A Concise Review on Dysregulation of LINC00665 in Cancers

Long Intergenic Non-Protein Coding RNA 665 (LINC00665) is an RNA gene located on the minus strand of chromosome 19. This lncRNA acts as a competing endogenous RNA for miR-4458, miR-379-5p, miR-551b-5p, miR-3619-5p, miR-424-5p, miR-9-5p, miR-214-3p, miR-126-5p, miR-149-3p, miR-379-5p, miR-665, miR-34a-5p, miR-186-5p, miR-138-5p, miR-181c-5p, miR-98, miR-195-5p, miR-224-5p, miR-3619, miR-708, miR-101, miR-1224-5p, miR-34a-5p, and miR-142-5p. Via influencing expression of these miRNAs, it can enhance expression of a number of oncogenes. Moreover, LINC00665 can influence activity of Wnt/β-Catenin, TGF-β, MAPK1, NF-κB, ERK, and PI3K/AKT signaling. Function of this lncRNA has been assessed through gain-of-function tests and/or loss-of-function studies. Furthermore, diverse research groups have evaluated its expression levels in tissue samples using microarray and RT-qPCR techniques. In this manuscript, we have summarized the results of these studies and categorized them in three sections, i.e., cell line studies, animal studies, and investigations in clinical samples.


Introduction
Long non-coding RNAs (lncRNAs) are a group of transcripts participating in the chromatin remodeling, transcriptional regulation of gene expression as well as posttranscriptional events, via different chromatin-associated routes and through interaction with other RNA molecules [1]. Their functions largely depend on their subcellular localization and interplays with DNA, RNA, and proteins [2]. In addition to their role in the modulation of chromatin function, they can influence the stability of mRNAs and their translation in the cytoplasm [2]. Their involvement in diverse biological and physiopathological functions has potentiated them as important participants in the pathoetiology of different disorders.
Long Intergenic Non-Protein Coding RNA 665 (LINC00665) is an RNA gene located on chr19:36,259,540-36,332,581. At least 33 splice variants have been identified for this gene (http://asia.ensembl.org/Homo_sapiens/Gene, accessed on 10 October 2022). This lncRNA has been shown to act as an oncogene in diverse malignancies. Function of this lncRNA has been assessed through gain-of-function tests and/or loss-of-function studies. Moreover, different groups have evaluated its expression levels in tissue samples using microarray and real time-quantitative polymerase chain reaction (RT-qPCR) techniques. Similar to some other lncRNAs, LINC00665 has been shown to encode a micropeptide. This micropeptide is called CIP2A-BP. Experiments in breast cancer cells have revealed that translation of CIP2A-BP is decreased by TGF-β. This micropeptide has an inhibitory role in the development of triple negative breast cancer possibly through suppression of PI3K/AKT/NF-κB signals and the subsequent down-regulation of matrix metalloproteinase (MMP)-2, MMP-9, and Snail levels [3].
Since this lncRNA has been dysregulated in different malignancies, it represents a potential target for therapeutic interventions. Thus, it is necessary to identify the functionally related molecules and pathways with this lncRNA in different tissues. In this manuscript, we have summarized the results of these studies and categorized them in three sections, i.e., cell line studies, animal model investigations, and investigations in clinical samples.

