| BackgroundCervical carcinoma is one of the most common malignant diseases among women.In recent years,with the popularity of cervical cancer screening and precancerous treatment,the incidence of cervical cancer has been decreased.But,in some countries and regions,the situation is still serious.As one of the women highly malignant tumors,CC has a very high mortality rate and also is a great threat to the health of women.Current studies suggest that the pathogenic factor of cervical cancer is the infection of High Risk Human papilloma virus(HR-HPV),and the'continuous,highly dangerous infection of it is the main cause led to canceration of cervical cells.However,some scholars believe that HR-HPV infection is not the only factor in the pathogenesis of cervical cancer but the host factors of the virus restrict occurrence and development of the disease.Therefore,in order to prevent and control cervical cancer,the role and mechanism of host factors in cervical cancer occurrence and development need to be studied.MicroRNAs(MiRNAS)can cause the degradation or translation inhibition of target gene mRNA.As an endogenous RNA with regulating function,MicroRNAS are non-coding,small-molecule,and single-stranded,whose active mechanism is to combine with non-translated region of mRNA3' so as to regulate gene expression at the post-transcription level.Many studies have proved that in the development process of tumors,miRNAs shows abnormal expression,and its biological function and mechanism in different tissues and cells are different,the characteristic of its biological effect is the differences of the tissues and cells.In addition,a number of different miRNAs can also apply to one target gene,and the same miRNA can affect many different target genes whcih cause the different biological function of it.Therefore,the miRNA and its target molecules can form huge,complex biological control network.To be sure,miRNAs play an important role in the development of cancer.In cervical cancer,it has been found many miRNA expression falling or rising,its participation in the occurrence,invasion and metastasis of cervical cancer,its regulation of the biological behavior of cervical cells can be done through different target genes.Therefore,the research of microRNAs in cervical cancer development is very significant for the prevention and control of cervical cancer.In previous studies,through cervical cancer samples,the correlation between Smad4 expression and the phase,metastasis and prognosis of tumor has been preliminarily proved,which showed that Smad4 is an important "potential" target in the study of the malignantly biological behavior of cervical cancer.But the cause of Smad4's influence on molecular mechanism of cervical cancer is unclear.In this study,the bioinformatics database will be adapted to predict the intersection of Samd4 and its target miRNAs is miR-205,and the correlation between the expression level of miR-205 and the clinicopathological factors of cervical cancer will be further examined.To apply relative experiments about genetic functions,the condition and effect of miR-205's abnormal expression in the abnormal biological behavior of CC cells have been studied to confirm the useful target genes and the useful biological mechanism.Part 1:The analysis about the relationship between miR-205 expression and clinicopathological factors in cervical cancerObjectiveTo determine the target gene of Smad4,and explore the relevance between miR-205 expression and clinicopathological factors in cervical cancer.MethodsSmad4 target genes were predicted by bioinformatics software.150 cases of cervical squamous cervical cancer and ME-180,Hela,CaSki,SiHa,MS751,C-33A and normal cervical cells Ectl/E6E7 were collected for this study.PCR was used to detect the expression level of miR-205.The luciferase reporter assay was used to examine the association between miR-205 and Smad4 mRNA,and the clinical and pathological data were collected to analyze the correlation between the expression level of miR-205 and the clinicopathological factors of cervical cancer.Results1.Database prediction was showed that the intersection of regulation Smad4 related miRNAs is miR-205.2.The expressiones of miR-205 in the ME-180?Hela?CaSki?MS751?SiHa?C-33A cervical cancer cell lines were 5.55?7.66?10.25?5.32?6.44?5.70,significantly increased than normai cervicai cell line Ectl/E6E7(1.41).The highest expression of miR-205 was showed in SiHa cell line.3.The Smad4 mRNA in the ME-180?Hela?CaSki?MS751?SiHa?C-33A cervical cell lines were 1109.74?1538.06?265.88?2574.97?1481.03?1462.83,significantly