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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (2): 210-220. doi: 10.19723/j.issn.1671-167X.2019.02.003

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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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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

CLC Number: 

  • R737.25

Table 1

Primers of LASS2/TMSG1 and its mutants"

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'

Figure 1

Four direct point mutations from serine to alanine of LASS2/TMSG1 LASS2/TMSG1 Mut1,S91A;LASS2/TMSG1 Mut2,S110A;LASS2/TMSG1 Mut3, S137A; LASS2/TMSG1 Mut4, S248A."

Figure 2

The influence of LASS2/TMSG1 gene and its mutations on LASS2/TMSG1 and ATP6V0C expression in prostate cancer cell lines PC-3M-1E8 A,Using semi-quantitative real-time RT-PCR, the expression of LASS2/TMSG1 mRNA in the LASS2/TMSG1 group and Mut1-Mut4 groups is significantly higher than that in the Vector group; and LASS2/TMSG1 mRNA in Mut1, Mut2 and Mut4 groups is higher than that in the LASS2/TMSG1 group; Moreover, ATP6V0C mRNA in LASS2/TMSG1 group is lower than that in Vector group, and ATP6V0C mRNA in Mut1-Mut4 groups is higher than that in LASS2/TMSG1 group (#P<0.01); B, Using Western blot, LASS2/TMSG1 protein in LASS2/TMSG1 group and Mut1-Mut4 groups is higher than that in Vector group; C, Western blot showed that ATP6V0C protein in LASS2/TMSG1 group is lower than that in Vector group, but ATP6V0C protein in Mut4 group is obviously higher than that in Vector group."

Figure 3

The influence of LASS2/TMSG1 gene and its mutations on proliferation in prostate cancer cell lines PC-3M-1E8 MTT test(A) and growth curve essay(B) showed growth ability in Mut2 group and Mut4 group is increasing compared with other groups from day 5,Mut2 group>Mut4 group>Mut3 group>Vector group>Mut1 group>LASS2/TMSG1 group in turn."

Figure 4

The influence of LASS2/TMSG1 gene and its mutations on anchor independent growth ability in prostate cancer cell lines PC-3M-1E8 Soft Agar colony formation experiment showed anchor independent growth ability in Mut2 group and Mut4 group is higher than other groups(#P<0.01), Mut4 group>Mut2 group>Mut3 group>Vector group>Mut1 group>LASS2/TMSG1 group in turn."

Figure 5

The influence of LASS2/TMSG1 gene and its mutations on cell migration in prostate cancer cell lines PC-3M-1E8 Cell migration(scratch repair rate within 24 hours) in Mut2 group and Mut4 group is increasing compared with LASS2/TMSG1 group(#P<0.01), Mut4 group> Vector group> Mut2 group>Mut1 group>Mut3 group>LASS2/TMSG1 group in turn."

Figure 6

The influence of LASS2/TMSG1 gene and its mutations on cell invasion in prostate cancer cell lines PC-3M-1E8 More cells passed through Matrigel in Vector group and Mut4 group compared with LASS2/TMSG1 group(#P<0.01), Vector group>Mut4 group>Mut2 group>Mut1 group>LASS2/TMSG1 group>Mut3 group in turn. ns, not significant."

Figure 7

The influence of LASS2/TMSG1 gene and its mutations on cell apoptosis in prostate cancer cell lines PC-3M-1E8 Apoptosis ratio in Mut2 group and Mut4 group is obviously higher than that in LASS2/TMSG1 group and Vector group(#P<0.01), Mut2 group(apoptosis rate:29.77%)>Mut4 group(15.15%)>Mut1 group(7.13%)>LASS2/TMSG1 group(7%)>Vector group(6.52%)>Mut3 group(5.06%)in turn."

Figure 8

The influence of LASS2/TMSG1 gene and its mutations on cell cycle in prostate cancer cell lines PC-3M-1E8 G0/G1 ratio in Mut1 group and Mut4 group is obviously higher than that in LASS2/TMSG1 group, Mut4 group (G0/G1 ratio: 85.46%)>Mut1 group(79.88%)>Mut2 group (65.50%)>Vector group (64.98%)>Mut3 group (60.96%)>LASS2/TMSG1 group (51.06%)in turn."

Figure 9

Double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 prote in and its mutations Double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, showing that LASS2/TMSG1 protein(green) and ATP6V0C protein(red) are mainly co-localized in plasma (yellow). The expression of ATP6V0Cin Mut4 group increased significantly compared to other groups, and the co-localization signal was increased evidently. DAPI, 4', 6-diamidino-2-phenylindole."

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