Cell Line Studies
LINC00665 has been shown to be up-regulated in acute myeloid leukemia (AML) cells parallel with the increase in expression of Dedicator Of Cytokinesis 1 (DOCK1) and decrease in expression of miR-4458. Knock-down of LINC00665 or DOCK1 has resulted in a significant decrease in proliferation, migration, and adhesion of these cells. On the other hand, suppression of miR-4458 has led to enhancement of these features and inhibition of apoptosis of AML cells. Mechanistically, LINC00665 could sponge miR-4458 and increase expression of DOCK1 through this route [4].
In breast cancer cells, LINC00665 has been found to act as a sponge for miR-379-5p, reducing the capacity of miR-379-5p to suppress expression of Lin-28 Homolog B (LIN28B). The impact of LINC00665 in induction of expression of LIN28B is associated with induction of progression of breast cancer and activation of epithelial-mesenchymal transition (EMT) program in these cells [5]. Another study in breast cancer cells has revealed attenuation of migration and invasion aptitude of these cells following LINC00665 silencing. Moreover, downregulation of this lncRNA has inhibited expressions of EMT-associated proteins in these cells [6]. LINC00665 can also influence progression of breast cancer via sponging miR-551b-5p [7]. LINC00665 silencing has also suppressed proliferation, migration, and invasive features of breast cancer cells, while it enhanced apoptosis. The effects of LINC00665 on these features of breast cancer cells are possibly exerted through sponging miR-3619-5p. Upregulation of miR-3619-5p has been shown to be similar to LINC00665 silencing at cellular level. Expression of β-catenin has been reduced following LINC00665 silencing and miR-3619-5p up-regulation, supporting the importance of LINC00665/miR-3619-5p/β-catenin axis in the progression of breast cancer [8].
An in vitro experiment in cervical cancer cells has revealed that short hairpin RNA (shRNA)-mediated knock down of LINC00665 can lead to reduction of cell viability, upregulation of E-cadherin level, down-regulation of N-cadherin, Vimentin and CTNNB1 levels, and suppression of migration and invasiveness of HeLa cells. The impact of LINC00665 on enhancement of EMT is possibly mediated via activation of WNT-CTNNB1/β-catenin signals [9].
A microarray-based assay in gemcitabine resistant cholangiocarcinoma cell lines has led to identification of LINC00665 among the top 10 over-expressed. Knock down of this lncRNA in chemoresistant cells has led to enhancement of gemcitabine effects, whereas up-regulation of LINC00665 has augmented gemcitabine resistance in chemosensitive cholangiocarcinoma cells. Chemoresistant cholangiocarcinoma cells have exhibited higher EMT and stemness features, and LINC00665 knock-down has inhibited sphere formation, migratory potential, invasiveness, and levels of EMT and stemness marker proteins. While Wnt/β-Catenin signals have been activated in chemoresistant cholangiocarcinoma cells, LINC00665 silencing has inhibited activity of this pathway. Mechanistically, LINC00665 can regulate expression of the nuclear transcriptional regulator of this pathway, i.e., BCL9L through sponging miR-424-5p. BCL9L down-regulation or miR-424-5p up-regulation has reduced resistance to gemcitabine, and decreased EMT, stemness, and Wnt/β-Catenin activity in chemoresistant cholangiocarcinoma cells [9]. In vitro experiments in colorectal cancer cells have shown the sponging role of LINC00665 on miR-9-5p and subsequent regulation of ATF1 as an important mechanism of carcinogenesis [10]. Moreover, LINC00665-mediated up-regulation of CTNNB1 has been demonstrated to result in activation of Wnt/β-catenin signals in colorectal cancer cells [11]. Finally, LINC00665 could stimulate proliferation and impede apoptosis of colorectal cancer cells through regulation of miR-126-5p expression [12]. Figure 1. Oncogenic role of LINC00665 in prostate cancer, colorectal cancer, breast cancer, and osteosarcoma. Detailed information about the assays is shown in Table 1. LINC00665 can regulate expression of the nuclear transcriptional regulator of this pathway, i.e., BCL9L through sponging miR-424-5p. BCL9L down-regulation or miR-424-5p up-regulation has reduced resistance to gemcitabine, and decreased EMT, stemness, and Wnt/β-Catenin activity in chemoresistant cholangiocarcinoma cells [9].
In vitro experiments in colorectal cancer cells have shown the sponging role of LINC00665 on miR-9-5p and subsequent regulation of ATF1 as an important mechanism of carcinogenesis [10]. Moreover, LINC00665-mediated up-regulation of CTNNB1 has been demonstrated to result in activation of Wnt/β-catenin signals in colorectal cancer cells [11]. Finally, LINC00665 could stimulate proliferation and impede apoptosis of colorectal cancer cells through regulation of miR-126-5p expression [12]. Figure 1. Oncogenic role of LINC00665 in prostate cancer, colorectal cancer, breast cancer, and osteosarcoma. Detailed information about the assays is shown in Table 1.    PKR, NF-κB signaling EK-293T, Huh-7 and HepG2, and SK-Hep1, pWPXL-LINC00665 LINC00665 was found to stabilize PKR by blocking its ubiquitination and involvement in the activation of NF-κB signaling.
In glioma cells, in vitro experiments have indicated down-regulation of TAF15 and LINC00665. Forced up-regulation of TAF15 has increased stability of LINC00665, suppressing malignant features in these cells. Moreover, MTF1 and YY2 transcription factor have been shown to be over-expressed in glioma cells, and their silencing has suppressed malignant behaviors of these cells. Up-regulation of LINC00665 could decrease stability of MTF1 and YY2 transcripts through interplay with STAU1, and STAU1 silencing has reversed LINC00665-mediated down-regulation of MTF1 and YY2. Finally, MTF1 or YY2 silencing has reduced expression of GTSE1 oncogene in glioma. Taken together, the TAF15/LINC00665/MTF1(YY2)/GTSE1 axis has been acknowledged as an important axis in the modulation of the malignant features of glioma cells [18]. Contrary to this study, another study has shown an oncogenic role for this lncRNA in glioma. This study has indicated that LINC00665 sponges miR-34a-5p to influence expression of AGTR1 [19].
In hepatocellular carcinoma, LINC00665 has been found to sponge miR-214-3p and increase expression of MAPK1, thus accelerating cell proliferation and Warburg effect [20]. Moreover, it can regulate the viability, apoptotic pathways, and autophagy of these cells through modulation of miR-186-5p/MAP4K3 [21] and Double-Stranded RNA-Activated Protein Kinase/NF-κB [22] axes. Another study in hepatocellular carcinoma cells has shown that LINC00665 silencing could decrease proliferation, migration, and invasion, whereas up-regulation the CIP2A-BP micropeptide enhances these features [36]. Figure 2. Role of LINC00665 in hepatocellular carcinoma, glioma, melanoma, gastric cancer, and lung cancer. Detailed information about the assays is shown in Table 1. [20]. Moreover, it can regulate the viability, apoptotic pathways, and autophagy of these cells through modulation of miR-186-5p/MAP4K3 [21] and Double-Stranded RNA-Activated Protein Kinase/NF-κB [22] axes. Another study in hepatocellular carcinoma cells has shown that LINC00665 silencing could decrease proliferation, migration, and invasion, whereas up-regulation the CIP2A-BP micropeptide enhances these features [36]. Figure 2. Role of LINC00665 in hepatocellular carcinoma, glioma, melanoma, gastric cancer, and lung cancer. Detailed information about the assays is shown in Table 1.

