Non-coding RNAs mediate chemotherapy resistance in ovarian cancer

With the advancement of next generation sequencing in past several years, a rising number of non-coding RNAs have been found as new actors to regulate gene expression. Non-coding RNAs not only play important roles in carcinogenesis, but also affect the clinical treatment strategies. They have been proved to be deeply correlated with chemoresistance in several cancers. Ovarian cancer is the leading cause of death in gynecological malignancy, with low 5-year survival rate. Most patients are identified when they have late-stage disease. This review mainly makes a compilation of the most relevant research literature in this field with the purpose of shedding light on the relation between ncRNAs and chemoresistance in ovarian cancer.


Introduction
As one of the most malignant cancers, ovarian cancer is the leading cause of death in gynecological malignancy, especially for epithelial ovarian cancer [1].The 5-year survival rate of ovarian cancer ranges from 30% to 90%.In total cases, over 90% of ovarian cancers are classified as epithelial ovarian cancer.The survival rate is usually close to 90% at early stages, while a large number of patients are diagnosed as late-stage disease [2,3].At present, the diagnostic tools mainly involve pelvic examination, transvaginal ultrasound, and serum cancer antigen 125 (CA125) measurements, and the standard therapy is surgery combined with neo adjuvant chemotherapy [4].Over the past decade, although major advances in the understanding of ovarian cancer have been achieved, tough early diagnosis and poor prognosis still exist.A large number of studies have revealed that non-coding RNAs (ncRNAs) are deeply involved in ovarian cancer progression, including chemoresistance [5,6].
With the development of next-generation sequencing (NGS), chemotherapy resistance has been closely related with nucleic acid, including both DNA and RNA.Besides, based on present studies, ncRNAs have been proved to play an important role in chemotherapy resistance, and even may be the solution of this problem in the future [7,8].
NcRNAs have been proved to regulate many biological processes, including affecting cancer prognosis and influencing response to chemotherapy.The variant expression of ncRNAs in chemotherapy-sensitive versus chemotherapy-resistant cells suggests that modulation of ncRNA expression may reduce or reverse chemotherapy resistance [9][10][11].Through participating in several signaling pathways, such as TGF-b/SMAD [12] and p53 signaling pathway [13], ncRNAs can function as potential prognostic biomarker and diagnostic tool in ovarian cancer.

ncRNAs and chemoresistance
Chemoresistance, classified as intrinsic and acquired, is common in chemotherapy.It can accelerate cancer progression and promote morality.Intrinsic resistance exists in certain chemo-naive patients, which causes the ineffectiveness of intended chemotherapy.During the course of treatment, acquired resistance can emerge when tumors are resistant to a particular drug [7].NcRNAs are RNA molecules that are not translated into protein, including highly abundant and functionally important RNAs such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as microRNAs, lncRNAs, circRNAs and tRFs [14].Large numbers of transcriptomic and bioinformatic studies suggest the existence of thousands of ncRNAs (Figure 1).
Optimal treatment of advanced ovarian cancer includes both surgical cytoreduction and platinum-based chemotherapy.A study demonstrated that patients with stages III and IV ovarian cancer who received chemotherapy had noninferior survival rate and less treatment-related mortality compared with those who received primary cytoreductive surgery [15]..As one of the critical problems in ovarian cancer therapy, nearly 20% of all ovarian cancer patients are resistant to the standard platinum-based chemotherapy.
Patients treated with chemotherapy will experience relapse and develop drug resistance, and the acquired resistance often faces with no other effective curative treatment [16,17].
As key point of many studies, compared with other ncRNAs, the function and regulation mechanism of miRNAs should be much clearer.Bioinformatics data show that miRNAs can regulate at least 20% to 30% of human genes.A single miRNA can regulate up to 100 different kinds of mRNAs, while more than 1000 kinds of mRNAs can be regulated by the same miRNA [22,23].An expression profiling study in a group of cisplatin-, paclitaxel-, and cyclosporin A-resistant ovarian cancer cells showed the expressions of let-7e, miR-30c, miR-125b, miR-130a and miR-335 in all resistant cell lines [24].

