Expressions of long non-coding RNA CASC11 and proto-oncogene c-myc in colorectal cancer and their correlation with recurrence and metastasis / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the expressions of long non-coding RNA (lncRNA) CASC11 and proto-oncogene c-myc in colorectal cancer and their correlation with recurrence and metastasis.Methods:The cancer tissues and paracancerous tissues of 90 colorectal cancer patients in Tangshan Union Medical College Hospital of Hebei Province from February 2016 to July 2018 were collected. Normal colon epithelial cell lines CCD841 and colorectal cancer cell lines SW480, SW620, HCT116, HT29, DLD-1 were cultured in vitro. Separated by the average value of relative expression level of CASC11 or c-myc mRNA in cancer tissues, patients were divided into the high expression and low expression of CASC11 or c-myc. The protein expressions of CASC11 and c-myc mRNA and c-myc were detected by using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot. Cell lines with the highest protein expressions of CASC11, c-myc mRNA and c-myc were used to do the subsequent experiments. The association of the expression levels of CASC11 and c-myc mRNA with the clinicopathological features was analyzed. Pearson correlation test was used to analyze the relationship between CASC11 and c-myc mRNA of cancer tissues. JASPAR software was used to analyze whether there were binding sites of CASC11 and c-myc gene. The wild-type and mutant-type CASC11 recombinant plasmids were constructed, and the relationship between c-myc and CASC11 was confirmed by using dual luciferase reporter gene assay. Cell lines with the highest expressions of CASC11 and c-myc were transfected with c-myc interference sequence plasmid (Sh-c-myc group) or the negative control sequence plasmid (Sh-NC group), and the conventional cultured blank control group (NC group). The proliferation of cells was detected by using methyl thiazolyl tetrazolium (MTT) method, the invasion and migration abilities of cells were detected by using Tanswell test, and the protein expressions of CASC11, c-myc mRNA and c-myc in cells of all groups were detected by using qRT-PCR and Western blot.Results:The protein expression levels of CASC11, c-myc mRNA and c-myc protein in cancer tissues were increased compared with those in paracancerous tissues, and the differences were statistically significant (all P < 0.05). Compared with CCD841 cells, the protein expression levels of CASC11, c-myc mRNA and c-myc in all colorectal cancer cell lines were increased, and the differences were statistically significant (all P < 0.05); the highest protein expressions of CASC11, c-myc mRNA and c-myc were found in SW480 cell lines which were used to do the subsequent experiments. Pearson correlation analysis showed that there was a positive correlation between CASC11 mRNA and c-myc mRNA in cancer tissues ( r = 0.494, P < 0.05). The high expression rate of CASC11 and c-myc mRNA in cancer tissues for patients with lymph node metastasis was higher than that for those without lymph node metastasis [73.7% (28/38) vs. 26.9% (14/52), 84.2% (32/38) vs. 23.1% (12/52)], the high expression rate of CASC11 and c-myc mRNA in cancer tissues for patients with TNM stage Ⅲ-Ⅳ was higher than that for those with TNM stage Ⅰ-Ⅱ [76.0% (38/50) vs. 10.0% (4/40), 72.0% (36/50) vs. 20.0% (8/40)], and the differences were statistically significant (all P < 0.05). JASPAR software showed that the binding sites were detected in CASC11 promoter region and c-myc gene; dual luciferase reporter gene assay results showed that the relative activity of SW480 cells co-transfected with Sh-c-myc and wild-type CASC11 plasmid was lower compared with that of SW480 cells co-transfected with Sh-NC and wild-type CASC11 plasmid ( P < 0.05). Compared with NC group and Sh-NC group, the expression level of CASC11 mRNA, the number of invasive and migratory cells of SW480 cells in Sh-c-myc group were decreased, while cell proliferation inhibition rate was increased, and the differences were statistically significant (all P < 0.05). Conclusions:CASC11 and c-myc mRNA are highly expressed in colorectal cancer tissues and cell lines, and binding sites can be detected in CASC11 promoter region and c-myc gene. The expressions of both have a correlation, and the down regulation of c-myc can inhibit the invasion and migration of colorectal cancer cells by inhibiting the expression of CASC11.