| Part1 The Expression and Mechanism Study of CHN1 in Cervical CancerBackground:Cervical cancer is the secondary gynecological malignant tumor worldwide,and it is also the eighth lethal cancer in China.Metastasis and invasion are key points to lead the mortality of cervical cancer,and EMT is now a universal explanation for the mechanisms of tumor metastasis.EMT and its regulatory factors are involved in tumor growth,apoptosis resistance,metastasis,and so on.A-chimeric protein(a-chimaerin,CHN1)is a ras-related protein,which plays an important role in the regulation of signal transduction and development.This study aims to detect the mechanism of CHN1 in the development and progression of cervical cancer,and reveals the relationship between CHN1,EMT,and related signaling pathways.Objectives:To investigate the connection between CHN1 expression and the clinic pathology of cervical cancer,and to clarify the effect of differential expression of CHN1 on tumor biological behavior.Meanwhile,to explore the CHN1-involved potential molecular mechanism during cervical cancer development.Methods:Firstly,the expression of CHN1 in cervical cancer and its adjacent tissues was detected by immunohistochemistry,and the pathological types and related parameters were analyzed to make sure the clinical significance of CHN1 as a biomarker.Then,the abilities of cell proliferation,invasion and migration were detected after constructed the CHN1-knockdown and overexpression cell lines.Thirdly,the expression of EMT related specific markers were detected in the transfected cell lines.After that,the effect of differential expressed CHN1 on tumorigenicity was tested through nude mice,and the expression of CHN1-related protein was confirmed by immunohistochemistry.Finally,the signal transduction pathway,which CHN1 might involved in,was analyzed by Western Blotting and immunofluorescence.Results:Immunohistochemistry showed that CHN1 was expressed in the cytoplasm of cervical cancer tissues.Compared with the paracancerous tissues,the positive rate of CHN1 in cancer tissues was 85.5%(P<0.01),and the positive rate was 43.4%(P<0.05)in the paracancerous tissues,with the statistically significant differences,respectively.The positively expressed CHN1 was not related to the age,tumor pathology and differentiation of patients with cervical squamous cell carcinoma(P>0.05).Survival curve single factor analysis showed that patients with CHN1 strong positive/positive expression had a lower survival time than CHN1 negative-expressed patients,with a statistically significant difference(P<0.05).In vitro CCK-8 experiment showed that the cell proliferation rate was significantly accelerated when CHN1 was overexpressed,and the proliferation ability of CHN1-knockdown cells was significantly decreased(P<0.01).The results of colony-forming assay told the same story that the colony forming ability of cervical cancer cells was significantly decreased when the expression of CHN1 was significantly increased,with the statistical significance(P<0.01).The results of invasion and migration showed that the invasion and migration ability of cervical cancer cells were significantly inhibited when the expression of CHN1 was disturbed,and the difference was statistically significant(P<0.01).In nude mice tumorigenesis,nude mice transplanted tumor was successfully constructed by using CHN1 overexpressing cell line and CHN1 interfering cell line.The tumor cells formed by the high expression of CHN1 were larger,and the expression of CHN1 was higher in the tumor compared to the control group by immunohistochemistry.Western Blotting results showed that high expression of CHN1 could induce the expression of EMT and epithelial markers in cervical cancer cells.The expression of interstitial markers was increased and the expression of EMT-related transcription factors was induced.In the cervical cancer cell line,high expression of CHN1 could activate Akt/GSK-3?signaling pathway,and the activation of this pathway is inhibited with the presence of the signal pathway specific inhibitor LY294002.Conclusion:The high expression of CHN1 in cervical cancer is related to the long-term prognosis of cervical cancer.At the cellular level,CHN1 can promote the occurrence of tumor biological behavior.CHN1 can stimulate the activation of EMT through Akt/GSK-3? and promotes the metastasis of cervical cancer by means of EMT.Part 2 The Expression and Mechanism Study of miR-205/CHN1 in Preeclampsia PlacentaBackground:Preeclampsia(PE)is an idiopathic hypertensive syndrome post 20-week pregnant.It is the leading cause of maternal bleeding,premature birth,abortion and fetal,and maternal mortality.MicroRNAs(miRNAs)are a class of endogenous,non-coding proteins,small RNAs.MiRNAs are involved in the regulation of cell proliferation,differentiation and apoptosis by participating in post-transcriptional regulation of gene expression.As a newly found miRNA,MicroRNA-205(miR-205)was proved highly expressed in the placenta and plasma of preeclampsia patients.The specific mechanisms between miR-205 and PE remain unknown.In this study,we focused on the expression detection of miR-205 and its target gene,and their direct connection in the clinical PE placenta samples and cell lines.and then to explore the effect of miR-205 on the biological function of trophoblast cells.Objective:To find out the potential mechanism of the miR-205's regulation role in preeclampsia samples and trophoblast cells,and then to detect the direct target role between miR-205 and CHN1.To explore the effect of miR-205-CHN1 on trophoblast cell functions,and to further reveal their specific mechanism during the development of pre-eclampsia.Methods:The expression of miR-205 and CHN1 in pre-eclampsia samples were detected by real-time PCR and immunohistochemistry.The potential binding sites between miR-205 and CHN1 was predicted through bioinformatics.The direct targeting effect of miR-205 on CHN1-3'UTR region was detected by double luciferase reporter system.The further confirmation of their binding sites was validated through real-time PCR and Western blotting.The effects of miR-205 and miR-205-CHN1 on villous trophoblast cells were studied by CCK-8 and transwell assays.Results:The results of real-time PCR showed that the expression of miR-205 in preeclampsia samples was significantly higher than that in normal controls;in contrast,the relative expression of CHN1 was significantly lower in patients with preeclampsia than in the normal control group,both with statistically significant differences(P<0.05);the results of immunohistochemistry proved that the expression of CHN1 in preeclampsia placenta paraffin sections was also lower than that in the normal maternal samples.According to the bioinformatics prediction,the region of CHN1-3'UTR has two conservative binding sites,which could bind with miR-205.After successfully constructed the wild-type and mutant vectors,the results of dual luciferase reporter system assay showed that there was only one binding site,which could connect miR-205 and CHN1.The results of real-time PCR showed that miR-205 was negatively regulated by the expression of CHN1;Western Blotting detection exhibited that the high expressed miR-205 correlated with reduced CHN1 expression.The in vitro functional detection showed that the increased expression of miR-205 was responsible for the attenuated ability of cell proliferation.The data of invasion and migration assay showed that high expressed miR-205 could reduce the invasion and migration and this phenomenon could be rescued by the addition of CHN1.Conclusion:miR-205 is highly expressed in the placental tissue of patients with preeclampsia.miR-205 can bind to the region of CHN1-3'UTR,to directly regulate the expression of CHN1 on both the mRNA and protein level.miR-205 could regulate CHN1 to influence the abilities of invasion and migration for trophoblast cells. |
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