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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (2): 210-220. doi: 10.19723/j.issn.1671-167X.2019.02.003

• 论著 • 上一篇    下一篇

肿瘤转移抑制基因LASS2/TMSG1 S248A突变体通过增加ATP6V0C表达促进前列腺癌的侵袭

张宽根1,周雨禾1,邵雅昆1,梅放1,由江峰1,刘北英2,裴斐1,3,∆()   

  1. 1. 北京大学基础医学院病理学系, 北京 100191
    2. 北京科技大学机械学院, 北京 100083
    3. 北京大学第三医院病理科, 北京 100191
  • 收稿日期:2018-08-20 出版日期:2019-04-18 发布日期:2019-04-26
  • 通讯作者: 裴斐 E-mail:peifei@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81572533);北京市自然科学基金(7182078)

Novel tumor metastasis suppressorgene LASS2/TMSG1 S248A mutant promotes invasion of prostate cancer cells through increasing ATP6V0C expression

Kuan-gen ZHANG1,Yu-he ZHOU1,Ya-kun SHAO1,Fang MEI1,Jiang-feng YOU1,Bei-ying LIU2,Fei PEI1,3,∆()   

  1. 1. Department of Pathology, Peking University School of Basic Medical Sciences, Beijing 100191China
    2. School of Mechanical Engineering, University of Science & Technology Beijing, Beijing 100083 China;
    3. Department of Pathology, Peking University Third Hospital, Beijing 100191,China
  • Received:2018-08-20 Online:2019-04-18 Published:2019-04-26
  • Contact: Fei PEI E-mail:peifei@bjmu.edu.cn
  • Supported by:
    the National Sciences Foundation of China(81572533);the National Sciences Foundation of Beijing(7182078)

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摘要：

目的: 探讨LASS2/TMSG1及其突变体在前列腺癌细胞增殖、迁移和侵袭中的分子作用机制。方法: 构建表达LASS2/TMSG1全长及其4个突变体的pcDNA3真核表达载体,并稳定转染到高转移潜能前列腺癌PC-3M-1E8细胞系中;采用qPCR和Western blot法鉴定稳定转染效果,并分析不同点突变体对LASS2/TMSG1和ATP6V0C表达量的影响;采用生长曲线测定、四甲基偶氮唑蓝(MTT)掺入实验、软琼脂集落形成实验、细胞划痕修复实验、Matrigel穿膜侵袭实验和流式细胞术研究LASS2/TMSG1及其4个点突变体的细胞生物学功能,并通过免疫双重荧光染色分析LASS2/TMSG1不同突变体和ATP6V0C的相互作用情况。结果: qPCR和Western blot检测显示LASS2/TMSG1 S248A组较LASS2/TMSG1野生型组ATP6V0C表达增加了3倍(P<0.05), 且免疫双重荧光染色结果显示LASS2/TMSG1 S248A组ATP6V0C表达明显增加;与LASS2/TMSG1野生型组相比,LASS2/TMSG1 S248A组的细胞增殖能力、锚着不依赖生长能力、细胞迁移能力 (细胞迁移率从35.3%±3.2%增加到70.3%±3%)和侵袭能力(穿膜细胞数从50.0±3.2增加到203.0±6.5)明显提高(P<0.05), G0/G1期比例增加(从51.0%增加到85.4%,P<0.05),但细胞凋亡率亦明显升高(从7%增加到15.1%,P<0.05)。结论: LASS2 /TMSG1第248位丝氨酸突变为丙氨酸后(S248A)能促进前列腺癌细胞增殖、迁移和侵袭能力,其分子机制可能是LASS2/TMSG S248A使ATP6V0C表达量增加,进而促进前列腺癌的侵袭,提示LASS2/TMSG1蛋白第248位丝氨酸是抑制前列腺癌侵袭的重要功能位点。

关键词: 前列腺癌, 点突变, 液泡型ATP酶, ATP6V0C, LASS2/TMSG1

Abstract:

