Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6.

BACKGROUND: Long non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of colorectal cancer (CRC). This study aims to examine the effects of lncRNA RP11-468E2.5 and its target genes (STAT5 and STAT6) on the biological activities of CRC cells via the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. METHODS: We initially screened the GEO database for differentially expressed lncRNAs related to CRC and then made a prediction of the implicated target genes. Then we collected CRC tissues and adjacent normal tissues from 169 CRC patients. Human CRC HCT116 and SW480 cells were treated with small interference RNA (siRNA) against RP11-468E2.5, AG490 (an inhibitor of the JAK/STAT signaling pathway), or both in combination. Next, we measured the effects of RP11-468E2.5 treatment on cellular activities such as cell viability, cycle distribution and cell apoptosis, and studied interactions among RP11-468E2.5, STAT5/STAT6, and the JAK/STAT signaling pathway. Finally, an in vivo tumor formation assay was performed to observe the effect of RP11-468E2.5 on tumor growth. RESULTS: The CRC-related gene microarray data showed low expression of RP11-468E2.5 in CRC surgical specimens. However, RP11-468E2.5 was confirmed to target STAT5 and STAT6, which participate in the JAK/STAT signaling pathway. CRC tissues showed lower expression of RP11-468E2.5, higher expression of STAT5, STAT6 and of the cell cycle marker Cyclin D1 (CCND1), compared to the findings in adjacent normal tissues. The treatment of siRNA against RP11-468E2.5 increased expression of JAK2, STAT3, STAT5, STAT6, CCND1 and Bcl-2 along with the extent of STAT3, STAT5 and STAT6 phosphorylation, while lowering expression of P21 and P27. Treatment with AG490 exhibited approximately opposite effects, whereas siRNA against RP11-468E2.5 treatment stimulated CRC cell proliferation and reduced cell apoptosis, while promoting cell cycle entry; AG490 treatment reversed these results. CONCLUSIONS: Altogether, we conclude that up-regulation of RP11-468E2.5 inhibits the JAK/STAT signaling pathway by targeting STAT5 and STAT6, thereby suppressing cell proliferation and promoting cell apoptosis in CRC.

Phospho-STAT5 expression is associated with poor prognosis of human colonic adenocarcinoma.

The signal transducer and activator of transcription-5 (STAT5) protein has been shown to play an important role in tumor progression through stimulating cell proliferation and preventing apoptosis. STAT5 activation has been observed in a variety of human tumors and cancer cell lines. However, it is not clear how activated STAT5 is expressed in colon cancer. In this study, we aimed to investigate phospho-STAT5 (activated form of STAT5) expression and its relationship with the clinicopathological factors and overall survival of patients with colonic adenocarcinoma. A total of 121 histological samples were selected for this study. Immunohistochemistry was used to detect the expression of phospho-STAT5. Analysis of the immunohistochemical staining was based on the proportion of stained cells in the field: positive, >15% stained cells, and negative, <15% stained cells. Survival times were analyzed using the Kaplan-Meier method, and the differences between groups were assessed with the log-rank test. A multivariate Cox regression model was used for prognostic power analysis. Expression of phospho-STAT5 was observed in the cytoplasms of colonic adenocarcinoma cells. Univariate analysis showed that phospho-STAT5 immunoreactivity was correlated with the depth of tumor invasion (P-value = 0.009), tumor-node-metastasis (TNM) stage (P-value = 0.048) and shorter overall survival times (P-value = 0.026). Lymph node metastasis, distant metastasis and TNM stage were associated with shorter overall survival times (P-value range from 0.003- < 0.001). Multivariate analysis showed that only distant metastasis was an independent predictor of overall survival time (P-value = 0.016). Our findings first demonstrate that phospho-STAT5 is frequently present and active in colonic adenocarcinoma and related to poor prognosis.

Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin.

To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombined and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.