MicroRNA-200b inhibits pituitary tumor cell proliferation and invasion by targeting PKCα
Authors: Jie Duan
Renzhao Kuang
Junwei Duan
Xiaohong Yin
Yuanchuan Wang
Shun Li
Long Zhao
Journal: Experimental and Therapeutic Medicine (2017)
Discipline: CLINICAL MEDICINE
Keywords: AtT-20 cells; apoptosis; cell proliferation; invasion; invasive pituitary
Affiliations: Department of Neurosurgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P.R. China.

Abstract:  The present study aimed to investigate the expression of miR-200b and protein kinase Cα (PKCα) in pituitary tumors and to determine whether miR-200b may inhibit proliferation and invasion of pituitary tumor cells. The regulation of PKCα expression was targeted in order to find novel targets for the treatment of pituitary tumors. In total, 53 pituitary tumor tissue samples were collected; these included 28 cases of invasive pituitary tumors and 25 cases of non-invasive tumors, in addition to 5 normal pituitaries. The expression level of miR-200b in the pituitary tumor tissue was detected by quantitative polymerase chain reaction (qPCR) and the expression of PKCα protein was detected by immunohistochemistry. A PKCα 3'untranslated region (UTR) luciferase vector was constructed and a dual luciferase reporter gene assay was employed in order to examine the effect of miR-200b on the PKCα 3'UTR luciferase activity. AtT-20 cells were transfected with miR-200b mimics, PKCα siRNA and miR-200b mimics + PKCα, and the changes in cellular proliferation, invasion and apoptosis were observed via MTT, Transwell assay and flow cytometric analysis. Furthermore, PKCα mRNA expression was determined by qPCR, and Western blotting was performed to detect the expression of PKCα protein. miR-200b revealed downregulation in invasive pituitary tumor tissue, and the expression level was significantly down-regulated compared with normal and non-invasive pituitary tumor tissue (P&lt;0.01). In addition, the positive rate of PKCα protein expression in invasive pituitary tumor tissues was significantly higher than in normal and non-invasive tissues (P&lt;0.01). PKCα protein levels are inversely correlated with miR-200b levels in invasive pituitary tumor tissues (r=-0.436, P=0.021). The dual luciferase reporter gene assay revealed that miR-200b could specifically bind to the 3'UTR of PKCα and significantly inhibit the luciferase activity by 39% (P&lt;0.01). Upregulation of miR-200b or downregulation of PKCα could suppress cell proliferation and invasion, and increase apoptosis of AtT-20 cells. It was revealed that PKCα siRNA could suppress both proliferation and invasion of AtT-20 cells and partially simulate the function of miR-200b. Expression of PKCα mRNA and protein decreased significantly in AtT-20 cells overexpressing miR-200b. Additionally, miR-200b was significantly down-regulated in invasive pituitary tumor tissue and inversely correlated with PKCα protein levels. In conclusion, miR-200b inhibited proliferation and invasiveness and promoted the apoptosis of pituitary tumor cells by targeting PKCα. The observations of the present study indicate that miR-200b and PKCα may serve as promising therapeutic targets for invasive pituitary tumors. 

Retracted: No

PubPeer comments:
  Figure 4. Cell apoptosis following 48 h transfection. The transfection of miR-200b mimics or PKCu03b1 siRNA led to the increase of cell death, whereas the simultaneous transfection of miR-200b mimics and PKCu03b1 led to significant alteration of cell apoptosis compared with the control . Differences between the NC and Control panels are minor and very structured. Differences between the Mimics, siRNA and Mix panel are also minor and confined to the right-hand edges. ![file] Reviewers and editors at ETM are evidently under the impression that this is what flow cytometry **should** look like. , 
 Fig 3B.  Proliferation rate and invasiveness of AtTu201120 cells transfected in different groups. ... (B) Transfection of miR-200b mimics or PKCu03b1 siRNA led to the decrease of cell invasion, whereas the simultaneous transfection of miR-200b mimics and PKCu03b1u2011Wt led to significant alteration of cell invasiveness compared with the control . ![file] The same panels have appeared previously and subsequently (variously rotated and relabeled) in at least four other papers, including Fig 4A B of [Bai et al (2016)] : ![] Fig 13 from [Lu et al (2018)] . ![] , 
 #2 Another appearance of 3B! ![file] Details match panels from Fig 4A of [Chen et al (2016)] . ![] ,