circSHKBP1通过靶向miR-125a-5p调控结肠癌细胞增殖和凋亡

CircSHKBP1 regulates colon cancer cell proliferation and apoptosis by targeting miR-125a-5p

背景 结肠癌是临床上常见的一种恶性肿瘤,有研究报道,环状RNA(circular RNA,circR NA)参与结肠癌的发生发展.其中,circSHKBP1作为癌基因促进癌症进展,因此我们假设circSHKBP1也参与结肠癌的发展.目的 探讨circSHKBP1对结肠癌细胞增殖及凋亡的影响及其可能作用机制.方法 选取本院2020-03/2020-07的69例结肠癌组织及癌旁组织标本,qRT-PCR检测circSHKBP1、miR-125a-5p的表达量;体外培养人结肠癌细胞HT29,随机分组:sh-NC组、sh-circSHKBP1组、miR-NC组、miR-125a-5p组、sh-circSHKBP1+anti-miR-NC组、shcircSHKBP1+anti-miR-125a-5p组;采用MTT法、平板克隆形成、和流式细胞术检测细胞增殖、克隆形成及凋亡;采用双荧光素酶报告验证miR-125a-5p过表达对野生型(wild-type,WT)载体wt-circSHKBP1的荧光素酶活性;采用Western blot检测b细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2),Bcl-2相关X蛋白(BCL-2-associated X protein,Bax)蛋白表达.结果 在结肠癌组织中circSHKBP1的表达上调(P<0.05),miR-125a-5p的表达下降(P <0.05);相对sh-NC组,沉默circSHKBP1显著降低结肠癌细胞的增殖率、提高凋亡率和Bax的表达,降低了细胞克隆形成数,减少Bcl-2的表达(P<0.05);miR-125a-5p过表达可降低wt-circSHKBP1的荧光素酶活性(P <0.05);与miR-NC组对照,miR-125a-5p组降低增殖率、提高凋亡率和Bax的表达,减少细胞克隆形成数,降低Bcl-2的表达(P<0.05);相对sh-circSHKBP1+anti-miR-NC组,shcircSHKBP1+anti-miR-125a-5p组提高了肿瘤细胞增殖率、降低了凋亡率和Bax的表达,增加了细胞克隆形成数,提高了Bcl-2的表达(P<0.05).结论 敲低circSHKBP1可通过靶向miR-125a-5p表达抑制结肠癌细胞增殖并促进细胞凋亡.

BACKGROUND Colon cancer is a common malignant tumor in clinical practice.It has been reported that circular RNAs(circRNAs)are involved in the occurrence and development of cancer.Among them,circSHKBP1 acts as an oncogene to promote cancer progression.Thus,we hypothesized that circSHKBP1might be also implicated in the development of colon cancer.AIM To explore the role of circSHKBP1 in colon cancer cell proliferation and apoptosis and the possible mechanism involved.METHODS Sixty-nine cancer tissues and matched adjacent normal tissues were selected from March 2020 to July 2020 at our hospital.The expression of circSHKBP1 and miR-125a-5p was detected by qRT-PCR.Human colon cancer cells(HT29) cultured in vitro were randomly divided into sh-NC group,sh-circS HKBP1 group,miR-NC group,miR-125a-5p group,sh-circS HKBP1 + anti-miR-NC group,and sh-circSHKBP1 + anti-miR-125a-5p group.MTT assay,colony formation experiment,and flow cytometry were used to detect cell proliferation,colony formation,and apoptosis,respectively.The dual luciferase reporter experiment was used to detect the impact of miR-125a-5p overexpression on the luciferase activity of the wild-type vector wt-circSHKBP1.Western blot was used to detect the protein expression of Bax and Bcl-2.RESULTS Compared with adjacent tissues,the expression of circS HKBP1 in colon cancer tissues was increased(P 0.05),while the expression of miR-125a-5p was decreased(P 0.05).Cell proliferation inhibition rate,apoptosis rate,and Bax protein level in the sh-circSHKBP1 group were increased(P 0.05),while the number of cell colonies(P 0.05) and Bcl-2 protein level were decreased(P 0.05).Overexpression of miR-125a-5p could reduce the luciferase activity of wtcircS HKBP1(P 0.05).Relative to the miR-NC group,miR-125a-5p reduced cell proliferation,increased apoptosis rate and Bax protein level(P 0.05),decreased the number of cell colonies(P 0.05),and reduced Bcl-2 protein level(P 0.05).Compared with the sh-circSHKBP1 + anti-miR-NC group,cell proliferation inhibition rate,apoptosis,and the protein level of Bax in the sh-circSHKBP1 + anti-miR-125a-5p group were decreased(P 0.05),while the number of cell colonies and Bcl-2 protein level were increased(P 0.05).CONCLUSION Knockdown of circSHKBP1 could inhibit colon cancer cell proliferation and promote apoptosis via up-regulating miR-125a-5p expression.