.MTT assayand transwell invasion assay were used to detect the proliferation and migration of cells.The nude mice model bearing tumorswas duplicated to further verify the effect of miR-130a-5p on the occurrence and development of gastric cancer in vivo.Results:(1)Compared with the adjacent tissues,the expression of miR-130a-5p in gastric cancer tissues was significantly increased(P<0.05).The expression level was correlated with TNM stage(P<0.0001),but not with age(P>0.05)and gender(P>0.05).In vitro cell experiments showed that the expression levels of miR-130a-5p in human gastric cancer cell lines BGC823,GC9811,HGC-27,MGC-803 and NCL-N87 were significantly higher than those in normal gastric epidermal cell lines GES-1(P<0.05).Further studies showed that miR-130a-5p could significantly promote the proliferation and migration of human gastric cancer cell line BGC823(P<0.05),and inhibit cell apoptosis(P<0.05).In addition,miR-130a-5p could significantly inhibit the expression of apoptotic-related Caspase 3 and Bax(P<0.05),and increase the expression level of Bcl-2 protein(P<0.05).(2)Compared with the adjacent tissues,the expression of CRMP4a in gastric cancer tissue was significantly decreased(P<0.05),while the expression of CRMP4b was significantly increased(P<0.05).There was a negative correlation between the expression of CRMP4a and CRMP4b(P<0.0001,r=-0.6726).In addition,CRMP4a and CRMP4b expression levels in cancer tissues were not significantly correlated with age(P=0.8543,P=0.5931)and gender(P=0.4515,P=0.4141),but CRMP4a expression level was negatively correlated with TNM stage(P<0.0001),and CRMP4b expression level was positively correlated with TNM stage(P<0.0001).Compared with GES-1 cells,CRMP4a was lower in gastric cancer cells(P<0.05),while CRMP4b washigher(P<0.05).Further experiments confirmed that overexpression of CRMP4a or knockdown of CRMP4b could significantly inhibit cell proliferation(P<0.05)and migration(P<0.05),and promote cell apoptosis(P<0.05).In addition,miR-130a-5p cound down-regulate the expression of CRMP4a,up-regulate the expression of CRMP4b,promote cell proliferation(P<0.05),migration(P<0.05),and inhibit cell apoptosis(P<0.05).(3)The expression level of RhoA in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.05),and the expression level of RhoA mRNA was positively correlated with TNM stage.In addition,RhoA was highly expressed in gastric cancer cell lines BGC823,C9811,HGC-27,MGC-803 and NCL-N87 compared withGES-1.In addition,RhoA inhibitor(Rhosin)could significantly inhibit the proliferation and migration of gastric cancer cells compared with control group(P<0.05).The results showed that CRMP4a could significantly inhibit the expression of RhoA in BGC823 cells(P<0.05),while CRMP4b had no significant effect on the expression of RhoA in BGC823 cells(P>0.05),and miR-130a-5p mimic and CRMP4a lentivirus super expression vector could significantly increase the expression of RhoA in BGC823 cells(P<0.05).Simultaneous transfection of CRMP4a lentivirus super expression vector could reverse the promotion effect of microRNA-130a-5p mimics on RhoA expression level.Conclusion:(1)miR-130a-5p plays an important role in the carcinogenesis and development of gastric cancer.(2)CRMP4a is an anti-oncogene and CRMP4b,playing opposite roles in the development of gastric cancer,is a pro-oncogene.miR-130a-5pcaninhibit the expression of CRMP4a,and CRMP4a can further inhibit the expression of CRMP4b and promote the development of gastric cancer.(3)In gastric cancer tissues and BGC823 cells,miR-130a-5p can prevent the inhibition of CRMP4a on RhoA expression and improve the expression of RhoA by inhibiting the expression of CRMP4a,resulting in promoting the proliferation and invasion of BGC823 cells,and inhibiting apoptosis. |
PDF Full Text Request