In this case-control study, we reported the significant association of ovarian cancer with the variants of circadian rhythm gene pathway, predominantly with already reported ovarian cancer risk associated circadian gene variants (BMAL1 / ARNTL rs475715 and rs1026071, PER3 rs228644, REV1 rs3792152, and TIMELESS rs7302060) [20]. We examined variation in the four most common genes of the circadian pathway (REV1, ARNTL/BMAL1, TIMELESS, and PER3) as prognosticators of ovarian cancer risk and invasiveness. We found that two out of five variants were associated with the risk of ovarian cancer. Specifically, the risk of ovarian cancer was associated with variant rs475715 of BMAL1/ARNTL & rs3792152 of REV1, whereas other variant rs228644 of PER3, rs7302060 of TIMELESS, and rs1026071 of ARNTL were found to be not associated with ovarian cancer in our studied region.
Biological clock in humans called circadian clock / circadian rhythm, autonomously oscillate with a period near 24 hours. The mechanism of the circadian clock is based on the positive/negative response circlets which are produced by core circadian clock genes. The monitoring feedback loop of circadian rhythm consists of PER, CRY, CLOCK, and BMAL1 proteins having a function of regulations in the transcription/translation process. The heterodimer (BMAL1/CLOCK) complex inhibiting or repressing the PER/CRY genes activity in the nucleus region where the monitoring feedback loop formation is completed after the complex (BMAL1/CLOCK) formation which regulates the transcription of Rev-erbα and Rora (nuclear receptors)[25].
Findings regarding the circadian rhythm seem to be initiated by both transcriptional and post-transcriptional mechanisms which induce gene expression [26, 27]. The transcription of PER and CRY genes is initiated by two transcription factors CLOCK and BMAL1 / ARNTL. After reaching the grave concentration the PER / CRY reduces the effect of CLOCK/ BMAL1 facilitated initiation of their particular genes in a negative feedback loop. It was found that both complex PER / CRY and CLOCK/ BMAL1 intricate with each other and bound to chromatin. The regular daily oscillations in clock gene is contributed by protein degradation, phosphorylation, and nuclear entry [28]. The regulation of CRY and PER gene expression generates genetic and biochemical evidence [29] but the regulation of the CLOCK/ BMAL1 gene is very much less known. Various studies reported that PER and CRY gene establishes a positive feedback loop in the process of BMAL1 transcription [28–30]
REV1 (REV1-DNA directed polymerase) is a nuclear receptor that acts as a transcriptional repressor in the circadian pathway, where activates and inhibits the transcription of the BMAL1 gene [31]. The BMAL1 transcription is regulated by REV-ERB alpha, thus it acts as a connector link through which components of negative and positive limb constitute to form a molecular link. It determines the length of the period and phase-shifting properties of the biological clock [30]. It was proved that BMAL1 deficient cells due to DNA damage lead to arrest in cell cycle and reveal a possible modulatory effect on tumor suppressor genes i.e. P53. It has been reported that the knockdown of the BMAL1 gene induces cell growth, reduced programmed cell death which appears to play a role in carcinogenesis [32].
Our study is the first replicative case-control association study of clock genes. Jin et al [20] reported that the gene expression of the BMAL1 gene has been controlled by cMYC where the overexpression of cMYC leads to the downregulation of BMAL1. So, it has been suggested that BMAL1 gene variants were significantly associated with the risk of ovarian cancer. The circadian gene variants are associated with prostate cancer which was reported in the GWAS study [33]. In some populations of the world, it has been reported that night workers have disturbed biological clocks where the findings of various studies proved the night working women’s and men’s are more prone to cancers (Breast, ovary, and prostate cancer) [19, 33–35]. Our results indicated that BMAL1 and REV1: rs475715 & rs3792152 both intronic variants, were associated with ovarian cancer risk. Functional prediction implicated that rs47515 has direct eQTL effect and regulates the expression of C2orf15, LIPT1, LYG1, MITD1, REV1, TSGA10, and TXNDC9 genes, whereas rs3792152 regulates the RN7SKP151 & BTBD10 with BMAL1 gene. These findings suggest that it is located within a region that directly affects expression potentially through the modulation of the histone markers in the enhancer region. Yeh et al, 2014 [36] proved that in the ovarian cancer cell (in CP70 and MCP2) the H3K27 (histone mark) is supplemented in the promoter region of ARNTL / BMAL1 gene, Whereas the presence of inhibitor (GSK126) of EZH2 region reestablished the expression of ARNTL/ BMAL1 gene in ovarian cancer cells (in CP70 and MCP2). They also confirmed that there is a sensitivity of chemotherapy drug (cisplatin) in ovarian cancer cells after increasing the expression of the ARNTL / BMAL1 gene. With these findings, it was confirmed that BMAL1 /ARNTL may act as a tumor suppressor by regulating the p53 tumor suppressor pathway in ovarian cancer [20, 36]. So, our results climax the implication of circadian rhythm gene variation in ovarian cancer susceptibility and suggest an early role for the BMAL1 and REV1 gene in ovarian cancer pathogenesis.
Our results also suggest that the circadian gene variant may play a significant role in the etiology of ovarian cancer. From the literature survey, the identification of a significant association between circadian genes and ovarian cancer is still unpredictable. To the best of our knowledge, the case-control association studies between circadian gene variants and ovarian cancer were investigated in very few studies (20,25–29). Few of them have found an association with ovarian cancer and other cancers at the variant level [20, 37–39]. Even though various previous research studies have implicated circadian genes in the progression of cancers in women. Nevertheless, our study possibly requires more variants of the circadian rhythm pathway to highlight significant associations between certain circadian genes and the risk of aggressive ovarian cancer.