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Archive > Volume 30 Issue 9 > 2023,30(9):762-770. DOI:10.3872/j.issn.1007-385X.2023.09.003 Prev Next
Basic Research
LINC00462 regulates clear renal cell carcinoma cells sensitivity to cisplatin by affecting their glycolysis via the MYC/ABCC3 axis
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Abstract:
Objective：：To investigate the effect of LINC00462 binding to transcription factor MYC to activate ABCC3 on cisplatin sensitivity in clear cell renal cell carcinoma (ccRCC) and its potential mechanisms. Methods: The expression of ABCC3, MYC and LINC00462 and their correlation in ccRCC were analyzed, and the enrichment pathway of ABCC3 was also analyzed. Human renal tubular epithelial cells (HK-2) and ccRCC cells (A-498, 786-O and Caki-2) were conventionally cultured. Nucleic acid sequences of si-LINC00462,si-LINC00462-NC, oe-ABCC3, oe-ABCC3-NC, si-ABCC3, si-ABCC3-NC, si-MYC and si-MYC-NC were transfected into A-498 or 786-O cells respectively, namely si-LINC00462 group, si-LINC00462-NC group, oe-ABCC3 group, oe-ABCC3-NC group, si-ABCC3 group,si-ABCC3-NC group, si-MYC group and si-MYC-NC group. 2-deoxy-D-glucose (2-DG) was used in the rescue experiment, and oe-NC+PBS group, oe-ABCC3+PBS group and oe-ABCC3+2-DG group were established. To investigate the effects of LINC00462/MYC/ABCC3 axis on the cisplatin sensitivity of ccRCC cells, si-NC+oe-NC group, si-LINC00462+oe-NC gorup, and si-LINC00462+oe-ABCC3 group were constructed. The expression of ABCC3, MYC and LINC00462 in ccRCC cells was detected by qPCR. CCK-8 method was used to measure cell viability and the IC50 of cisplatin inhibiting the proliferation of ccRCC cells. WB was used to detect the expression of glycolytic metabolism-related proteins. Seahorse XP96 was used to analyze the extracellular acidification rate (ECAR) and oxygen consumption rate(OCR) in the cells of different treatment groups. The levels of pyruvate, lactic acid and ATP in the cells were detected by corresponding kits.The binding relationship between ABCC3 and MYC was verified by double luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay, and the binding relationship between LINC00462 and MYC was verified using RNA-binding protein immunoprecipitation (RIP) experiment. Results: Database analysis and qPCR experiments showed that ABCC3 was highly expressed in ccRCC tissues and cells and was enriched in the glycolytic pathway. Knockdown of ABCC3 could promoted the sensitivity of A-498 cells to cisplatin, while overexpression of ABCC3 could reduce the sensitivity of 786-O cells to cisplatin. ABCC3 could inhibit the sensitivity of A-498 cells to cisplatin through promoting glycolysis. 2-DG treatment reversed the inhibitory effect of ABCC3 overexpression on cisplatin sensitivity of ccRCC cells. MYC could directly bind to ABCC3, and LINC00462 could recruit MYC. Knockdown of LINC00462 inhibited the expression of ABCC3, suppressed aerobic glycolysis of ccRCC cells and increased their sensitivity to cisplatin; however, further over-expression of ABCC3 reversed the effects of LINC00462 knockdown on inhibiting aerobic glycolysis and promoting cisplatin sensitivity in ccRCC cells. Conclusion：： LINC00462 promotes glycolysis of ccRCC cells and further inhibits their sensitivity to cisplatin by recruiting the transcription factor MYC to activate ABCC3 expression.
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