Proteomics Identifies LUC7L3 as a Prognostic Biomarker for Hepatocellular Carcinoma

Round 1

Reviewer 1 Report

Hou et al. through various bioinformatics analyses identified altered splicing indicates poor HCC patient survival and reported LUC7L3 as a new biomarker for HCC. Following LUC7L3 knockdown experiments in HCC lines validated LUC7L3 positively controls HCC proliferation in vitro. Overall although it is an interesting study, there are serious concerns about the study that prevent it from being considered for publication at its current format.

It remains unclear if LUC7L3 participates into the observed deregulated splicing alternations in HCC, and if observed LUC7L3 function in regulating HCC cell line growth in vitro is due to its roles in regulating splicing. In addition, the title is confusing by suggesting LUC7L3 is alternatively spliced in HCC but overall study didn’t examine this at all. In addition to LUC7L3, are RBMX and DDX17 also involved in RNA splicing and why they were identified from RNA-splicing alteration oriented assays? If splicing is deregulated in HCC, are deficiency in splicing or increased splicing events associated with HCC prognosis?

Typos and grammar mistakes should be further eliminated from manuscript.

Author Response

Thank you very much for your valuable time reviewing our manuscript and giving favorable consideration. Please find the detailed responses in the file and the corresponding revisions in the manuscript outlined in yellow highlights.

Author Response File: Author Response.pdf

Reviewer 2 Report

Hou et al identified LUC7L3 as a prognostic biomarker for HCC with a clear research strategy and writing. It would be interesting to know if the LUC7L3 is a better biomarker than some other HCC biomarkers, such as Alpha-fetoprotein (AFP), AFP-L3 and des-γ-carboxy prothrombin (DCP), etc. in the screening for the HCC, which could be added to the discussion. And what about the level of LUC7L and LUC7L2? Do they keep unchanged in the HCC vs. Normal tissues? Also, in

Figure 5: Please mention that the proliferation assays were done in triplicate in figure legends and *** depicts P value <0.001; please add a schematic depiction of the three LUC7L3 siRNA locus in the LUC7L gene; show with immunoblotting whether or not these siRNAs would affect the level of LUC7L and LUC7L2 other than that of LUC7L3. Need some more experiments if the level of LUC7L or LUC7L2 was changed as well.

Figure 6: it would be great if you would use immunoblotting to validate several target genes with your LUC7L3 knock down experiments.

Figure 7: please add PRM2 immunoblotting with your LUC7L3 knock down experiments to validate the correlation of LUC7L3 and PRM2.

Some minor changes - line 47, 153, 155: full name for NASH, NAFLD, CAA and FA; line 238: mRNA outgrowth or mRNA export from nucleus?

Author Response

Thank you very much for your valuable time reviewing our manuscript and giving favorable consideration. Please find the detailed responses below in red font and the corresponding revisions in the manuscript outlined in yellow highlights.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed my raised concerns. I am of the opinion to publish the revision version of the study.

Language can be further polished. The revised version has been improved.

Reviewer 2 Report

The authors have added the suggested control blots and provided some comparisons with other known HCC biomarkers in the discussion. Their responses are very satisfactory to me.