LncRNAs
Long non-coding RNAs (lncRNAs) is a kind of single-stranded RNA molecule with the length of over 200 nt.lncRNAs have been found to exist in every branch of life with organismal complexity, which is better correlated with the diversity and size of ncRNAs expression repertoires than with that of protein-coding genes.lncRNAs engage in numerous important biological processes such as imprinting genomic loci, shaping chromosome conformation and allosterically regulating enzymatic activity.
Based on the location, lncRNAs can be classified into sense lncRNAs, anti-sense lncRNAs, intronic lncRNAs, intergenic lncRNAs and divergent lncRNAs.lncRNAs were initially discovered as mRNA-like transcripts that do not encode proteins, but further studies revealed that features of lncRNAs distinguish them from mRNAs.
Specific expression of lncRNAs is also proved to coordinate cell state and differentiation.The overexpression, deficiency and mutant of lncRNAs has been found in a series of human diseases.
As a classical lncRNAs, HOX antisense intergenic RNA (HOTAIR) was found to link DNA damage response and chemoresistance in ovarian cancer [38].This process could also cause the activation NF-κB signaling pathway.In related study, upregulation of HOTAIR could induce platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs. primary ovarian tumors.This suggested that a NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DNA damage response and contributes to chemotherapy resistance in ovarian and other cancers [39].
BC200 was also reported to be associated with ovarian cancer.It was observed that carboplatin could upregulate the expression of BC200 in cell lines, and that the inhibition of BC200 decreased the sensitivity of the cells to the drug.So, BC200 appears to serve a role in the mediation of carboplatin-induced ovarian cancer cell death [40].

CircRNAs
Circular RNAs (circRNAs) were discovered as a special novel type of endogenous non-coding RNAs, and have been a new research hotspot in recent years.
Different from linear RNAs, circRNAs form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome.Some studies have demonstrated that thousands of endogenous circRNAs exist in mammalian cells, and most of them are generated from exonic or intronic sequences.In the process of circRNAs biogenesis, RNA-binding proteins (RBPs) are necessary which can regulate circRNAs formation [41][42][43][44][45][46].
Recent studies revealed that the biological function of circRNAs is abundant.
They can affect alternative splicing of transcripts, regulate translation process of some mRNAs, modulate the expression of parental genes, and even function as miRNAs sponges [47][48][49].Some reports found that circRNAs are widely involved in diseases, including atherosclerotic vascular disease, neurological disorders, prion diseases and cancers [50][51][52].This means, in the near future, circRNAs can be diagnostic or prognostic biomarkers in the clinical treatment.
CircRNAs CircRNA_001059 and circRNA_000167 were the two largest nodes in circRNA/miRNA co-expression network [53].Although no studies have verified the relationship between circRNAs and chemoresistance, circRNAs are widely involved in many biological processes and interact with miRNAs, which means that circRNAs may also affect the chemoresistance in cancer.

tRFs
tRNA-derived RNA fragments (tRFs) is a class of non-coding single-stranded RNA with the length of 14-35 nt, which always derive from 5' end or 3' end of tRNAs in the particular environment [54].Some tRNA cleavages can be generated from mature tRNAs with the length of 30-35 nt, which are called tRNA halves.The rest of tRNA cleavages can be classified into three groups: 3' U tRFs, 3' CCA tRFs and 5' tRFs.3' U tRFs can be generated during the maturation of tRNA in the nucleus, while 3' CCA tRFs and 5' tRFs can be generated at any time from mature tRNAs [55,56].
Up to now, it hasn't been found any direct relationship between tRFs and chemotherapy resistance.However, tRFs have been proved to be closely connected with stress condition [57].Chemotherapy resistance or sensitivity is much likely to be associated with cancer microenvironment, and then affect the stress condition in vivo.
This means, due to the change of microenvironment, tRFs may play a key role in chemotherapy resistance, which is believed to be the research hotspot in the future.

The crosstalk between ncRNAs in ovarian cancer
The transcription process is completed in vivo, from DNA to RNA, and as the first step of proteins biosynthesis, it is also the synthesis procedure of mRNAs and other ncRNAs (tRNAs, rRNAs, etc).
As the most abundant RNAs, tRNAs occupy nearly 15% of total RNAs in humans.A large amount of reports show that tRNAs can be cleaved into small molecule fragments under certain stress condition, called tRFs.At present, the biological function of tRFs still keeps unclear, and limited research indicate tRFs have a close relationship with RNA silence and Argonaute family proteins.Besides, tRFs were once found to regulate the progression of breast cancer via YBX1 [58].
Compared to tRFs, the mechanism of miRNAs and lncRNAs is much clearer.
Through binding to the 3'-untranslated region (3'-UTR) of mRNAs, miRNAs can cause mRNA degradation and inhibit translation.In many signaling pathways, miRNAs also play important roles, and then have close correlation with critical diseases [59][60][61].
As a kind of typical ncRNAs, lncRNAs can be combined with both mRNAs and miRNAs, as well as perform gene silencing on them.The latest research shows the new function of lncRNAs that they can encode some proteins in organisms, which is different from traditional research [62].Since circRNAs aren't sensitive to nuclease, they are more stable than linear RNAs.
Recent studies reveal that circRNAs and lncRNAs are both competing endogenous RNAs (ceRNA), and can compete with microRNA response elements (MREs) to regulate the expression of transcriptosome.CircRNAs can affect miRNAs directly or indirectly, and then function as miRNAs sponges to mediate target genes.This means circRNAs may play key roles in chemotherapy resistance through the interaction with miRNAs [63,64].