decreased than normai cervicai cell line Ectl/E6E7(2810.72),and the the lowest expression level was showed in SiHa cell line.4.Luciferase reporter assay showed that luciferase activity was importantly lower in the miR-205 group than control groups.5.150 cases of cervical cancer tissues showed that the expression level of miR-205(1.366 ±0.054)was significantly higher than normal cervical epithelium(0.286±0.015).6.1n the 75 cases of mir-205 low expression groups,60 cases were positive for Smad4 protein expression,in the 75 cases of mir-205 high expression groups,9 cases were positive for Smad4 protein expression.There was a significant negative correlation between the expression of miR-205 and Smad4 protein.7.The high expression of miR-205 was significantly correlated with high FIGO stage,lymphatic metastasis,deep interstitial infiltration,vaginal wall involvement and positive vascular space.Conclusionwe use bioinformatics way to predict the possible interaction between miR-205 and Smad4:Luciferase reporter system confirmed the direct interaction between miR-205 and Smad4.The expression of miR-205 and Smad4 showed a negative correlation both in the cell lines and cervical cancer tissue.The analysis of clinical and pathological factors of cervical cancer showed the miR-205 may play an important role in cervical cancer cell proliferation,invasion and metastasis.Part 2:the effect of miR-205 on the biological behavior of cervical cancer cellsObjectiveTo investigate the effects of miR-205 on the proliferation,apoptosis and invasion of cervical cancer cells.MethodsOn the basis of the decreased expression of miR-205 in the SiHa to decrease the expression of Smad4 by using slow virus interference,and use MTT to check the multiplication capacity,FCM to check the mortality,and Trans well to check the alteration of invasive ability.Also,Westernblot was used to check the expression of TGF-??MMP?Bcl-2 proteins.Results1.After SiHa being transfected by miR-205 inhibitor for 72h,the multiplication capacity of cancer cells is decreasedand the inhabitation rate is approximately 36.1%.Compared with negative control and non-treatment groups,the difference has statistical significance(P<0.05)?2 After SiHa being transfected by miR-205 inhibitor for 72h,The mortality rate of.cancer cell is increased from3.18±0.51%to 8.48±0.62%.Compared with negative control and non-treatment groups,the difference has statistical significance(P<0.001)?3.After SiHa being transfected by miR-205 inhibitor for 72h,the metastased cancers cells are(158.31 ± 22.15).which is significantly lower than negative control group(301.24± 19.40)and non-treatment group(326.79±30.25),the difference has statistical significance(P<0.001)?ConclusionDown regulation of miR-205 expression can inhibit the proliferation and apoptosis of cervical cancer cells,and decrease the invasiveness of tumor cells.Part 3:The mechanism of the effect of Smad4 on the biological behavior of cervical cancer by miR-205ObjectiveTo reveal the molecular mechanism of miR-205 in the process of cervical carcinogenesis.MethodsOn the basis of reducing the expression of miR-205 in SiHa cell line,the expression of Smad4 was decreased by lentivirus interference.The proliferation of cervical cancer cell line Siha was detected by MTT.The apoptosis of cells was found by flow cytometry.The invasion of cells was detected by Transwell.The TGF-?,MMP and Bcl-2 protein expression was tested by westemblot.Results1.After the SiHa cell expression Smad4 was down regulated on the basis of inhibition of miR-205,the proliferation of cells was enhanced compared with miR-205 inhibition group.2.After the SiHa cell expression Smad4 was down regulated on the basis of inhibition of miR-205,the apoptosis rate was significantly lower than miR-205 inhibition group,the apoptosis cells were(7.93±0.23)and(2.92±0.24).3.After the SiHa cell expression Smad4 was down regulated on the basis of inhibition of miR-205,the cell invasion ability was significantly enhanced(315.31±14.17)compared with miR-205 inhibition group(164.03 ± 22.23).4.Compared with the control group after miR-205 inhibition,the expression of Smad4,TGF-?,Bcl-2 and MMP-2 expression were significantly decreased.After expression Smad4 was down regulated on the basis of inhibition of miR-205,the expression of TGF-?,Bcl-2 and MMP-2 was up regulated.ConclusionMiR-205 could regulate the expression of TGF-?,Bcl-2 and MMP-2 by regulating the biological function of Smad4,and could affect biological behavior of cervical cancer... |
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