LINC00839调节miR-17-5p/WEE1轴对结直肠癌细胞增殖、凋亡和迁移的影响

Effects of LINC00839 on proliferation,apoptosis,and migration of colorectal cancer cells by regulating the miR-17-5p/WEE1 axis

目的探究长链非编码RNA LINC00839(LINC00839)调节微小RNA-17-5p(miR-17-5p)/丝氨酸/苏氨酸蛋白激酶1(WEE1)轴对结直肠癌(CRC)细胞增殖、凋亡和迁移的影响。方法检测CRC细胞系中LINC00839、miR-17-5p、WEE1 mRNA表达水平,筛选最佳细胞系,将LS513细胞分为对照组(Control组)、LINC00839敲低阴性对照组(si-NC组)、LINC00839敲低组(si-LINC00839组)、LINC00839敲低+miR-17-5p抑制表达阴性对照组(si-LINC00839+NC inhibitor组)、LINC00839敲低+miR-17-5p抑制表达组(si-LINC00839+miR-17-5p inhibitor组)。qRT-PCR检测细胞中LINC00839、miR-17-5p和WEE1 mRNA表达水平;双荧光素酶报告基因实验验证LINC00839、miR-17-5p和WEE1之间的靶向关系;MTT检测细胞活力;Edu检测细胞增殖;划痕实验检测细胞的迁移能力;流式细胞仪检测细胞凋亡;Western blot检测WEE1、PCNA、Bax、Bcl-2、caspase-3、E-cadherin、N-cadherin蛋白水平。结果在CRC细胞系中LINC00839、WEE1 mRNA高表达,miR-17-5p低表达,选择LS513细胞进行实验。双荧光素酶报告基因实验证实miR-17-5p与LINC00839、WEE1存在靶向关系,LINC00839敲低可上调miR-17-5p表达,下调WEE1表达。敲低LINC00839能显著降低LS513细胞活力、Edu阳性率、划痕愈合率及PCNA、Bcl-2、N-cadherin和WEE1蛋白表达(P<0.05),显著增加Bax、caspase-3、E-cadherin蛋白表达(P<0.05)。抑制miR-17-5p的表达能逆转LINC00839敲低对LS513细胞恶性生物学行为的抑制作用(P<0.05)。结论LINC00839在CRC细胞中上调,敲低LINC00839可能通过调控miR-17-5p/WEE1轴抑制CRC细胞增殖和迁移,促进细胞凋亡。

Objective To investigate the effects of long non-coding RNA LINC00839 on the proliferation,apoptosis,and migration of colorectal cancer(CRC)cells by regulating the microRNA-17-5p(miR-17-5p)/serine/threonine protein kinase 1(WEE1)axis.Methods The expression levels of LINC00839,miR-17-5p,and WEE1 mRNA in CRC cell lines were detected to screen the optimal cell line.LS513 cells were separated into Control group,LINC00839 knockdown negative control group(si-NC group),LINC00839 knockdown group(si-LINC00839 group),LINC00839 knockdown+miR-17-5p inhibition negative control group(si-LINC00839+NC inhibitor group),and LINC00839 knockdown+miR-17-5p inhibitor expression group(si-LINC00839+miR-17-5p inhibitor group).qRT-PCR was applied to detect the expression levels of LINC00839,miR-17-5p,and WEE1 mRNA in cells.Dual luciferase reporter gene experiment was applied to verify the targeting relationship between LINC00839,miR-17-5p,and WEE1.MTT was applied to detect cell viability.Edu was applied to detect cell proliferation.Scratch experiment was applied to detect the migration ability of cells.Flow cytometry was applied to detect cell apoptosis.Western blot was applied to detect protein levels of WEE1,PCNA,Bax,Bcl-2,caspase-3,E-cadherin,and N-cadherin.Results In the CRC cell line,LINC00839 and WEE1 mRNA were highly expressed,while miR-17-5p was low expressed.LS513 cells were selected for the experiment.Dual luciferase reporter gene experiment confirmed a targeted relationship between miR-17-5p and LINC00839,WEE1.Knocking down LINC00839 up-regulated miR-17-5p expression and down-regulated WEE1 expression.Knocking down LINC00839 greatly reduced LS513 cell viability,Edu positive rate,scratch healing rate,and expression of PCNA,Bcl-2,N-cadherin,and WEE1 proteins(P<0.05),while greatly increased the expression of Bax,caspase-3,and E-cadherin proteins(P<0.05).Inhibiting the expression of miR-17-5p was able to reverse the inhibitory effect of LINC00839 knockdown on the malignant biological behavior of LS513 cells(P<0.05).Conclusion LINC00839 is up-regulated in CRC cells,and knocking down LINC00839 may inhibit CRC cell proliferation and migration by regulating the miR-17-5p/WEE1 axis,promoting cell apoptosis.