| BackgroundProstate cancer(PCa)is a common malignant tumor in male.In China,the mortality and prevalence of prostate cancer have steadily increased over the past ten years.It is a hot topic to find more sensitive tumor markers and molecules,thereby improving the detection rate of prostate cancer and improving the prognosis of tumors.Therefore,studying the molecular mechanism underneath the prostate cancer and finding the target for diagnosis and treatment of prostate cancer in molecular level is currently the focus of research in prostate cancer.Recently,many studies have found that microRNA is involved in the formation,development and metastasis of prostate cancer.microRNA plays a key role in many biological processes,such as cell proliferation,growth and apoptosis.It is found that in many diseases,microRNA can even be used as a potential biological target for diagnosing diseases,contributing to the diagnosis and treatment of diseases.Although microRNA is expected to become an important index for early diagnosis,pathological grading and prognosis of malignant tumor,there are still many microRNAs waiting to be uncovered.The specific function of most microRNAs is not yet clear.Other studies have shown that the abnormal regulation of microRNAs affects the metastasis of tumor.Its mechanism may be related to many links.The future microRNA has bright prospects in the clinical diagnosis and treatment of tumors.Many studies have confirmed that there are many abnormalities in the expression of microRNA in prostate cancer.Some scholars have found that the expression of microRNA-125b and let-7 is down-regulated in prostate cancer,and its expression profile is related to the grade of prostate cancer.microRNA-17-3p can inhibit the development of prostate cancer,its expression is significantly reduced in the more aggressive prostate cancer.In addition,studies have shown that the down-regulation of microRNA-221 expression in metastatic prostate cancer seems to be related to the invasive ability of prostate cancer,which may be used as a marker for the invasive ability of prostate cancer in the future.These suggest that there is a close relationship between microRNA and the pathogenesis of prostate cancer.microRNA-212,a micro RNA,is a hot spot in recent research of cancer.microRNA-212 belongs to a family,which is located on the human 17p13.3 chromosome.A number of studies have found that microRNA-212 is involved in the regulation of a variety of tumors.In gastric cancer,head and neck cancer,non-small cell lung cancer,nasopharyngeal cancer,and hepatocellular carcinoma,there was abnormal expression of microRNA-212.A number of studies have found that microRNA-212 may be involved in the regulation of tumors.To date,the mechanism of action of microRNA-212 in the development of prostate cancer remains unclear.ObjectiveThis study examined the expression of microRNA-212 in prostate cancer tissues and cells,and the relationship between microRNA-212 and the biological behavior of prostate cancer cells,trying to find evidence of microRNA-212 in prostate cancer regulation,and further explore the mechanism of prostate cancer occurrence and development.Methods1.Prostate cancer tissues and normal prostate tissues were obtained from patients undergoing radical prostatectomy.Human prostate cancer cell lines(DU 145,PC3,22RV1 and LNCaP)and normal prostate epithelial cell(RWPE-1)were incubated under the standard conditions recommended by the providers.In order to study the role of microRNA-212 in the occurrence and progression of prostate cancer,we first used qRT-PCR to detect the expression of microRNA-212 in prostate cancer tissues.2.Further,we detected the expression of microRNA-212 in four kinds of prostate cancer cell lines(DU145,PC3,22RV1 and LNCaP)and normal prostate epithelial cell(RWPE-1)using qRT-PCR method.3.In order to further study the biological function of microRNA-212 in prostate cancer,we transfected microRNA-212 mimics plasmid or NC negative control plasmid in two kinds of cells with relatively low expression of microRNA-212 and DU145,and PC3.After 48 hours of transfection of the corresponding plasmid,we used the qRT-PCR method to detect the expression of microRNA-212.The transfected cells were further tested by MTT kit,Transwell