Knockdown of Circular RNA Hsa_circ_0000714 Can Regulate RAB17 by Sponging miR-370-3p to Reduce Paclitaxel Resistance of Ovarian Cancer Through CDK6/RB Pathway
Authors: Xiaoyun Zhao
Bin Zhang
Shuo Li
Ying Yuan
Min Guo
Yifu Guan
Journal: OncoTargets and Therapy (2020)
Discipline: CLINICAL MEDICINE
Keywords: RAB17; hsa_circ_0000714; miR-370-3p; ovarian cancer; paclitaxel resistance.
Affiliations: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110122, People's Republic of China., Department of Microbiology and Cell Biology, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, People's Republic of China.

Abstract:  Purpose: Paclitaxel resistance in ovarian cancer has become an urgent clinical problem. This study investigated the regulatory effects of RAB17 on the non-coding RNA network of the paclitaxel-resistant ovarian cancer cell A2780/PTX. Methods: Microarray analysis was used to identify differentially expressed genes in paclitaxel-resistant cell A2780/PTX compared to the parent paclitaxel-sensitive cell A2780. Quantitative real-time PCR and Western blot were used to measure the expression of related mRNAs and proteins. The CCK8 assay was used to determine cell survival ratios and drug resistance indices in ovarian cancer cells. The clone forming assay was used to analyze the cell clone proliferation. Flow cytometry was used to analyze the cell cycle. Dual-luciferase reporter gene assays evaluated the relationship between the genes. Results: RAB17 is highly expressed in A2780/PTX cells. RAB17 knockdown increased the cell sensitivity to paclitaxel, inhibited proliferation, and caused cell cycle arrest in the G1 phase in A2780/PTX. Western blot confirmed that RAB17 influenced cell behavior by activating the CDK6/RB signaling pathway. Bioinformatics analyses identified RAB17 as a new target by the microRNA miR-370-3p, and the latter was predicted to interact with circular RNA hsa_circ_0000714. Hsa_circ_0000714 indeed acted as a miRNA sponge for miR-370-3p allowing its regulation of RAB17 expression. This regulation was accomplished through the CDK6/RB signaling pathway. Conclusion: Hsa_circ_0000714 acts as a sponge for miR-370-3p, and regulates RAB17 expression through the CDK6/RB signaling pathway, which plays a role in the malignant progression of the paclitaxel-resistant ovarian cancer cell A2780/PTX. 

Retracted: No

PubPeer comments:
 Fig 11.  C: protein bands of each factor in ASMCs of normal mice in each group detected by western blot analysis ... E: protein bands of each factor in ASMCs of asthma mice in each group detected by western blot analysis Bands seem to have been reused. ![file] , 
 Fig 11E again, at right. At left, Fig. 6B from [Zhou et al (2018)] .  ELISA and western blot analysis were applied to determine the miR- 495 expression and mRNA and protein expression of NLRP3, TNF-u03b1, IL-1u03b2, IL-18 and caspase-1. The loading controls seem familiar, though labeled as beta-actin and GAPDH. ![file] Despite the stylistic similarities in the WBs, there are no authors in common. , 
 Fig. 10A and C.  Overexpression of miR-485 promotes apoptosis of ASMCs in mice. A, B:: the cell apoptosis of ASMCs of normal mice in each group; C, D: the cell apoptosis of ASMCs of asthmatic mice in each group . ![file] The two red-boxed panels are very similar in the lower left corners. Here they are on their own: ![file] Black-white reversing the miR-485-mimic panel and superimposing it on the siRNA-smurf2 panel (enlarging it as well) dramatises the similarity. White and black points are cells unique to the former or latter; cells present in both panels cancel out. ![file] , 
 #3 Fig. 10A again.  Overexpression of miR-485 promotes apoptosis of ASMCs in mice. A, B:: the cell apoptosis of ASMCs of normal mice in each group . ![file] Several panels are recognisable from Fig. 9C of [Wu et al (2018)] .  Cell apoptosis rate and cell cycle of detrusor cells in the control, diabetic, miR-27a mimics, mimics control, miR-27a inhibitors, inhibitors control, siPRKAA2 and inhibitors + siPRKAA2 groups using flow cytometry. ...C, cell apoptosis in each group . ![file] , 
 Fig 9A, C:  Overexpression of miR-485 arrests cells at G1 phase of ASMCs in mice. A, B: the cell cycle proportion of ASMCs of normal mice in each group; C, D: the cell cycle proportion of ASMCs of asthmatic mice in each group . ![file] From what is visible of the distributions behind the G1 S G2 populations fitted to them, the same distributions seem to appear in A and C (though fitted with slightly different populations). , 
 #3 Fig. 10A again.  Overexpression of miR-485 promotes apoptosis of ASMCs in mice. A, B:: the cell apoptosis of ASMCs of normal mice in each group . ![file] This time, note the similarity to Fig 9(a) of [Yu et al (2019)] . Resolution is not perfect, but good enough for the purpose. ![file] , 
 I am trying to understand the quantitative PCR in this paper and am hoping the authors can assist. Specifically, I do not understand how the primers listed in Table 1 could amplify some of the given targets. The sequence of mature miR-485-5p is AGAGGCUGGCCGUGAUGAAUUC. To amplify a mature miRNA, a special RT procedure must first be used, but there is no indication of this in methods. After RT, amplification is done with a forward primer (typically all or most of the target mature miRNA) and a reverse primer complementary to a sequence introduce during RT. However, Table 1 lists the forward and reverse primers: 1) F: CCAAGCTTCACCCATTCCTAACAGGAC 2) R: CGGGATCCGTAGGTCAGTTACATGCATC It does not seem that any part of the mature miR-485 sequence would be recognized and amplified by these primers, even if RT had been done successfully. I also checked the U6 primers, since U6 is used as the control for miR-485. 1) F: CTCGCTTCGGCAGCACACATATAC 2) R: ACGCTTCACGAATTTGCGTGTC The reverse primer should work well for mouse U6. The forward primer is unlikely to work, since it includes numerous 3 nucleotides that do not match mouse U6. It is ok to have 5 nucleotides on a primer, for example to introduce tags or restriction sites, but 3 nucleotides will interfere with amplification. I have not looked at the other primer sets, but these findings suggest to me that the miRNA U6 results should be revisited. , 
 [Retracted] . ----------------- The article u201cMicroRNA-485 Modulates the TGF-u03b2 Smads Signaling Pathway in Chronic Asthmatic Mice by Targeting Smurf2u201d [Cell Physiol Biochem 2018;51(2):692-710, DOI: 10.1159 000495327] by Jing Wang, Hong-Yan Li, Hu-Shan Wang and Zhen-Bo Su has been retracted by the Editor. The retraction has been approved by the corresponding author, on behalf and with the approval of all authors, and the following explanation has been submitted: u201cWe studied airway epithelial cells other than studying airway smooth muscle cells, but we are sorry to say that we can not get the same trend of result as well as the smooth muscle of trachea. As the main cellular basis of asthma model is airway smooth Muscle cells and airway epithelial cells, but the two kinds of cells can not reach the same conclusion, we have no choice but to contact you to retract the published article.u201d ,