The Preliminary Study About Ars2/miR-21Signaling Pathways Involved In Regulation Of Proliferation And Invasive Capability In Cholangiocarcinomas

BACKGROUND AND OBJECTSCholangiocarcinoma is the second most common in the hepatobilliary malignanciesand the incidence rate has been gradually rising in recent years. The main feature of thedisease is continual infiltrative growth at the primary site, and resulting biliary tractobstruction. Surgical resection is the only curative treatment. Unfortunately, the disease isoften discovered very late in patients and consequently the therapeutic results are poor. IfCCA could be diagnosed at an early stage, the therapeutic efficacy for the disease could begreatly enhanced. MicroRNA-21(miR-21) has been reported recently to be increased inCCA cell lines and specimens, and could be an early diagnostic marker with favorablesensitivity and specificity. However, the detection methods of microRNAs are not widelyused in the clinical diagnosis in many hospitals. Therefore, more studies are required on theregulation factors that increase miR-21in CCA to determine more suitable diagnosticmarkers.Ars2was first cloned by screening for cDNAs that could confer sodium arseniteresistance in a hamster cell line. However, the protein has remained poorly characterized.Several lines of evidence supported the role of Ars2in RNAi function in miR biogenesisand cell proliferation. In addition, it has recently been found that Ars2depletion candecrease levels of several miRNAs, including miR-21, let-7, and miR-155. Therefore, wehypothesized that Ars2also exists in CCA and affects miR-21and its down-stream pathwayproteins. As clear direct targets of miR-21, the phosphatase and tension homolog deleted onchromosome10(PTEN) and Programmed Cell Death4(PDCD4) are specificanti-oncogenes which have been wildly investigated in many kinds of malignant cancersincluding CCA and found low-expression to affect the biological behavior of cancer cellssuch as proliferation. So, in the current study, we sought to determine whether Ars2isover-expressed in CCA specimens and cell line, and whether it could affect the expression of PTEN and PDCD4by the miR-21pathway.METHODS AND MATERIALS1. Eighteen CCA specimens and paired normal tissues were obtained at surgeriesperformed at the Hepatobilliary Surgery Department of Southwest Hospital (11men and7women), ranging from51to67years of age (mean,56.78±5.91). Clinically, the patientswere9extrahepatic CCAs and9intrahepatic CCAs. These patients underwent aCCA-eradication therapy procedure. Paired normal tissues contained in the specimens wereconfirmed to be histologically free of tumors. QBC939cells were generated at SouthwestHospital from an extrahepatic CCA patient. Human Intrahepatic Biliary Epithelial Cell(HIBEC, purchased from ATCC).2. Ars2,PTEN and PDCD4immunolabeling. We used an immunohistochemical assayfor the detection of Ars2, PTEN and PDCD4protein levels according to the protocals.Appropriate positive and negative controls were included. Immunostaining was classifiedas follows:-, no immunostaining above the cutoff level;+, low expression (the percentageof positive cells between1%and33%);++, moderate expression (the percentage of positivecells between34%and66%);+++, strong expression (the percentage of positive cells over67%).3. Real-time PCR assays and Northern blots for mature MiR-21and normalized to U6expression. Quantitative qRT-PCR was taken for Ars2, PTEN and PDCD4mRNAexpression in the specimens and CCA cells.4. Transduction with the shArs2lentiviral vector. Lentiviral shRNA-expression vectorwas purchased from Sigma-Aldrich. TurboGFP shRNA was introduced into the cells as anegative control. Both total RNA and protein were harvested72h after transfection. Weobserved the changes of proliferation, invasion and downstream molecules afterknockdown Ars2in QBC939and REB cells by MTS cell proliferation assays, in vivo tumorformation assays, western blots, qRT-PCR and Immunofluorescence.5. Transfection of mir-21mimics and control. Hsa-mir-21RNA mimics was purchasedfrom Sigma. A non-human miRNA predicted to not hybridize with the human genome wasselected as the negative control. The QBC939and RBE cell lines