| BackgroundGallbladder Carcinoma(GBC)is a common malignant tumor of the biliary system.However,it's often discovered at clinical middle-late stage with relatively poor prognosis,predominantly due to having no specific clinical manifestations and metastasis easily in initial stage.GBC tends to transfer early to liver,adjacent viscera and happening distant metastasis,causing GBC patients lose the chance of radical surgery after diagnosis that causing to the prognosis of postoperative 5-year survival rate less than 5%.It's very important for GBC patients prognosis to improve the early diagnosis level and receive early treatment.Therefore,through research at the molecular level,and discusses the gallbladder biological behavior,seeking biomarkers to improve the early diagnosis,genetic and molecular target therapy disease and prognosis is of great significance in such aspects as evaluation.So,combining existing basic research with clinical application,looking for the biomarker which can be used for early diagnosis and molecular targeting treatment is currently one of the key research directions.miRNA is a kind of endogenous non-coding RNA which contains about 22 nucleotides,and mainly through matches to targeted m RNA 3 'untranslated region(3'UTR)incomplete complementary pairing to suppress the translation of m RNA,mediate gene regulation after transcription.PTEN is discovered first phosphatase activity of tumor suppressor genes.PTEN gene encoding protein with lipid phosphatase and protein phosphatase dual specificity phosphatase activity,is closely related to many kinds of cancer.This team found in GBC mi RNA-21 expression level is closely related to the expression of PTEN,and further confirmed that the target gene PTEN in gallbladder for mi RNA-21,mi RNA-21 may inhibit PTEN genes affect the development of gallbladder carcinoma;In our research,we found the mi RNA-21 expression level is closely related to cellular tight junction protein expression in GBC,hint mi RNA-21 may participate in the invasion and migration in GBC,which offers the clue for further studying the biological function mechanism of mi RNA-21 in GBC laid the theoretical foundation.Methods1.Constructed knockdown mi RNA-21 stable line in gallbladder cancer GBC-SD cell.1)Detect cellular tight junction protein Occludin,ZO 1 protein expression respectively in m RNA and protein level in mi RNA-21 knockdown stable line.2)Overexpress/Inhibits mi RNA-21 in GBC-SD cell line,detect the PTEN level respectively in protein and m RNA level.2.Transwell analysis to check the ability of invasion and migration in GBC knockdown mi RNA-21 cell.3.Double luciferase experimental detected the mechanism of PTEN interacting with mi RNA-21.4.Collect GBC clinical specimens,using in situ hybridization and Immunohistochem--irstry detected 67 cases paraffin embedded specimens of primary gallbladder carcinoma the expression of mi RNA-21,PTEN protein.5.We used Pearson Chi-Square test to examined the parameters relationships between PTEN,mi RNA-21 expression and clinicopathologic.The Kaplan-Meier method was used to make mi RNA-21 overall and disease-free survival curves and examined the correlations between PTEN expression.In order to evaluate survival data,we used the Univariate and multivariate Cox regression analyses.Results:1.Constructed mi RNA-21 knockdown stable line in GBC-SD cell.:1)Compared with control group,the level of PTEN up-regulated expression while the expression of occluding,ZO-1were higher at protein level.2)The expression of occludin,ZO – 1 was risen at m RNA level.2.Confirm mi RNA-21 is negatively related to the expression of PTEN in GBC.1)Overexpress mi RNA-21 could inhibit the expression of PTEN.2)The suppression of mi RNA-21 in GBC cell can cause the expression of PTEN was upregulation not only at the m RNA level but also the protein level.3)Knockdown mi RNA-21 in Gallbladder cells reduced the ability of invasion and migration in GBC cell.4)3 'UTR is the target sequence that mi RNA-21 mutually combined with PTEN.5)The analysis of postoperative survival of patients with gallbladder carcinoma show that the expression of mi RNA-21 was inversely correlated with survival and prognosis.Conclusion:1.The expression of mi RNA-21 is closely related to the level of tight junction protein and PTEN in the Gallbladder carcinoma.Knockdown mi RNA-21 in Gallbladder cells reduced the ability of invasion and migration in GBC cell.2.The 3'UTR is the target sequence that mi RNA-21 mutually combined with PTEN,mi RNA-21 can be involved ingthe development of GBC by inhibiting the function of PTEN.The high expression of mi RNA-21 in gallbladder carcinoma indicates that the survival time is short,and it is an independent prognostic factor.mi RNA-21 perhaps can be a target gene for the treatment of gallbladder carcinoma. |
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