In this study, Xu and co-workers analyzed the role of circular RNA (circRNA) in cholangiocarcinoma (CCA). The authors specifically evaluated the expression of the circRNA circ-0001649 (a.k.a circSHPRH). A comprehensive analysis has been carried out showing a decreased expression of circ-0001649 in both CCA cell lines and tissues. Gain and loss of function approaches were performed in order to better understand the molecular mechanisms regulated by circ-0001649. Thus, the authors reported that the inhibition of circ-0001649 expression positively regulates CCA cells migration, invasion and proliferation and attenuates apoptosis of CCA cell lines. Interestingly, the tumor-suppressive action of circ-0001649 was also assessed in vivo on mouse xenograft models. Overall, this manuscript provides some interesting information. However, the whole study in the present form is simple and descriptive and needs further investigation to get a better insight into the regulatory functions of circ-0001649. I have some suggestions that if properly addressed may increase the significance and overall message of this otherwise commendable study.

1- In the introductory section, the rationale of studying specifically circ-0001649 in CCA should be better clarified.

2- In the method section, it should better clarified whether the term non-tumorous tissues designate normal liver cancer tissues or normal cholangiocytes. In this instance, normal cholangiocytes are the most relevant control.

3- In the method section, the quantification method for circRNA should be further detailed. Notably, the authors should precise whether they enriched their total RNA pool in circRNA by degrading linear RNA through RNAse treatment.

4- It is well established that multiple circRNA isoforms are produced from the same parental transcript. It is not clear whether or not the pair of divergent primers used for circ-0001649 can amplify additional isoforms. Authors should provide the method used to isolate this specific isoform and subsequently sequence it (Fig. 1).

5- CircRNA are well-known for their capability to sponge miRNA, acting as competitive endogenous RNA preventing them to bind their natural targets. Therefore, a full mechanistic part could have been added to identify the tumor suppressive miRNA sponged by circ-0001649. Additionally, the miRNA targets dysregulated by the overexpression or the inhibition of circ-0001649 could have been identified by WB analyses.

6- It would have been valuable to associate circ-0001649 expression with additional clinical features, such as overall survival and relapse free survival.