that were transduced withthe shArs2-expressing vector were co-transfected with100nM of miR-21mimics and itscontrol. Then western blots and qRT-PCR were carried out to observe the changes of PTEN, PDCD4and pAKT.6. Transfection of Ars2plasmid and control that were constructed by semi-chemicalsynthesis and were prechased from GeneCopoeia Co. The QBC939and HIBEC cell lineswere transduced with10μg of the Ars2full-length cDNA plasmid or empty vector controlinto approximately5x105cells in20cm2cell culture flasks. After successfully transfection,we detected the related downstream molecules by western blots, qRT-PCR and northernblots. To certify weather the exogenous Ars2had its biological functions, we took theimmunoprecipitation to observe it could combined with Drosha or CBP-80or not.Results1. In immunohistochemical results, overexpression of Ars2was observed in18tumorsamples, but reduced expression or no expression was observed in the paired normal tissues.There was a significant difference by the chi-squared test (P<0.01). The low or loss ofeither PTEN or PDCD4was identified in the cancer specimens but was present in the pairednormal sections. A significant negative difference was identified between the protein levelof PTEN/PDCD4and Ars2by the chi-squared test (P<0.01). Quantitative RT-PCR assaysshowed all tumors displayed higher Ars2and miR-21levels than the normal specimens.However, the results of the PTEN and PDCD4qRT-PCR showed there was no difference intheir tumor expression levels compared to the normal specimens. A line regression resultshowed that the R-squared value comparing the expression of Ars2mRNA and miR-21was0.469(P<0.001, n=36) in CCA and paired normal tissues; however, there were nosignificant correlations between Ars2/PTEN/PDCD4and miR-21/PTEN/PDCD4. Ars2mRNA levels correctly diagnosed all of the18cancers and18paired normal specimens,yielding a sensitivity of95%and a specificity of100%. The area under the ROC curve(AUROC) was0.995.2. Ars2knockdown suppresses proliferation in QBC939and RBE cell lines. TheshArs2-2hairpin suppressed the proliferation of both the QBC939and RBE lines byapproximately75%and65%, respectively, compared with the shVec-infected control cells;notably, the shArs2-1hairpin caused a smaller decrease in Ars2protein levels and had amodest effect on suppressing cell proliferation. In the in vivo tumor formation assays, bothshArs2-2-expressing QBC939and RBE cells could not form tumors in nude mice whileshVec-expressing QBC939and RBE cells formed. 3. Ars2depletion by the shArs2-2hairpin reduced miR-21levels by73%and65%inQBC939and RBE, respectively. Additionally, the depletion of Ars2by the shArs2-1hairpinreduced miR-21levels in QBC939and RBE by42%and34%, respectively. Similar resultswere observed by northern blot. PTEN and PDCD4protein levels increase after knockdownof Ars2but are reduced after co-transfection with miR-21mimics. Furthermore, weinvestigated the expression level of pAkt, which is inhibited by PTEN, and found theopposite results of PTEN.4. Expression levels of PTEN, PDCD4, and miR-21do not change after transfectionwith the Ars2expression plasmid in QBC939and HIBEC cells. To further study whetherthe exogenous Ars2had activity in cells, we performed an immunoprecipitation with theHIBEC and found that cells overexpressing Ars2could pull down more Drosha, but notCPB80; however, CBP80could not pull down more Ars2and Drosha in HIBECoverexpressing Ars2. These results suggest that exogenous Ars2had intracellular activitybut could not play its role in miR biogenesis alone.ConclussionIn this study, we described for the first time that Ars2may have a relationship withmiR-21in both CCA primary specimens and cell lines. The knockdown of Ars2increasedthe expression of PTEN and PDCD4proteins via decreasing miR-21levels in QBC939andRBE cells; however, Ars2overexpression did not display the opposite effect. Although Ars2is a protein that has not been extensively studied, our work indicated that its depletioncontributes to inhibiting cell proliferation. We also described its role in regulating themiR-21/PTEN and PDCD4signaling pathways in CCA. This suggests that Ars2can be anearly diagnostic marker and access to treatment.