Lnc-TALC promotes O6-methylguanine-DNA methyltransferase expression via regulating the c-Met pathway by competitively binding with miR-20b-3p

Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.

1. I am very concerned with the large volume of data presented. Just to give an example, Figures 5,6,7 and 8 have more than 10-14 panels. If this paper is being revised to address additional comments/suggestions, it will continue to lack focus that is needed for a good, solid paper to move the field forward. If the authors care about this they might want to read the following article in Nature, entitled "Publish houses of brick, not mansions of straw".  Figure 2C, what is the effect of kd of the lncRNA in untreated condition?

Its not clear what lnc-TALC is.
What is the Gene symbol? Is there only one isoform? When I used the coordinates from Figure 3C, it looks like there are many isoforms? However, the authors show only one transcript. 5. What is the number of molecules of lnc-TALC, miR-20-3p and MET mRNA in these cells?
Reviewer #3 (Remarks to the Author): Summary: In this study, the authors showed that a long non-coding RNA (temozolomideassociated LncRNA in glioblastoma recurrence)(lnc-TALC) competed and bound to miR-20b-3p. MiR-20b-3p inhibits the activation of Receptor Tyrosine Kinase, c-Met. c-Met induces the activation of Akt which in turn phosphorylates and inactivates the tumor suppressor FOXO3. c-Met also activates MAPK/Stat3/p300 pathway. Stat3/p300 complex modifies the Histone 3 in MGMT promoter and induces MGMT expression in TMZ resistant GBM cells. Methyl Guanine Methly Transferase (MGMT) is a DNA repair enzyme that removes methyl groups on DNA. Temozolomide (TMZ) is the frontline chemotherapy drug for GBM. TMZ alkylates DNA and causes DNA damage in cancer cells which if left unrepaired, leads to apoptosis and cell death. However, activation of DNA repair enzyme, MGMT and removal of alkyl group on DNA and repair of DNA by this enzyme leads to TMZ resistant GBM. The authors suggest that because lnc-TALC regulates these pathways through miR-20b-3p, lnc-TALC can serve as a new target for therapy of TMZ-resistant GBM. Overall, this is an interesting study focusing on a novel long non-coding RNA that sheds light to a potential mechanism that explains TMZ-resistant GBM. The paper will need some editing to improve the readability. I was confused a number of times during reading of the manuscript. Specific comments: 1. The current title is not very clear. The authors may want to consider alternative that specifies miR020b-3p since that is the proposed direct target. 2. Introduction; line 45: The sentence "For example, the pancreatic cancer risk variant of LINC00673 creates a miR-1231 binding site and interferes with PTPN11 degradation"8. This sentence needs to be expanded a little bit more as it is a supporting evidence of importance of embedded miRNAs in long non-coding RNAs that regulate different signaling pathways. In reference 8, the authors showed that LINC00673 is a tumor suppressor and diminishes SRC-ERK oncogenic signaling. 3. Introduction; line 66: "…… development and progression by stimulating the PI3K/AKT, -catenin signaling pathways, among others". No references were provided at the end of the sentence. A reference for each of these pathways should be provided. 4. Materials and Methods: Line 211: "The levels of miRNA RNA were normalized to U6". The word "RNA" should be deleted. U6 SnRNA (small nuclear RNA) is a more accurate term. Lines 212 to 217: It is not necessary to mention the PCR condition. Also if the quantitative real time PCR was used, why at the end the PCR products were run on a 4.8% agarose gel? 5. Results; Line 331-332, Figure 4E: The authors did not explain well how they knocked down lnc-TALC by CRISPR-Cas9. Line 401. The sentence "The RIP assay showed that lnc-TALC and MET could bind Ago2 (Fig. 5B-D)". This should be changed to " ……lnc-TALC and c-MET transcript could bind Ago2". Figure 5B: In this figure, the RNA and RNA binding protein should be labelled. The figure seems to depict the RIP assay in general. Line 402; Figure 5B-D should be separated and each figure should be explained separately e.g. Figure 5B, 5C, 5D because each figure contains results that needs to be explained. In general, figures and results should be better explained. In Figure 9, the phosphorylated FOXO3 is shown in the nucleus, and its degradation in the cytoplasm. This does not seem to be consistent with the literature which suggests that phosphorylation of FOXO3 takes place in the cytoplasm by phosphorylated AKT and subsequently the phosphorylated FOXO3 gets ubiquitinated and degraded in the cytoplasm [(References (12) and (32) in this article)]. The authors suggest that lnc-TALC can be targeted for therapy of TMZ-resistance GBM, but no original Figure 8A and 8B were merged into the revised Figure 8A, making the conclusion that knockdown of lnc-TALC restores TMZ sensitivity in TMZ-resistant GBM xenografts much more intelligible.

Revised Figure 8:
Our results show that lnc-TALC could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy in GBM. Thank you for your valuable suggestions for improving the quality of our work. (1) We increased the font size of the original Figure 1B-D, Figure 2B and Figure 4A (the revised Figure S4A), 4B (the revised Figure 4A), 4C (the revised Figure S4B); removed some unimportant or illegible labels from the original Figure 1C and Figure 4A (the revised Figure   S4A), 4B (the revised Figure 4A); and added additional labels to the original Figure 4C (the revised Figure S4B).

Revised Figure 1
Revised Figure S4: (2) We added the names of resistant cells in the revised Figure 2A, the figure legend, and the manuscript. Thank you very much.

