DNAJC12 promotes lung cancer growth by regulating the activation of β‑catenin

Abstract

Lung cancer has become the leading cause of cancer‑associated mortality worldwide. However, the underlying mechanisms of lung cancer remain poorly understood. DnaJ heat shock protein family (HSP40) member C12 (DNAJC12) is a type III member belonging to the HSP40/DNAJ family. The role of DNAJC12 in numerous types of cancer has been previously reported; however, the effect of DNAJC12 in lung cancer remains unknown. The results of the present study indicated that DNAJC12 may be involved in lung cancer proliferation and migration by regulating the β‑catenin signaling pathway. Data generated in the present study and from The Cancer Genome Atlas revealed that the DNAJC12 expression levels were significantly upregulated in lung cancer tissues compared with non‑cancer lung tissues. The expression of DNAJC12 was subsequently knocked down in A549 and NCI‑H1975 lung cancer cells using lentiviral transfections and further experiments demonstrated that the knockdown of DNAJC12 inhibited the proliferation, colony formation, migration and invasion of lung cancer cells. The results of flow cytometric assays also revealed that the knockdown of DNAJC12 induced the apoptosis of lung cancer cells. In addition, the effects of DNAJC12 knockdown on the in vivo growth of lung cancer cells were observed. Signaling pathway analysis revealed that the knockdown of DNAJC12 expression suppressed the phosphorylation of p65 NF‑κB, downregulated the expression levels and inhibited the subsequent activation of β‑catenin, and downregulated the expression levels of vimentin. Rescue experiments demonstrated that the overexpression of β‑catenin, but not that of NF‑κB or vimentin, reversed the effects of DNAJC12 knockdown on the proliferation and invasion of lung cancer cells. On the whole, the findings of the present study suggest that DNAJC12 may play a crucial role in lung cancer tumorigenesis by regulating the expression and activation of β‑catenin. Therefore, DNAJC12 may represent a novel target for the treatment of lung cancer.

Keywords: DnaJ heat shock protein family (Hsp40) member C12; apoptosis; lung cancer; migration; proliferation; β‑catenin.

Figure 1

DNAJC12 expression levels are upregulated in human lung cancer tissues. (A) Fold change in DNAJC12 expression levels in 15 lung cancer tissues compared with paired adjacent non-cancer lung tissues were analyzed using reverse transcription-quantitative PCR. The fold change in the expression levels of each patient are presented. (B) Analysis of data from part (A) was performed using a paired Student's t-test. (C) Fold change in DNAJC12 expression levels in lung cancer tissues compared with paired non-cancer lung tissues obtained from TCGA database; n=57. (D) Analysis of data from part (C) was performed using a paired Student's t-test. ^***P<0.001. (E) Expression levels of DNAJC12 in lung cancer tissues compared with unpaired non-cancer lung tissues obtained from TCGA database. Control tissues, n=43; lung cancer tissues, n=498. ^***P<0.001. Data were analyzed using an unpaired Student's t-test. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; TCGA, The Cancer Genome Atlas.

Figure 2

Knockdown of DNAJC12 in lung cancer cells. (A) Relative mRNA expression levels of DNAJC12 were analyzed in the four lung cancer cell lines, A549, NCI-H1299, NCI-1975 and 95D by RT-qPCR. Fluorescence images demonstrating the infection efficiency of lentivirus expressing shDNAJC12 or shCtrl following 24 h of infection in (B) A549 and (C) NCI-H1975 cells. RT-qPCR and western blot analysis were used to determine DNAJC12 knockdown efficiency in (D) A549 and (E) NCI-H1975 cells transfected as described in parts (B and C), respectively. ^***P<0.001. Data were analyzed using a Student's t-test. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin RNA; Ctrl, control.

Figure 3

Knockdown of DNAJC12 inhibits the proliferation of lung cancer cells. HCS and MTT assays were used to determine the effects of DNAJC12 on A549 and NCI-H1975 cell proliferation. (A and B) Representative images of the HCS assay using A549 cells with/without DNAJC12 knockdown (A) and the statistical result (B). The relative cell number was analyzed using a one-way ANOVA. ^***P<0.001. (C and D) MTT assay was used to demonstrate that DNAJC12 knockdown inhibited the proliferation of (C) A549 and (D) NCI-H1975 cells with/without DNAJC12 knockdown. The relative cell number was analyzed using a one-way ANOVA. ^***P<0.001. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; HCS, high-content screening.

