[目的]探讨HPV18致癌基因E6选择性剪接在宫颈癌细胞中的潜在作用。[方法]HPV18感染或过表达HPV18致癌基因E6后，MTT法检测宫颈癌细胞C-33 A的增殖水平、逆转录PCR和琼脂糖凝胶电泳检测E6选择性剪接产物表达水平、免疫印迹检测E7蛋白表达水平。高通量测序HPV18感染或不感染的C-33 A细胞后调控E6选择性剪接的关键分子。[结果]过表达E6并感染HPV18的C-33 A细胞的增殖水平(2。35±0。37vs1。27±0。28)显著上升，且高于未过表达E6(1。27±0。28vs0。68±0。11)(P＜0。05)。HPV18感染和过表达SRSF3后，选择性剪接产物E6*Ⅰ水平上升(0。25±0。03 vs 0。65±0。13)、E7蛋白表达水平(0。20±0。04 vs 0。77±0。18)上升。敲低SRSF3和过表达miR-1208 时，C-33 A 细胞的增殖水平(1。30±0。18 vs 0。65±0。13，1。75±0。27 vs 0。75±0。13)显著下降(P＜0。05)。与只过表达E6相比，E6与SRSF3共表达、E6过表达同时干扰miR-1208均能够显著促进C-33 A细胞的增殖水平(0。65±0。13 vs 1。65±0。40，0。59±0。11 vs 1。70±0。24)(P＜0。05)。敲低 SRSF3 后，选择性剪接产物 E6*Ⅰ 水平下降(0。15±0。02 vs 0。57±0。15)、E7 蛋白表达水平下降(0。20±0。04 vs 0。77±0。18)(P＜0。05)。干扰 miR-1208 后，选择性剪接产物 E6*Ⅰ 水平上升(1。12±0。25 vs 2。56±0。33)、SRSF3(0。15±0。03 vs 0。75±0。15)和 E7 蛋白表达水平上升(0。65±0。11vs0。98±0。20);过表达miR-1208后，选择性剪接产物E6*Ⅰ水平下降(1。85±0。34 vs 1。15± 0。20)、SRSF3(1。33±0。14vs0。20±0。05)和 E7 蛋白表达水平下降(0。88±0。13 vs 0。50±0。10)(P＜0。05)。此外，miR-1208靶向SRSF3 mRNA的3'端非编码区。[结论]miR-1208靶向SRSF3 mRNA的3'端非编码区并减少SRSF3的蛋白表达水平。SRSF3能够促进HPV18致癌基因E6的选择性剪接和E7蛋白的表达。HPV18感染后操纵miR-1208/SRSF3/E6/E7调控轴促进宫颈癌细胞的增殖。
Role of HPV18 oncogene E6 alternative splicing in cervical cancer cells
[Objective]To explore the potential role of HPV18 oncogene E6 alternative splicing in cervical cancer cells.[Meth-od]After HPV18 infected or overexpressed HPV18 oncogene E6,MTT method was used to detect the proliferation level of cervi-cal cancer cell C-33A,reverse transcription PCR and agarose gel electrophoresis were used to detect the expression level of E6 alternative splicing products,and Western Blotting was used to detect the expression level of E7 protein.High throughput se-quencing of key molecules regulating E6 alternative splicing after HPV 18 infected or uninfected C-33A cells.[Result]The proliferation level of C-33A cells overexpressing E6 and infected with HPV18(2.35±0.37 vs 1.27±0.28)was significantly higher than that of cells overexpressing E6(1.27±0.28 vs 0.68±0.11)(P＜0.05).After HPV18 infection and overexpres-sion of SRSF3,the level of alternative splicing product E6*Ⅰ increased(0.25±0.03 vs0.65±0.13),and the level of E7 protein expression increased(0.20±0.04 vs 0.77±0.18).When SRSF3 was knocked down and miR-1208 was overex-pressed,the proliferation level of C-33A cells(1.30±0.18 vs 0.65±0.13,1.75±0.27 vs 0.75±0.13)decreased signifi-cantly(P＜0.05).Compared with over expression of E6,co expression of E6 and SRSF3,and over expression of E6 simultane-ously interfering with miR-1208 could significantly promote the proliferation of C-33A cells(0.65±0.13 vs 1.65±0.40,0.59±0.11 vs 1.70±0.24)(P＜0.05).After knockdown of SRSF3,the level of alternative splicing product E6*Ⅰ de-creased(0.15±0.02 vs 0.57±0.15),and the level of E7 protein expression decreased(0.20±0.04 vs 0.77±0.18)(P＜0.05).After interfering with miR-1208,the level of alternative splicing product E6*Ⅰ increased(1.12±0.25 vs 2.56± 0.33),SRSF3(0.15±0.03vs0.75±0.15)and E7 protein expression increased(0.65±0.11 us 0.98±0.20);After overex-pression of miR-1208,the levels of alternative splicing product E6*Ⅰ decreased(1.85±0.34 vs 1.15±0.20),SRSF3(1.33±0.14 vs 0.20±0.05)and E7 protein decreased(0.88±0.13 vs 0.50±0.10)(P＜0.05).[Conclusion]miR-1208 targets the 3'non-coding region of SRSF3 mRNA and reduces the protein expression level of SRSF3.SRSF3 can promote the alternative splicing of HPV18 oncogene E6 and the expression of E7 protein.Manipulating the miR-1208/SRSF3/E6/E7 reg-ulatory axis to promote the proliferation of cervical cancer cells after HPV18 infection.
HPV18oncogene E6alternative splicingcervical cancer cellsmiR-1208SRSF3
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