Wang Keming,  DingJie,  Lian Yifan,  Shi Yongguo,  Lin Yan,  Wang Zhaoxia,  Li Juan

Nanjing, China Second Affiliated Hospital, NanjingMedical UniversityDepartment of Oncology

Objective: Colorectalcancer (CRC), as the third most common cancer affecting the gastrointestinaltract, is a common type of cancer worldwide, and is associated with a highmortality rate due to its rapid progression and advanced tumor presentation atthe time of diagnosis. The early detection of CRC is significantly beneficialto improve the probability of survival. Therefore, a better understanding ofthe mechanisms that result in CRC and the identification of a new molecularmarker or factor that can be used to create novel diagnostic and therapeuticstrategies is urgently needed for patients with CRC. Long non-coding RNAs(LncRNAs, > 200 nucleotides in length) are a class of newly discoverednon-coding RNA molecules with limited or no protein-coding capacity. Recently, aberrantexpression of long noncoding RNAs (lncRNAs) has frequently been reported incancer studies, including those of colorectal cancer. They may function asoncogenes or anti-oncogenes similar to protein-coding genes, and theirdysregulated expression is significantly correlated with carcinogenesis. Theunderlying molecular mechanisms by which lncRNAs exert their functions arecomplex, diverse and exist at various levels during the development of cancer, includingthe epigenetic, transcriptional, post-transcriptional and translational levels.For instance, lncRNA HOTAIR is upregulated in CRC and may be a critical elementin metastatic progression as a result of its interaction with PRC2 (PolycombRepressive Complex 2). Loc554202 is a 2166-bp transcript on human chromosome9p21.3, the expression of which is dysregulated in breast and lung cancercells. A previous study demonstrated that Loc554202 regulates the proliferationand migration of breast cancer cells, and CpG island methylation plays animportant role in silencing the Loc554202 genes. However, the functions ofLoc554202 in CRC were previously unknown. Method: Expression ofLoc554202 was analyzed in 48 colorectal cancer tissue samples, five humancolorectal cancer cell lines by quantitative real-time polymerase chainreaction (qRT-PCR). Over-expression and RNA interference (RNAi) approaches wereused to investigate the biological functions of Loc554202. The effect of Loc554202on proliferation was evaluated by MTT and colony formation assays, and cellapoptosis was evaluated by hochest stainning. Colorectal cancer cellstransfected with pCDNA3.1 –Loc554202 were injected into nude mice to study theeffect of Loc554202 on tumorigenesis in vivo. qRT-PCR, western-blotting andimmunohistochemistry were used to evaluate the mRNA and protein expression ofapoptosis-related factors. Differences between groups were tested forsignificance using Student’s t-test (two-tailed). Result: We providedthe first evidence that lncRNA Loc554202 was significantly down regulated incolorectal cancer tissues compared with adjacent normal tissues, and lowexpression of the lncRNA in CRC patients was associated with an increased tumorsize and advanced TNM stage. Additionally, we treated CRC cells with a DNAdemethylating agent (5-aza-CdR) to determine the influence of DNA methylationin these cells, and found that the Loc554202 expression was significantlyincreased in the 5-aza-CdR treated HCT116 and DLD1 cells compared with controlcells. Our subsequent studies showed that overexpression of Loc554202 decreasedcell proliferation, caused a dramatic decrease in colony formation, promotedsignificant arrest in the G0/G1-phase and an obvious increase in apoptosis inthe HCT116 and DLD1 cells. To determine whether the over expression ofLoc554202 could affect tumorigenesis, pCDNA-Loc554202 and empty vectortransfected HCT116 cells were inoculated into male nude mice. However, thetumors formed in the pCDNA-Loc554202 group were dramatically smaller than thosein the empty vector group. We also examined the HE staining of tumor tissuesand found that karyopyknosis and a shape change were present in the tumorsamples treated with pCDNA-Loc554202, and these findings were not noted in thenegative control samples. The tumors developed from pCDNA-Loc554202 cellsdisplayed significantly increased cleaved caspase-3 staining compared withtumors formed from empty vector transfected cells. These results indicate thatLoc554202 is significantly associated with the proliferation and apoptosis ofcolorectal cancer cells in vivo. To confirm the role of caspase activation inLoc554202 induced apoptosis, we treated HCT116 and DLD1 cells with a generalcaspase inhibitor, Z-VAD-FMK (10mmol/L). The pretreatment with Z-VAD-FMKdecreased the Loc554202 induced apoptosis rate detected by flow cytometry.Consistent with this finding, the results of a qRT-PCR analysis and westernblot analysis showed that the mRNA levels of Bax, caspase-3 and caspase-9 andthe protein levels of Bax, cleaved caspase-3, cleaved caspase-9 weresignificantly increased in pCDNA-Loc554202 treated cells, whereas the mRNA andprotein levels of Bcl-2 were decreased. These findings indicate that Loc554202induces CRC cell apoptosis at least partly through the activation of specificcaspase cleavage cascades. Conclusion: We have shown that Loc554202 isdownregulated in colorectal cancer tissues, and provide the first evidence thatits low expression was significantly associated with the tumor size andadvanced TNM stage in CRC patients. In addition, treatment with a DNAdemethylating agent (5-aza-CdR) significantly increased the Loc554202expression in CRC cells, indicating that CpG island methylation plays animportant role in silencing the Loc554202 genes. Moreover，theoverexpression of Loc554202 decreased colorectal cancer cell proliferation andinduced apoptosis in vitro，hinderingtumorigenesis in vivo. Finally, we showed that Loc554202 regulated cellapoptosis at least partly through the activation of specific caspase cleavagecascades. Together，ourfindings suggest that lncRNA Loc554202 acts as a tumor-inhibiting factor in CRC,and could be a candidate prognostic biomarker or a target for new cancertherapies. However, further studies in a larger number of samples andinvestigations of the other possible mechanisms of action are required.

Key Words: Loc554202 colorectal