and flow cytometry to detect cell function changes.4.Using TargetScan and microRNAanda to analyze the possible target gene of microRNA-212 by bioinformatics.The target gene of microRNA-212 is verified by rescue experiment.5.We established an allograft tumor model to detect the effect of microRNA-212 on the proliferation of prostate cancer in vivo.Results1.Compared with the normal adjacent tissues,the expression of microRNA-212 in the tumor tissues of the prostate cancer was significantly decreased.The expression of microRNA-212 in four kinds of prostate cancer cell lines(DU145,PC3,22RV1 and LNCaP)and normal prostate epithelial cell(RWPE-1)were detected in vitro.The results showed that the expression of microRNA-212 in prostate cancer cells was significantly down regulated.2.We transfected microRNA-212 mimics plasmids or NC negative control plasmids in two kinds of cells-DU145 and PC3 cells,which expressed relatively low microRNA-212.MTT kit and Transwell test showed that the DU145 and PC3 cells transfected with microRNA-212 mimics had lower proliferation rate.Meanwhile,the up regulation of microRNA-212 inhibited the migration and invasion of DU145 and PC3 cells.The results of flow cytometry showed that the apoptosis of DU145 and PC3 cells transfected with microRNA-212 mimics was significantly up-regulated.However,DU 145 and PC3 cells transfected with microRNA-212 inhibitor showed higher proliferation,migration and invasion ability,and lower apoptosis rate.3.TargetScan and microRNAanda bioinformatics analysis showed that MAPK1 was a possible target for microRNA-212.Luciferase reporter gene test showed that microRNA-212 significantly reduced the luciferase activity of pmicroRNAGLO-MAPK1-3’-UTR Wt.The results showed that microRNA-212 could directly target the 3’-UTR binding site of MAPK1.Then we transfected microRNA-212 mimics and microRNA-NC into prostate cancer cell DU 145 and PC3 cells.The over expressed microRNA-212 inhibited MAPK1 mRNA(P<0.05)and protein(P<0.05)expression.In prostate cancer,MAPK1 is a direct target gene for microRNA-212.4.The mRNA expression of MAPK1 in the prostate cancer tissues was significantly up-regulated.The Spearman correlation analysis showed that the expression of MAPK1 mRNA was negatively correlated with the expression of microRNA-212.Further Western blot results showed that the protein expression of MAPK1 in prostate cancer was significantly higher than that of its corresponding normal adjacent tissue.microRNA-212 mimics was transfected into prostate cancer cell DU145 and PC3 cells,and empty vector(pcDNA3.1)or MAPK1 over expression vector(pcDNA3.1-MAPK1)were added.Western blot experimental analysis showed that in DU 145 and PC3 cells,the downregulation of MAPK1 induced by microRNA-212 overexpression could be recovered by simultaneous transfection of pcDNA3.1-MAPK1.MTT,Transwell and flow cytometry showed that the expression of MAPK1 significantly reversed the effect of microRNA-212 on the proliferation,invasion and apoptosis of prostate cancer cells.5.The in vivo tumorgenesis experiment showed that compared with NC group,the tumor were significantly reduced in nude mice inoculated with cells transfected by microRNA-mimics.Ki67 expression was localized in the nucleus and reflected the proliferation of tumor cells.Immunohistochemical staining showed that the Ki67 expression level of nude mice transfected with microRNA-212 mimics was significantly lower than that of microRNA-NC group.Western blot experiments showed that transfection of microRNA-212 mimics.Groups of nude mouse tumors expressed lower levels of MAPK1(P<0.05).Conclusion1.The expression of microRNA-212 in prostate cancer tissue is significantly decreased.microRNA-212 can inhibit the biological behavior of prostate cancer,that is,inhibit the proliferation,invasion and apoptosis of prostate cancer cells.2.In prostate cancer,MAPK1 is a direct target gene for microRNA-212.microRNA-212,through its downstream target gene MAPK1,inhibits the proliferation,invasion and apoptosis of prostate cancer cells.We believe that the microRNA-212/MAPK1 signaling pathway may serve as a new theoretical basis for the treatment of prostate cancer and to serve the clinic in the future. |
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