Objective: LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by Department of Pathology,Peking University of Basic Medical Sciences. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of vacuolar-ATPase (ATP6V0C). In this study, we explored the effect of LASS2/TMSG1 and its mutants on proliferation, migration and invasion of human prostate cancer cells and its molecular mechanism.Methods: We constructed four LASS2/TMSG1 mutants and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The stable transfectants were identified by qPCR and Western blot through analyzing the expression of LASS2/TMSG1 and ATP6V0C, the cell biology functions of LASS2/TMSG1 and its four mutants were studied using growth curve,MTT assay, soft agar colony formation assay, wound migration assay, Matrigel invasion study and flow cytometry. Furthermore, immunofluorescence was used to analysis the interaction of LASS2/ TMSG1 mutants and ATP6V0C.Results: LASS2/TMSG1 mRNA and protein in LASS2/TMSG1 group and Mut1-Mut4 groups were higher than that in Vector group; Western blot showed that ATP6V0C protein in LASS2/TMSG1 wild group was lower than that in Vector group, but ATP6V0C protein in LASS2/TMSG1 S248A group was obviously higher than that in Vector group. MTT test and growth curve assay showed growth ability in LASS2/TMSG1 S248A group was increasing compared with other groups from day 5. Soft Agar colony formation experiment showed anchor independent growth ability in LASS2/TMSG1 S248A group was higher than those in the other groups (P<0.05), Cell migrations (from 35.3%±3.2% to 70.3%±3%) in LASS2/TMSG1 S248A group was increasing compared with LASS2/TMSG1 wild group (P<0.01), and more cells passed through Matrigel in LASS2/TMSG1 S248A group compared with LASS2/TMSG1 wild group (from 50±3.2 to 203±6.5, P<0.01), the apoptosis rate in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 7% to 15.1%, P<0.05), and the G0/G1 ratio in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 51.0% to 85.4%). Furthermore, double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, the expression of ATP6V0C in LASS2/TMSG1 S248A group increased significantly compared with the other groups.Conclusion: LASS2/TMSG1 S248A promotes proliferation, migration and invasion of prostate cancer cells through increasing ATP6V0C expression, suggesting that aa248-250 is an important function site for LASS2/TMSG1 in invasion suppression of prostate cancer cells.

Key words: Prostate cancer, Mutants, Vacuolar ATPase, ATP6V0C, LASS2/TMSG1

中图分类号: 

  • R737.25

表1

LASS2/TMSG1基因全长及其定位点突变体引物序列"

Gene Primer
LASS2/TMSG1 Forward: 5'-GGG ACA AGT TTG TAC AAA AAA GCA GGC TTA ATG CTC CAG ACC TTG TAT GAT TA-3'
Reverse: 5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA GTC ATT CTT ACG ATG GTT GT-3'
M1: S91A Forward: 5'-AAC ATT TCT ACC TGA CCG CTG GCA AGC AGC CCA AGC-3'
Reverse: 5'-GCT TGG GCT GCT TGC CAG CGG TCA GGT AGA AAT GTT-3'
M2: S110A Forward: 5'-CTG GCG GCC AGC GAG CCC GCT CT-3'
Reverse: 5'-AGA GCG GGC TCG CTG GCC GCC AG-3'
M3: S137A Forward: 5'-GGT AAA ATG TGA ATC TCC AGG CGG CTT CTC GGA ACT TCT TG-3'
Reverse: 5'-CAA GAA GTT CCG AGA AGC CGC CTG GAG ATT CAC ATT TTA CC-3'
M4: S248A Forward: 5'-CCG ATT ACC TGC TGG AGG CAG CCA AGA TGT TTA AC-3'
Reverse: 5'-GTT AAA CAT CTT GGC TGC CTC CAG CAG GTA ATC GG-3'

图1

LASS2/TMSG1 4个定点突变体位置"

图2

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8表达LASS2/TMSG1和ATP6V0C的影响"

图3

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8增殖能力的影响"

图4

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8细胞锚着不依赖能力的影响"

图5

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8细胞迁移能力的影响"

图6

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8细胞侵袭能力的影响"

图7

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8细胞凋亡的影响"

图8

LASS2/TMSG1全长及其点突变体对前列腺癌细胞系PC-3M-1E8细胞周期的影响"

图9

免疫荧光双重染色直接观察LASS2/TMSG1全长及其点突变体和 ATP6V0C 之间的共定位"

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