Revised Figure 2:
(3) Single-sample GSEA (ssGSEA) 2 , an extension of Gene Set Enrichment Analysis (GSEA) 3 , calculates separate enrichment scores for each pairing of a sample and gene set. Each ssGSEA enrichment score represents the degree to which genes in a particular gene set are coordinately up-or down-regulated within a sample. In this manner, ssGSEA projects a single sample's gene expression profile from the space of a single gene onto the space of gene sets. The ssGSEA projection transforms data to a higher-level (pathways instead of genes) space representing a more biologically interpretable set of features 4 . We have described this method in the methods section of the revised manuscript.

(4) The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and
CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material 5 . The CRISPR/Cas9 system has been shown to be a powerful genome-editing tool in a variety of organisms 6 . There are three types of CRISPR/Cas systems. The type II system uses single effector enzymes, such as Cas9, to cleave dsDNA, and the DNA double-strand cutting process can be completed only by the combination of Cas9 protein and sgRNA 7,8,9 . Because of this unique feature, the CRISPR/Cas type II system has been extensively utilized studied as a genetic engineering tool from bacteria to mammals 9 . The CRISPR/Cas9 system has been used to successfully knock down various lncRNAs 10,11,12,13,14 . Application of the CRISPR/Cas9 system for knocking down genes has been mastered and exemplified in our previous studies 15,16 . Therefore, we designed a strategy to knockdown lnc-TALC using two pairs of sgRNAs, and the sgRNA sequences are shown in Supplementary Table 8 The word "RNA" should be deleted. U6 SnRNA (small nuclear RNA) is a more accurate term.
Lines 212 to 217: It is not necessary to mention the PCR condition. Also if the quantitative real time PCR was used, why at the end the PCR products were run on a 4.8% agarose gel?
Response: Thank you for these suggestions. We have deleted the word "RNA" in the sentence "The levels of miRNA RNA were normalized to U6". U6 SnRNA (small nuclear RNA) has been used in the revised manuscript instead of "U6". We regret our vague description in the methods. PCR products derived from ChIP-PCR were run on a 4.8% agarose gel to quantitatively compare differences among groups based on the gray scale. We have revised the description of the experimental method in the revised manuscript.

Results
; Line 331-332, Figure 4E: The authors did not explain well how they knocked down lnc-TALC by CRISPR-Cas9.

Response:
We appreciate your comment. The CRISPR/Cas9 system has been declared to successfully knock down various lncRNAs 10,11,12,13,14 . Application of the CRISPR/Cas9 system for knocking down genes has been mastered and validated in our previous studies 15,16 .
Therefore, we designed a strategy to knock-down lnc-TALC using two pairs of sgRNAs, and the sgRNA sequences are shown in Supplementary Table 8 2. Figure 5B: In this figure, the RNA and RNA binding protein should be labelled. The figure seems to depict the RIP assay in general.

Response:
Thank you for your suggestion. We have labelled the RNA and RNA binding protein in the revised Figure 5B. A RIP assay was performed as reported previously by our team 15 . The supernatant of cell lysate was incubated with protein A/G magnetic beads coated with anti-Ago2 antibody. The beads were then washed, and RNA was isolated and processed for PCR analysis of lnc-TALC and c-MET transcript. Thank you again for your precise suggestion. Figure 5B-D should be separated and each figure should be explained separately e.g. Figure 5B, 5C, 5D because each figure contains results that needs to be explained. In general, figures and results should be better explained.

Response:
We thank you for your careful review. The legends and results of the original Figure 5B-D (the revised Figure 5A-B) have been separately described and further explained in the revised manuscript. The revised Figure 5A is a schematic of the RIP-PCR assay.
Supernatants of cell lysate were incubated with protein A/G magnetic beads coated with anti-Ago2 antibody. Beads were then washed, and RNA was isolated and processed for PCR analysis of lnc-TALC and the c-MET transcript. The revised Figure 5B  4. In Figure 9, the phosphorylated FOXO3 is shown in the nucleus, and its degradation in the cytoplasm. This does not seem to be consistent with the literature which suggests that phosphorylation of FOXO3 takes place in the cytoplasm by phosphorylated AKT and subsequently the phosphorylated FOXO3 gets ubiquitinated and degraded in the cytoplasm [(References (12) and (32) in this article)].
Response: Thank you for your crucial suggestions. We have carefully checked the references for phosphorylated FOXO3 induction by activated AKT, which is ubiquitinated and degraded in the cytoplasm 21,30 . We have revised Figure 9 according to the above biological process. We thank you again for your careful and critical comments.

Revised Figure 9:
5. The authors suggest that lnc-TALC can be targeted for therapy of TMZ-resistance GBM, but no such effort such as siRNA approach was shown.

Response:
We thank you for your suggestion. In the present study, we used the siRNA to knock-down the 10 selected lncRNAs and validated the RNA expressive level (Fig. S2B) for further loss-of-function analysis in TMZ-resistant GBM cells. Knockdown of lnc-TALC inhibited TMZ-resistance in two types of resistant cells ( Fig. 2A). In addition, we stably knocked down lnc-TALC in TMZ-resistant cells using the CRISPR-Cas9 system (Fig. S2C).
Knockdown of lnc-TALC in resistant GBM cells significantly decreased cell viability, promoted apoptosis, and inhibited cell colony formation and proliferation in response to TMZ treatment ( Fig. 2C-F). We also evaluated the therapeutic value of lnc-TALC in vivo (Fig. 7E).
Bioluminescent imaging revealed that knockdown of lnc-TALC efficiently restored the sensitivity of TMZ-resistant xenografts to TMZ treatment (Fig. 7F). Mice receiving a combined treatment exhibited much smaller tumor volume than others (Fig. 7G) and exhibited a significantly prolonged lifespan (Fig. 7H). Thank you again for your valuable suggestions.