ERCC6L That Is Up-regulated In High Grade Of Renal Cell Carcinoma Enhances Cell Viability In Vitro And Promotes Tumor Growth In Vivo Potentially Through Modulating MAPK Signalling Pathway

Objective: Renal cell carcinoma(RCC),which is one of the most diagnosed urological malignancies worldwide,is usually associated with abnormality in both genetic and cellular processes.In the present study,through analyzing The Cancer Genome Atlas(TCGA)dataset,we screened out ERCC6 L as a candidate gene that is potentially related to the development of RCC based on its increased expression in cc RCC tissues compared with normal kidney tissues as well as its possible relevance to cancer prognosis.Methods: By using RT-PCR,it was confirmed that the m RNA expression of ERCC6 L was upregulated in RCC tissues as compared to normal controls in 28 pared samples.In addition,the immunohistochemistry study in a tissue microarray(TMA)containing 150 cc RCC samples showed that the staining score of ERCC6 L was positively correlated with the Fuhrman grade of cancers.Next,when the expression of ERCC6 L was lowered by specific sh RNA,the cell viability was significantly inhibited in 786-O and Caki-1 cells,while the apoptosis was induced accordingly.At the same time,RCC cells those were transfected with sh RNA targeting to ERCC6 L grew significantly slower than parental cells in immunodeficient mice.Results: To screen for genes that are abnormally expressed in RCC,we examined the RNA-seq data in TCGA database.The TCGA database contained 530 cc RCC cases in total.After simple cluster analysis,we found that the m RNA expression profile of 69 pairs of samples exhibited good consistency with the other samples in their group(Tumor and Paratumor).In comparison to the normal tissues,the m RNA expression of numerous genes was apparently upregulated in the tumor samples,including the ERCC6 L gene.After searching for published articles,gene annotation and uncharted gene function,we observed that ERCC6 L plays an important role in cell mitosis.However,few studies investigated its function in diseases.Survival analysis demonstrated that ERCC6 L was significantly associated with clinical prognosis.To validate the expression of ERCC6 L in RCC,RNA derived from 28 pairs of cancer–normal matched tissue samples were subjected to a real-time PCR assay.Consistent to the results in the above TCGA dataset,the m RNA expression of ERCC6 L in our sample set was significantly increased in RCC compared to normal controls.To further evaluate the clinical significance of ERCC6 L,a TMA containing 150 RCC samples was used to detect ERCC6 L protein expression.As expected,ERCC6 L protein was detected at a stable level in most tumor samples,although at different levels.To investigate the role of ERCC6 L in cancer cells,a Celigo analysis and MTT assay were performed and revealed that cells transfected with lentivirus carrying sh-ERCC6 L were less viable than cells treated with sh-Ctrl.Fluorescence-activated cell sorting and Annexin V-allophycocyanin(APC)showed that depletion of ERCC6 L m RNA inhibits cell viability partially through the induction of apoptosis in RCC cells.To further test the effect of ERCC6 L on tumor growth in vivo,we established a xenograft model using sh-ERCC6 L and sh-Ctrl transfected 786-O cells in nude mice.Continuous measurement of the tumor volume revealed significantly slower tumor growth in the sh-ERCC6 L group than the control group.To explore the potential mechanism of ERCC6 L on RCC cells,an m RNA microarray analysis was performed in 786-O cells with or without ERCC6 L knockdown with sh RNA.We identified a total of 327 upregulated and 445 downregulated genes following ERCC6 L silencing.Subsequently,the canonical pathway analysis using IPA revealed that the downregulation of ERCC6 L potentially suppressed some fundamental biological signaling axis,such as G1/S checkpoint regulation signaling,calcium signaling and extracellular signal-regulated kinase(ERK)/MAPK signaling(all P values < 0.05).Notably,co-expression analysis demonstrated that the transcriptional level of ERCC6 L negatively correlated with dual specificity phosphatase 1(DUSP1).Conclusion: The present study provides preliminary evidence of the promoting effect of ERCC6 L on the cell viability of RCC in vitro and in vivo.ERCC6 L protein was detected at a stable level in most tumor samples,although at different levels.The ERCC6 L expression was associated tumor grades.Further studies of the mechanisms underlying the regulation of ERCC6 L may provide novel therapeutic targets in RCC.