Animal Studies
Experiment in xengraft models of breast, colorectal, gastric, liver, lung, and prostate cancers as well as cholangiocarcinoma, endometrial carcinoma, and melanoma have confirmed that up-regulation of LINC00665 increases tumor burden, while its silencing decreases tumor weight (Table 2). Thus, these studies consistently point to the oncogenic effects of LINC00665.

Studies in Clinical Samples
LINC00665 has been shown to be over-expressed in breast cancer tissue samples in association with poor prognosis of breast cancer patients [6]. Similarly, up-regulation of LINC00665 in breast cancer samples has been correlated with tumor size and TNM stages in another cohort of breast cancer patients [8]. Over-expression of LINC00665 has also been associated with poor prognosis and resistance of cholangiocarcinoma patients to chemotherapy [9]. In liver cancer, expression of LINC00665 has also been elevated, which noticeably designated poor prognosis. Moreover, up-regulation of LINC00665 has been associated with further development of the tumors, which has been closely correlated with clinical diagnosis. Accuracy of LINC00665 levels in prediction of overall survival has also been verified by ROC curve analyses [36].
LINC00665 can also affect response to a number of anticancer drugs including gemcitabine, apatinib, and gefitinib. Thus, therapies targeted against LINC00665 are expected to decrease tumor volume, reduce malignant features, and improve the response of cancer cells to both chemotherapeutic drugs and targeted therapies. Although the results of investigations in animal models of diverse kinds of cancers have been promising, these results have not been validated in clinical settings due to several obstructions, particularly regarding safety and bioavailability issues. Moreover, LINC00665 has interactions with a wide range of biomolecules including miRNAs. Identification of other LINC00665 targets, particularly possible tissue-specific targets, is a prerequisite for the design of more specific therapeutic modalities.
An important note about this lncRNA is its ability in the production of a functional micropeptide. The encoded micropeptide by this lncRNA can promote progression of breast cancer and hepatocellular carcinoma. Future studies should assess the impact of this micropeptide in other types of cancers and find the possible functional relationship between lncRNA and micropeptide. Notably, expression of this micropepetide is regulated by TGF-β [3].
Finally, in spite of adequate data about the prognostic impact of LINC00665, there is no data about its role in diagnostic approaches. Thus, future studies should evaluate whether LINC00665 levels can separate patients with malignancies from normal subjects.
In conclusion, LINC00665 is an lncRNA that affects several aspects of carcinogenesis, particularly in response to both chemotherapeutic agents and small molecules that are used in targeted cancer therapy. Therefore, this lncRNA is a candidate for designing novel drugs for the treatment of cancer.