The Role Of CASC2 In Renal Cell Carcinoma And Its Regulatory Mechanism

Renal cell carcinoma(RCC) is one of the most popular malignant tumor in urinary system, and its incidence accounts for 3% in all malignant tumor. Clear cell renal cell carcinoma(cc RCC) and papillary renal cell carcinoma are the most common types of RCC. Their proportion in RCC are 60%-85% and 7%-14%, respectively. RCC originates in the renal tubular epithelial cells. 30% patients of localized renal cell carcinoma suffer from the postoperative local recurrence or distant metastasis, which are the major causes of death among RCC patients, but their mechanisms are still unclear.Cancer susceptibility candidate 2(CASC2), a lnc RNA whose gene is located at chromosome 10q26, was originally identified as a downregulated gene in endometrial cancer and acted as a tumor suppressor gene as well. Previous studies have demonstrated that exogenous expression of CASC2 significantly inhibits the growth of undifferentiated endometrial cancer cells and suppress glioma cell metastasis. However, little is known currently regarding the expression and function of CASC2 in RCC. Micro RNAs(miRNA) are non-coding RNA molecules, which have important regulatory roles in various biological processes, including proliferation, migration, invasion, apoptosis and cell cycle distribution. Studies have shown that lnc RNAs are new miRNAs targets. This study, using RCC tissue specimens and in vitro test, detected the expression level of CASC2 in RCC tissues and paired normal tissues and human RCC cell lines and confirmed that CASC2 was downregulated in human RCC tissues and human RCC cell lines and acted as a tumor suppressor gene as well. The miR-21 was able to decrease the expression of CASC2 in human RCC cell lines. Furthermore, overexpression of miR-21 partly abrogated CASC2-mediated inhibition of RCC cell proliferation and migration. Therefore, CASC2 may be considered as a novel target for the treatment of RCC and a marker of prognosis.Part?The clinical significance of CASC2 expression in renal cell carcinomaObjective: To investigate the expression of CASC2 in cc RCC and paired adjacent normal tissues, to investigate the expression of CASC2 expression in RCC lines, and to explore whether CASC2 plays an important role in RCC.Methods: CASC2 expression was measured in 32 pairs of cc RCC tissues and adjacent normal tissues by Real-time Quantitative PCR( q PCR). Then the relationship among CASC2 expression, pathological staging and prognosis of patients were analyzed. CASC2 expression was measured in three cell lines of 786-O, A498 and HEK 293 by q PCR, then results were compared and analyzed based on the experiments.Results: q PCR results showed that: CASC2 expressions in RCC tissues and adjacent non-tumor tissues were different. CASC2 expressions in RCC were obviously lower than the result of the adjacent non-tumor tissues. The difference is statistically significant. Analysis of the relationship between CASC2 expression and pathological staging indicated that: Low levels of CASC2 expressions were observed in 44.8%(13/29) patients with low primary tumour stages(p T1+p T2) and 66.7%(2/3) patients with high primary tumour stages(p T3+p T4). Kaplan-Meier analysis revealed that lower levels of CASC2 expression in cc RCC tissues were significantly associated with decreased overall survival. CASC2 expression in cell lines 786-O, A498 were statistically significantly lower than HEK 293 cell lines.Conclusion: CASC2 expressions in RCC tissues and in RCC cell lines are decreased obviously, which indicates that CASC2 plays the role of tumor suppressor genes in RCC. CASC2 expressions in RCC tissues are associated with pathological staging, lower levels of CASC2 expression in RCC tissues predicting poor prognosis.Part? Overexpression of CASC2 decreases the proliferation and migration of renal cell carcinoma cellsObjective: To explore the influence of overexpression of CASC2 in RCC cells, and to verify the function of CASC2 in RCC.Methods: Overexpression vector pc DNA3.1(+)-CASC2 was constructed by pc DNA3.1, then up-regulation of CASC2 expression levels were induced by pc DNA3.1(+)-CASC2 in RCC cell lines(786-O and A498), MTT and wound scratch assays were used to observe the change of cell proliferation and migration.Results: After post-transfection with the pc DNA3.1(+)-CASC2 overexpression vector, the optical density(OD) of 786-O cells was analyzed and indicated that relative cell proliferation was significantly decreased, and overexpression of CASC2 markedly inhibited the migration of 786-O and A498 cells.Conclusion: Overexpression of CASC2 inhibits the proliferation and migration of RCC cells, and CASC2 plays the role of tumor suppressor gene in RCC.Part? miR-21 can specifically bind itself to CASC2 and regulate CASC2's functionObjective: To search for effective targeted relative miRNAs through bioinformatics analysis for CASC2, and then to confirm its specific binding ability with CASC2 and its regulating function.Methods: The potential miR-21 binding sites of CASC2 predicted by computer-aided algorithms were obtained from miRanda(http://www.microrna.org) and miRcode(http://www.mircode.org). Dual-luciferase reporter assays was to confirm whether CASC2 was a direct target gene of miR-21, and then to confirm whether miR-21 can interfere CASC2-mediated inhibition of 786-O and A498 cell proliferation and migration.Results: Bioinformatics analysis and dual-luciferase reporter assays confirm that CASC2 is a direct target gene of miR-21. The miR-21 mimic is able to decrease the expression of CASC2 in 786-O and A498 cells. The relative luciferase activity was significantly inhibited by miR-21 in the CASC2-WT vector-transfected cells, whereas miR-21 exhibited no inhibitory effect in cells transfected with the CASC2-MUT vector. Overexpression of miR-21 partly abrogated CASC2-mediated inhibition of 786-O and A498 cell proliferation and migration.Conclusion: CASC2 containing miR-21 binding sites, is a direct target gene of miR-21.Overexpression of miR-21 partly abrogated CASC2-mediated inhibition of RCC cell proliferation and migration. CASC2 maybe has other molecular pathways to suppress tumor.