Chemoresistance and precision medicine
The development of modern medical technology is gradually revealing the mechanisms of critical diseases.Cancer is thought as a 'genetic disease', and with the advancement of NGS and HGP, the therapeutic strategy for cancer has gradually moved towards the era of precision medical.In this context, the roles of RNAs will become much more important.
Among of all ncRNAs, miRNAs have the most potential to be the diagnostic biomarker and therapeutic drug.For example, as the most famous miRNAs, miR-34 play important roles in breast cancer and ovarian cancer, through targeting EGFR, Bcl-2, CCND1 and so on [65][66][67].Compared with other ncRNAs, the mechanisms of miRNAs are much clearer, whose biological function is performed through participating in several signaling pathways.The abundant features make it possible for miRNAs to be key members in precision medical.
As a kind of novel ncRNAs, the research on tRFs is still at start stage.Since the similarity with miRNAs in structure, tRFs were once thought as one kind of miRNAs.
Besides, the generation of tRFs is closely related with stress condition, which means they are more sensitive to cancer microenvironment, and it is more possible for them to be diagnostic biomarker.
The reports that circRNAs can function as ceRNA make it to be hotspots in the research field of cancers.Because of closed loop structure, circRNAs can be away from the effect of exonuclease.Due to its stability, the existence of circRNAs in cells and human blood is long-term.In this circumstance, circRNAs are thought as the most suitable early diagnostic biomarker by some researchers.
Based on the unique features, lncRNAs can be combined with other ncRNAs and mRNAs, functioning as gene silencing.The biological mechanism of lncRNAs is full of complexity, and they can even encode proteins.In many instances, lncRNAs can be the bridge of the crosstalk between other ncRNAs.
With the advancement of precision medical, the relationship between ncRNAs and diseases will be the research focus, including cancers and chemotherapy resistance.The total kinds of ncRNAs have researched new heights, but it is believed that there still exists other types of ncRNAs, and they can form a RNA world.will help us to characterize them.Moving forward, with these and other future techniques we will surely uncover even more fascinating and unique features and functions of ncRNAs.It can be expected that ncRNA-based therapeutics will play an important role in the future of clinical medicine [68].

Future perspectives
Based on present studies, miRNAs have the most abundant functions in ovarian cancer chemoresistance.Several miRNAs have been identified to be remarkably up/down-regulated in chemoresistant ovarian cancer, including let-7i, miR-27a, miR-23a, miR-449b, miR-21 and so on [69].They are associated with poor prognosis and short survival in chemoresistant ovarian cancer patients.Besides, lncRNA also play key roles in chemresistance.They are closely linked with DNA damage and repair.Contrast to miRNAs and lncRNAs, the biological function of circRNAs and tRFs in chemoresistance still keeps unclear.In view of their relationship with miRNAs and lncRNAs, they are also believed to contribute to chemoresistance in ovarian cancer [70].
Although many breakthroughs have been made on regulatory mechanism of sncRNAs, a significant number of them remain unknown.For example, tRFs were once thought of as a kind of miRNAs, but later, they were affirmed to be a type of cleavage product from tRNAs, different from miRNAs.So, for us, it is still necessary to be absorbed into the studies of ncRNAs, just not only to correct any possible mistakes, but also to get more achievements for the development of physianthropy.

XIST
RNAs are a classical kind of lncRNAs, whose model for gene silencing mechanism has been established.Based on its own three-dimensional structure, X chromosome can bind XIST RNA to the nearby binding sites, and in turn XIST RNA can change the structure of X chromosome, which eventually makes X chromosome silent.CircRNAs are a new class of RNAs which is different from traditional linear RNAs.It has a closed loop structure and is abundant in eukaryotic transcription.Most of circRNAs are composed of exon sequences, which are conserved in different species and show expression specificity in different tissues and developmental stages.

Figure 1 :
Figure 1: The relationship between ncRNAs and chemotherapy resistance in ovarian cancer
have recently been identified as miRNA sponges and involve in various biological processes.Limited research showed that circRNAs play some roles in the radioresistance of esophageal cancer.Reported by Wu et al, among the detected Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted:

preprints.org) | NOT PEER-REVIEWED | Posted: 4 October 2017 doi:10.20944/preprints201710.0021.v1 unique
regulatory mechanisms, alternative forms of biogenesis and functional structured RNA domains, ncRNAs can be classified into several types, and distinguished from each other.Current deep RNA-sequencing and advanced epigenomic technologies are accelerating the rate of discovering new ncRNAs, this ncRNAs have myriad roles in cells, from controlling the architecture of whole chromosomes and acting as a regulator of gene expression to affecting the translation of genetic codons into protein sequences.With the help of different features, including Preprints (www.