Figure 4

Knockdown of DNAJC12 reduces the colony formation of lung cancer cells. (A) A549 and (B) NCI-H1975 cells with/without DNAJC12 knockdown were subjected to a colony formation assay and the number of colonies was analyzed 2 weeks later. DNAJC12 knockdown reduced the number of colonies formed in both lung cancer cell lines. ^***P<0.001. Data were analyzed using a Student's t-test. DNAJC12 knockdown reduced the size of the colonies from (C) A549 and (D) NCI-H1975 lung cancer cells. Representative colony size of A549 cells is shown in (A) and the representative colony size of NCI-H1975 cells is shown in (B). DNAJC12, DnaJ heat shock protein family (Hsp40) member C12.

Figure 5

Knockdown DNAJC12 represses lung cancer cell growth in vivo. (A) Microscopy images of xenograft tumors grown in nude mice that were transplanted with 1×10⁷ shCtrl- and shDNAJC12-transfected A549 cells. (B) Tumor volume was monitored for 42 days after transplantation. ^***P<0.001. Data were analyzed using a one-way ANOVA. The maximum tumor diameter obtained was 14.85 mm. The maximum tumor volume obtained was 1,247.66 mm³. (C) Tumors were weighed immediately after sacrificing the mice. ^***P<0.001. Data were analyzed using a Student's t-test. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; sh, short hairpin RNA; Ctrl, control.

Figure 6

Knockdown of DNAJC12 inhibits the migration and invasion of lung cancer cells. The knockdown of DNAJC12 inhibited the migration of (A) A549 and (B) NCI-H1975 lung cancer cells. Number of migratory cells per field were analyzed. ^***P<0.001. Data were analyzed using a Student's t-test. Invasion of A549 and NCI-H1975 cells with/without DNAJC12 knockdown was analyzed using a Matrigel invasion assay. DNAJC12 knockdown inhibited the invasion of (C) A549 and (D) NCI-H1975 lung cancer cells. Number of invasive cells per field were analyzed. ^***P<0.001. Data were analyzed using a Student's t-test. Migration of A549 or NCI-H1975 cells with/without DNAJC12 knockdown was analyzed using a wound-healing assay. The knockdown of DNAJC12 inhibited the migration of (E) A549 and (F) NCI-H1975 lung cancer cells. The migratory rate was analyzed at the end of the experiment. ^***P<0.001. Data were analyzed using a Student's t-test. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12.

Figure 7

Knockdown of DNAJC12 induces the apoptosis of lung cancer cells. (A) A549 and (B) NCI-H1975 cells were infected with lentivirus expressing shDNAJC12 or shCtrl for 24 h, then the cells were harvested and subjected to apoptosis analysis with flow cytometry. Knockdown of DNAJC12 was discovered to induce the apoptosis of A549 and NCI-H1975 cells, respectively. ^***P<0.001. Data were analyzed using a Student's t-test. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; short hairpin RNA; Ctrl, control.

Figure 8

DNAJC12 regulates NF-κB, β-catenin and vimentin signaling pathways. Total protein extracted from A549 cells with/without DNAJC12 knockdown was analyzed using western blotting. (A) DNAJC12 knockdown reduced the phosphorylation of NF-κB p65 in lung cancer cells. (B) DNAJC12 knockdown reduced the phosphorylation and downregulated the expression levels of β-catenin in lung cancer cells. (C) DNAJC12 knockdown downregulated vimentin expression levels in lung cancer cells. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12.

Figure 9

β-catenin is involved in the effect of DNAJC12 on lung cancer cells. (A) RT-qPCR analysis was used to determine DNAJC12 knockdown efficiency and overexpression efficiency of CTNNB1, VIM and RELA in A549 cells. ^***P<0.001. Data were analyzed using one-way ANOVA followed by a Tukey's post hoc test. (B) A549 cells with knocked down DNAJC12 expression were infected with lentivirus overexpressing p65, β-catenin, or vimentin. Cells were analyzed using a HCS assay. The overexpression of β-catenin reversed the DNAJC12-induced inhibition of proliferation. (C) Cells were infected with lentivirus overexpressing β-catenin and proliferation was analyzed using an MTT assay. The overexpression of β-catenin reversed the DNAJC12-induced inhibition of proliferation. (D) Cells were infected with lentivirus overexpressing β-catenin and invasion was analyzed using a Transwell invasion assay. The overexpression of β-catenin reversed the migration of A549 lung cancer cells. ^**P<0.01. Data were analyzed using a one-way ANOVA. DNAJC12, DnaJ heat shock protein family (Hsp40) member C12; HCS, high-content screening.